Improving the degradation behavior and in vitro biological property of nano-hydroxyapatite surface- grafted with the assist of citric acid.

PubMed

Jiang, Liuyun; Jiang, Lixin; Xiong, Chengdong; Su, Shengpei

2016-10-01

To obtain ideal nano-hydroxyapatite(n-HA) filler for poly(lactide-co-glycolide) (PLGA), a new surface-grafting with the assist of citric acid for nano-hydroxyapatite (n-HA) was designed, and the effect of n-HA surface-grafted with or without citric acid on in vitro degradation behavior and cells viability was studied by the experiments of soaking in simulated body fluid (SBF) and incubating with human osteoblast-like cells (MG-63). The change of pH value, tensile strength reduction, the surface deposits, cells attachment and proliferation of samples during the soaking and incubation were investigated by means of pH meter, electromechanical universal tester, scanning electron microscope (SEM) coupled with energy-dispersive spectro-scopy (EDS), fluorescence microscope and MTT method. The results showed that the introduction of citric acid not only delayed the strength reduction during the degradation by inhibiting the detachment of n-HA from PLGA, but also endowed it better cell attachment and proliferation, suggesting that the n-HA surface-grafted with the assist of citric acid was an important bioactive ceramic fillers for PLGA used as bone materials. Copyright © 2016 Elsevier B.V. All rights reserved.

Intracellular trafficking of hyaluronic acid-chitosan oligomer-based nanoparticles in cultured human ocular surface cells.

PubMed

Contreras-Ruiz, Laura; de la Fuente, María; Párraga, Jenny E; López-García, Antonio; Fernández, Itziar; Seijo, Begoña; Sánchez, Alejandro; Calonge, Margarita; Diebold, Yolanda

2011-01-27

Nanoparticles are a promising alternative for ocular drug delivery, and our group has proposed that they are especially suited for ocular mucosal disorders. The goal of the present study was to determine which internalization pathway is used by cornea-derived and conjunctiva-derived cell lines to take up hyaluronic acid (HA)-chitosan oligomer (CSO)-based nanoparticles (HA-CSO NPs). We also determined if plasmids loaded onto the NPs reached the cell nucleus. HA-CSO NPs were made of fluoresceinamine labeled HA and CSO by ionotropic gelation and were conjugated with a model plasmid DNA for secreted alkaline phosphatase. Human epithelial cell lines derived from the conjunctiva and the cornea were exposed to HA-CSO NPs for 1 h and the uptake was investigated in living cells by fluorescence microscopy. The influence of temperature and metabolic inhibition, the effect of blocking hyaluronan receptors, and the inhibition of main endocytic pathways were studied by fluorometry. Additionally, the metabolic pathways implicated in the degradation of HA-CSO NPs were evaluated by lysosome identification. There was intracellular localization of plasmid-loaded HACSO NPs in both corneal and conjunctival cells. The intracellular presence of NPs diminished with time. HA-CSO NP uptake was significantly reduced by inhibition of active transport at 4 °C and by sodium azide. Uptake was also inhibited by blocking hyaluronan receptors with anti-CD44 Hermes-1 antibody, by excess HA, and by filipin, an inhibitor of caveolin-dependent endocytosis. HA-CSO NPs had no effect on cell viability. The transfection efficiency of the model plasmid was significantly higher in NP treated cells than in controls. HA-CSO NPs were internalized by two different ocular surface cell lines by an active transport mechanism. The uptake was mediated by hyaluronan receptors through a caveolin-dependent endocytic pathway, yielding remarkable transfection efficiency. Most of HA-CSO NPs were metabolized within 48 h. This uptake did not compromise cell viability. These findings further support the potential use of HA-CSO NPs to deliver genetic material to the ocular surface.

HA and double-layer HA-P2O5/CaO glass coatings: influence of chemical composition on human bone marrow cells osteoblastic behavior.

PubMed

Ferraz, M P; Fernandes, M H; Santos, J D; Monteiro, F J

2001-07-01

Human osteoblastic bone marrow derived cells were cultured for 28 days onto the surface of a glass reinforced hydroxyapatite (HA) composite and a commercial type HA plasma sprayed coatings, both in the "as-received" condition and after an immersion treatment with culture medium during 21 days. Cell proliferation and differentiation were analyzed as a function of the chemical composition of the coatings and the immersion treatment. Cell attachment, growth and differentiation of osteoblastic bone marrow cells seeded onto "as-received" plasma sprayed coatings were strongly affected by the time-dependent variation of the surface structure occurring during the first hours of culture. Initial interactions leading to higher amounts of adsorbed protein and zeta potential shifts towards negative charges appeared to result in surface structures with better biological performance. Cultures grown onto the pretreated coatings showed higher rate of cell proliferation and increased functional activity, as compared to those grown onto the corresponding "as-received" materials. However, the cell behavior was similar in the glass composite and HA coatings. The results showed that the glass composites present better characteristics for bone cell growth and function than HA. In addition, this work also provide evidence that the biological performance of the glass composites can be modulated and improved by manipulations in the chemical composition, namely in the content of glass added to HA. Copyright 2001 Kluwer Academic Publishers

MC3T3-E1 cell response to stainless steel 316L with different surface treatments.

PubMed

Zhang, Hongyu; Han, Jianmin; Sun, Yulong; Huang, Yongling; Zhou, Ming

2015-11-01

In the present study, stainless steel 316L samples with polishing, aluminum oxide blasting, and hydroxyapatite (HA) coating were prepared and characterized through a scanning electron microscope (SEM), optical interferometer (surface roughness, Sq), contact angle, surface composition and phase composition analyses. Osteoblast-like MC3T3-E1 cell adhesion on the samples was investigated by cell morphology using a SEM (4h, 1d, 3d, 7d), and cell proliferation was assessed by MTT method at 1d, 3d, and 7d. In addition, adsorption of bovine serum albumin on the samples was evaluated at 1h. The polished sample was smooth (Sq: 1.8nm), and the blasted and HA coated samples were much rougher (Sq: 3.2μm and 7.8μm). Within 1d of incubation, the HA coated samples showed the best cell morphology (e.g., flattened shape and complete spread), but there was no significant difference after 3d and 7d of incubation for all the samples. The absorbance value for the HA coated samples was the highest after 1d and 3d of incubation, indicating better cell viability. However, it reduced to the lowest value at 7d. Protein adsorption on the HA coated samples was the highest at 1h. The results indicate that rough stainless steel surface improves cell adhesion and morphology, and HA coating contributes to superior cell adhesion, but inhibits cell proliferation. Copyright © 2015. Published by Elsevier B.V.

Adsorption of Amorphous Silica Nanoparticles onto Hydroxyapatite Surfaces Differentially Alters Surfaces Properties and Adhesion of Human Osteoblast Cells

PubMed Central

Kalia, Priya; Brooks, Roger A.; Kinrade, Stephen D.; Morgan, David J.; Brown, Andrew P.; Rushton, Neil; Jugdaohsingh, Ravin

2016-01-01

Silicon (Si) is suggested to be an important/essential nutrient for bone and connective tissue health. Silicon-substituted hydroxyapatite (Si-HA) has silicate ions incorporated into its lattice structure and was developed to improve attachment to bone and increase new bone formation. Here we investigated the direct adsorption of silicate species onto an HA coated surface as a cost effective method of incorporating silicon on to HA surfaces for improved implant osseointegration, and determined changes in surface characteristics and osteoblast cell adhesion. Plasma-sprayed HA-coated stainless steel discs were incubated in silica dispersions of different concentrations (0–42 mM Si), at neutral pH for 12 h. Adsorbed Si was confirmed by XPS analysis and quantified by ICP-OES analysis following release from the HA surface. Changes in surface characteristics were determined by AFM and measurement of surface wettability. Osteoblast cell adhesion was determined by vinculin plaque staining. Maximum Si adsorption to the HA coated disc occurred after incubation in the 6 mM silica dispersion and decreased progressively with higher silica concentrations, while no adsorption was observed with dispersions below 6 mM Si. Comparison of the Si dispersions that produced the highest and lowest Si adsorption to the HA surface, by TEM-based analysis, revealed an abundance of small amorphous nanosilica species (NSP) of ~1.5 nm in diameter in the 6 mM Si dispersion, with much fewer and larger NSP in the 42 mM Si dispersions. 29Si-NMR confirmed that the NSPs in the 6 mM silica dispersion were polymeric and similar in composition to the larger NSPs in the 42 mM Si dispersion, suggesting that the latter were aggregates of the former. Amorphous NSP adsorbed from the 6 mM dispersion on to a HA-coated disc surface increased the surface’s water contact angle by 53°, whereas that adsorbed from the 42 mM dispersion decreased the contact angle by 18°, indicating increased and decreased hydrophobicity, respectively. AFM showed an increase in surface roughness of the 6 mM Si treated surface, which correlated well with an increase in number of vinculin plaques. These findings suggest that NSP of the right size (relative to charge) adsorb readily to the HA surface, changing the surface characteristics and, thus, improving osteoblast cell adhesion. This treatment provides a simple way to modify plasma-coated HA surfaces that may enable improved osseointegration of bone implants. PMID:26863624

Physical Properties and Cellular Responses to Crosslinkable Poly(Propylene Fumarate)/Hydroxyapatite Nanocomposites

PubMed Central

Lee, Kee-Won; Wang, Shanfeng; Yaszemski, Michael J.; Lu, Lichun

2008-01-01

A series of crosslinkable nanocomposites has been developed using hydroxyapatite (HA) nanoparticles and poly(propylene fumarate) (PPF). PPF/HA nanocomposites with four different weight fractions of HA nanoparticles have been characterized in terms of thermal and mechanical properties. To assess surface chemistry of crosslinked PPF/HA nanocomposites, their hydrophilicity and capability of adsorbing proteins have been determined using static contact angle measurement and MicroBCA protein assay kit after incubation with 10% fetal bovine serum (FBS), respectively. In vitro cell studies have been performed using MC3T3-E1 mouse pre-osteoblast cells to investigate the ability of PPF/HA nanocomposites to support cell attachment, spreading, and proliferation after 1, 4, and 7 days. By adding HA nanoparticles to PPF, the mechanical properties of crosslinked PPF/HA nanocomposites have not been increased due to the initially high modulus of crosslinked PPF. However, hydrophilicity and serum protein adsorption on the surface of nanocomposites have been significantly increased, resulting in enhanced cell attachment, spreading, and proliferation after 4 days of cell seeding. These results indicate that crosslinkable PPF/HA nanocomposites are useful for hard tissue replacement because of excellent mechanical strength and osteoconductivity. PMID:18403013

Influence of multilayer rhBMP-2 DNA coating on the proliferation and differentiation of MC3T3-E1 cells seeded on roughed titanium surface.

PubMed

Jiang, Qiao-Hong; Liu, Li; Shen, Jian-Wei; Peel, Sean; Yang, Guo-Li; Zhao, Shi-Fang; He, Fu-Ming

2012-10-01

For bone morphogenetic protein (BMP) gene therapy to be a viable approach for enhancing implant osseointegration clinically, requires the development of efficient nonviral delivery vectors that can coat the implant. This study evaluated a multilayer cationic liposome-DNA complex (LDc) coating as a delivery vehicle for recombinant human BMP-2 (rhBMP-2). Multilayered coatings, comprising hyaluronic acid (HA) and LDc, were fabricated onto titanium using a layer-by-layer (LBL) assembly technique. Preosteoblastic MC3T3-E1 cells were cultured on the roughened titanium surfaces coated with multilayers of HA/LDc, or on uncoated or HA/liposome only surfaces as controls. The amount of rhBMP-2 secreted by the MC3T3-E1 cells and the effect of the various surfaces on cell viability, proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and calcium deposition were evaluated. Messenger RNA levels of OC, ALP, Runx2, and Osx were also investigated. The results demonstrated that rhBMP-2 protein secreted into culture medium at 3 days was significantly higher than control groups. MC3T3-E1 cells cultured on the HA/LDc coating displayed significantly higher ALP activity and OC secretion at 7 days and 14 days culture, respectively. MC3T3-E1 cells cultured on HA/LDc upregulated expression of the osteoblast differentiation markers, especially on days 12 for OC and on days 6 and 12 for ALP and Osx. In conclusion, MC3T3-E1 cell cultured on the multilayer HA/LDc coating surface can secret rhBMP-2 protein and the protein levels were effective in inducing early osteogenic differentiation. Copyright © 2012 Wiley Periodicals, Inc.

Silver oxide-containing hydroxyapatite coating supports osteoblast function and enhances implant anchorage strength in rat femur.

PubMed

Eto, Shuichi; Miyamoto, Hiroshi; Shobuike, Takeo; Noda, Iwao; Akiyama, Takayuki; Tsukamoto, Masatsugu; Ueno, Masaya; Someya, Shinsuke; Kawano, Shunsuke; Sonohata, Motoki; Mawatari, Masaaki

2015-09-01

Antibacterial silver with hydroxyapatite (Ag-HA) is a promising coating material for imparting antibacterial properties to implants. We previously reported that 3% (w/w) silver with HA (3% Ag-HA) has both antibacterial activity and osteoconductivity. In this study, we investigated the effects of Ag-HA on the in vitro osteoblast function and the in vivo anchorage strength and osteoconductivity of implants. Production of the osteoblast marker alkaline phosphatase, but not cytotoxicity, was observed in cells of the osteoblast cell line MC3T3-E1 cultured on the 3% Ag-HA-coated surface. These results were similar to those observed with silver-free HA coating. In contrast, a significant high level of cytotoxicity was observed when the cells were cultured on a 50% Ag-HA-coated surface. The anchorage strength of implants inserted into the femur of Sprague-Dawley (SD) rats was enhanced by coating the implants with 3% Ag-HA. On the 3% Ag-HA-coated surface, both metaphyseal and diaphyseal areas were largely covered with new bone and had adequate osteoconductivity. These results suggest that 3% Ag-HA, like conventional HA, promotes osteogenesis by supporting osteoblast viability and function and thereby contributes to sufficient anchorage strength of implants. Application of 3% Ag-HA, which combines the osteoconductivity of HA and the antibacterial activity of silver, to prosthetic joints will help prevent postoperative infections. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Nano hydroxyapatite-blasted titanium surface affects pre-osteoblast morphology by modulating critical intracellular pathways.

PubMed

Bezerra, Fábio; Ferreira, Marcel R; Fontes, Giselle N; da Costa Fernandes, Célio Jr; Andia, Denise C; Cruz, Nilson C; da Silva, Rodrigo A; Zambuzzi, Willian F

2017-08-01

Although, intracellular signaling pathways are proposed to predict the quality of cell-surface relationship, this study addressed pre-osteoblast behavior in response to nano hydroxyapatite (HA)-blasted titanium (Ti) surface by exploring critical intracellular pathways and pre-osteoblast morphological change. Physicochemical properties were evaluated by atomic force microscopy (AFM) and wettability considering water contact angle of three differently texturized Ti surfaces: Machined (Mac), Dual acid-etching (DAE), and nano hydroxyapatite-blasted (nHA). The results revealed critical differences in surface topography, impacting the water contact angle and later the osteoblast performance. In order to evaluate the effect of those topographical characteristics on biological responses, we have seeded pre-osteoblast cells on the Ti discs for up to 4 h and subjected the cultures to biological analysis. First, we have observed pre-osteoblasts morphological changes resulting from the interaction with the Ti texturized surfaces whereas the cells cultured on nHA presented a more advanced spreading process when compared with the cells cultured on the other surfaces. These results argued us for analyzing the molecular machinery and thus, we have shown that nHA promoted a lower Bax/Bcl2 ratio, suggesting an interesting anti-apoptotic effect, maybe explained by the fact that HA is a natural element present in bone composition. Thereafter, we investigated the potential effect of those surfaces on promoting pre-osteoblast adhesion and survival signaling by performing crystal violet and immunoblotting approaches, respectively. Our results showed that nHA promoted a higher pre-osteoblast adhesion supported by up-modulating FAK and Src activations, both signaling transducers involved during eukaryotic cell adhesion. Also, we have shown Ras-Erk stimulation by the all evaluated surfaces. Finally, we showed that all Ti-texturing surfaces were able to promote osteoblast differentiation up to 10 days, when alkaline phosphatase (ALP) activity and osteogenic transcription factors were up-modulated. Altogether, our results showed for the first time that nano hydroxyapatite-blasted titanium surface promotes crucial intracellular signaling network responsible for cell adapting on the Ti-surface.Biotechnol. Bioeng. 2017;114: 1888-1898. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Quantitative analysis of osteoblast behavior on microgrooved hydroxyapatite and titanium substrata.

PubMed

Lu, Xiong; Leng, Yang

2003-09-01

The effects of implant surface topography and chemistry on osteoblast behavior have been a research focus because of their potential importance in orthopedic and dental applications. This work focused on the topographic effects of hydroxyapatite (HA) and titanium (Ti) surface that had identical micropatterns to determine whether there was synergistic interaction between surface chemistry and surface topography. Surface microgrooves with six different groove widths (4, 8, 16, 24, 30, and 38 microm) and three different groove depths (2, 4, and 10 microm) were made on single crystalline silicon wafers using microfabrication techniques. Ti and HA thin films were coated on the microgrooves by radio-frequency magnetron sputtering. After that, human osteoblast-like cells were seeded and cultured on the microgrooved surfaces for up to 7 days. The cells' behavior was examined using scanning electron microscopy after cells were fixed and dehydrated. Statistical analysis was based on quantitative data of orientation angle, evaluating the contact guidance, and form index, describing cell shape or cell morphology changes. The contact guidance and cell shape changes were observed on the HA and Ti microgrooves. No difference in orientation angle between HA and Ti microgrooves was found. This might suggest that surface chemistry was not a significant influence on cell guidance. However, the form index analysis indicated an interaction between topographic effects and surface chemistry. Thus, conclusions about surface topographic effects on cell behavior drawn from one type of material cannot simply be applied to another type of material. Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 677-687, 2003

Development and characterization of titanium-containing hydroxyapatite for medical applications.

PubMed

Huang, J; Best, S M; Bonfield, W; Buckland, Tom

2010-01-01

Hydroxyapatite containing levels of titanium (TiHA) of up to 1.6 wt.% has been produced via a chemical co-precipitation route. The distribution of Ti was seen by transmission electron microscopy/energy-dispersive X-ray analysis to be uniform throughout as-prepared nanosized TiHA particles (20 nm x 100 nm). The incorporation of Ti into the HA structure was found to influence the ceramic microstructure on sintering and the grain size was found to decrease from 0.89 microm with HA to 0.63 microm with 0.8 wt.% TiHA (0.8 TiHA) and 0.45 microm with 1.6 wt.% TiHA (1.6 TiHA). Rietveld refinement analysis showed that there was a proportional increase in both the a and c axis with incorporation of Ti into the HA lattice structure, leading to an increase in the cell volume with the addition of Ti. Fourier transform-Raman analysis showed a slight increase in the ratio of O-H/P-O peaks on TiHA, in comparison with HA. A bone-like apatite layer was formed on the surface of TiHA after immersion in simulated body fluid for 3 days, which demonstrated the high in vitro bioactivity of TiHA. In vitro culture with primary human osteoblast (HOB) cells revealed that TiHA was able to support the growth and proliferation of HOB cells in vitro, with a significantly higher cell activity being observed on 0.8 TiHA after 7 days of culture in comparison with that on HA. Well-organized actin cytoskeletal protein was developed after 1 day of culture, and an increase in cell filopodia (attachment) was observed on TiHA sample surfaces. The results indicate that TiHA has great potential for biomedical applications.

Detachment strength of human osteoblasts cultured on hydroxyapatite with various surface roughness. Contribution of integrin subunits.

PubMed

Kokkinos, Petros A; Koutsoukos, Petros G; Deligianni, Despina D

2012-06-01

Hydroxyapatite (HA) has been widely used as a bone substitute in dental, maxillofacial and orthopaedic surgery and as osteoconductive bone substitute or precoating of pedicle screws and cages in spine surgery. The aim of the present study was to investigate the osteoblastic adhesion strength on HA substrata with different surface topography and biochemistry (pre-adsorption of fibronectin) after blocking of specific integrin subunits with monoclonal antibodies. Stoichiometric HA was prepared by precipitation followed by ageing and characterized by SEM, EDX, powder XRD, Raman spectroscopy, TGA, and specific surface area analysis. Human bone marrow derived osteoblasts were cultured on HA disc-shaped substrata which were sintered and polished resulting in two surface roughness grades. For attachment evaluation, cells were incubated with monoclonal antibodies and seeded for 2 h on the substrata. Cell detachment strength was determined using a rotating disc device. Cell detachment strength was surface roughness, fibronectin preadsorption and intergin subunit sensitive.

Improved cell viability and hydroxyapatite growth on nitrogen ion-implanted surfaces

NASA Astrophysics Data System (ADS)

Shafique, Muhammad Ahsan; Murtaza, G.; Saadat, Shahzad; Uddin, Muhammad K. H.; Ahmad, Riaz

2017-08-01

Stainless steel 306 is implanted with various doses of nitrogen ions using a 2 MV pelletron accelerator for the improvement of its surface biomedical properties. Raman spectroscopy reveals incubation of hydroxyapatite (HA) on all the samples and it is found that the growth of incubated HA is greater in higher ion dose samples. SEM profiles depict uniform growth and greater spread of HA with higher ion implantation. Human oral fibroblast response is also found consistent with Raman spectroscopy and SEM results; the cell viability is found maximum in samples treated with the highest (more than 300%) dose. XRD profiles signified greater peak intensity of HA with ion implantation; a contact angle study revealed hydrophilic behavior of all the samples but the treated samples were found to be lesser hydrophilic compared to the control samples. Nitrogen implantation yields greater bioactivity, improved surface affinity for HA incubation and improved hardness of the surface.

Effect of micro-nano-hybrid structured hydroxyapatite bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway.

PubMed

Mao, Lixia; Liu, Jiaqiang; Zhao, Jinglei; Chang, Jiang; Xia, Lunguo; Jiang, Lingyong; Wang, Xiuhui; Lin, Kaili; Fang, Bing

2015-01-01

The surface structure of bioceramic scaffolds is crucial for its bioactivity and osteoinductive ability, and in recent years, human periodontal ligament stem cells have been certified to possess high osteogenic and cementogenic differential ability. In the present study, hydroxyapatite (HA) bioceramics with micro-nano-hybrid surface (mnHA [the hybrid of nanorods and microrods]) were fabricated via hydrothermal reaction of the α-tricalcium phosphate granules as precursors in aqueous solution, and the effects of mnHA on the attachment, proliferation, osteogenic and cementogenic differentiations of human periodontal ligament stem cells as well as the related mechanisms were systematically investigated. The results showed that mnHA bioceramics could promote cell adhesion, proliferation, alkaline phosphatase (ALP) activity, and expression of osteogenic/cementogenic-related markers including runt-related transcription factor 2 (Runx2), ALP, osteocalcin (OCN), cementum attachment protein (CAP), and cementum protein (CEMP) as compared to the HA bioceramics with flat and dense surface. Moreover, mnHA bioceramics stimulated gene expression of low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin, which are the key genes of canonical Wnt signaling. Moreover, the stimulatory effect on ALP activity and osteogenic and cementogenic gene expression, including that of ALP, OCN, CAP, CEMP, and Runx2 of mnHA bioceramics could be repressed by canonical Wnt signaling inhibitor dickkopf1 (Dkk1). The results suggested that the HA bioceramics with mnHA could act as promising grafts for periodontal tissue regeneration.

Surface characterization of colloidal-sol gel derived biphasic HA/FA coatings.

PubMed

Cheng, Kui; Zhang, Sam; Weng, Wenjian

2007-10-01

Hydroxyapatite (HA) powders are ultrasonically dispersed in the precursor of fluoridated hydroxyapatite (FHA) or fluorapatite (FA) to form a "colloidal sol". HA/FA biphasic coatings are prepared on Ti6Al4V substrate via dip coating, 150 degrees C drying and 600 degrees C firing. The coatings show homogenous distribution of HA particles in the FA matrix. The relative phase proportion can be tailored by the amount of HA in the colloidal sol. The surfaces of the coatings consist of two kinds of distinct domains: HA and FA, resulting in a compositionally heterogeneous surface. The biphasic coating surface becomes increasingly rougher with HA powders, from around 200 nm of pure FA to 400-600 nm in Ra of biphasic coatings. The rougher biphasic HA/FA surfaces with chemically controllable domains will favor cell attachment, apatite layer deposition and necessary dissolution in clinical applications.

Antimicrobial and osteogenic properties of a hydrophilic-modified nanoscale hydroxyapatite coating on titanium.

PubMed

Murakami, Asuka; Arimoto, Takafumi; Suzuki, Dai; Iwai-Yoshida, Misato; Otsuka, Fukunaga; Shibata, Yo; Igarashi, Takeshi; Kamijo, Ryutaro; Miyazaki, Takashi

2012-04-01

Hydroxyapatite (HA)-coated titanium (Ti) is commonly used for implantable medical devices. This study examined in vitro osteoblast gene expression and antimicrobial activity against early and late colonizers of supra-gingival plaque on nanoscale HA-coated Ti prepared by discharge in a physiological buffered solution. The HA-coated Ti surface showed super-hydrophilicity, whereas the densely sintered HA and Ti surfaces alone showed lower hydrophilicity. The sintered HA and HA-coated Ti surfaces enhanced osteoblast phenotypes in comparison with the bare Ti surface. The HA-coated Ti enabled antimicrobial activity against early colonizers of supra-gingival plaques, namely Streptococcus mitis and Streptococcus gordonii. Such antimicrobial activity may be caused by the surface hydrophilicity, thereby leading to a repulsion force between the HA-coated Ti surface and the bacterial cell membranes. On the contrary, the sintered HA sample was susceptible to infection of microorganisms. Thus, hydrophilic-modified HA-coated Ti may have potential for use in implantable medical devices. From the Clinical Editor: This study establishes that Hydroxyapatite (HA)-coated titanium (Ti) surface of implanted devices may result in an optimal microenvironment to control and prevent infections and may have potential future clinical applications. Copyright © 2012 Elsevier Inc. All rights reserved.

The influence of rat mesenchymal stem cell CD44 surface markers on cell growth, fibronectin expression, and cardiomyogenic differentiation on silk fibroin - Hyaluronic acid cardiac patches.

PubMed

Yang, Ming-Chia; Chi, Nai-Hsin; Chou, Nai-Kuan; Huang, Yi-You; Chung, Tze-Wen; Chang, Yu-Lin; Liu, Hwa-Chang; Shieh, Ming-Jium; Wang, Shoei-Shen

2010-02-01

Since MSCs contain an abundant of CD44 surface markers, it is of interesting to investigate whether CD44 on rat MSC (rMSCs) influenced cell growth, fibronectin expression and cardiomyogenic differentiation on new SF/HA cardiac patches. For this investigation, we examined the influences of rMSCs with or without a CD44-blockage treatment on the aforementioned issues after they were cultivated, and further induced by 5-aza on SF and SF/HA patches. The results showed that the relative growth rates of rMSCs cultured on cultural wells, SF/HA patches without or with a CD44-blockage treatment were 100%, 208.9+/-7.1 (%) or 48.4+/-6.0 (%) (n=3, for all), respectively, after five days of cultivations. Moreover, rMSCs cultivated on SF/HA patches highly promoted fibronectin expressions (e.g., 1.8x10(5)/cell, in fluorescent intensity) while cells with a CD44-blockage treatment markedly diminished the expressions (e.g., 1.1x10(4)/cell, in fluorescent intensity) on same patches. For investigating possible influences of CD44 surface markers of rMSCs on their cardiomyogenic differentiation, the expressions of specific cardiac genes of cells were examined by using real-time PCR analysis. The results indicated that 5-aza inducing rMSCs significantly promoted the expressions of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P<0.01 or better, n=3) on SF/HA patches compared with those expressions on SF patches and for cells with a CD44-blockage treatment on SF/HA patches. Furthermore, the intensity of the expressions of cardiotin and connexin 43 of 5-aza inducing rMSCs were markedly higher than those of cells with a CD44-blockage treatment after they were cultured on SF/HA patches. Through this study, we reported that CD44 surface markers of rMSCs highly influenced the proliferations, fibronectin expressions and cardiomyogenic differentiation of rMSCs cultivated on cardiac SF/HA patches.

The mechanism for keratinocyte detaching from pH-responsive chitosan.

PubMed

Chen, Yi-Hsin; Chang, Shao-Hsuan; Wang, I-Jong; Young, Tai-Horng

2014-11-01

In this study, we compared the detachment ratio of HaCaT and Hs68 cells from pH-responsive chitosan surface by raising medium pH from 7.20 to 7.65 for 60 min. The detachment ratio of elongated Hs68 cells was over 75%, but that of round-shaped HaCaT cells was less than 50%, even extending the incubation time to 6 h or enhancing the cytoskeletal contractile force with the Rho activator CN01. However, the addition of 2 mm of EDTA into the medium at pH 7.65 could effectively detach HaCaT cells (detachment ratio > 90%), indicating that the calcium ion played an important role in the detachment process. Therefore, the family of Ca(+2)-dependent integrin receptors was examined by RT-PCR, real-time PCR and immunocytochemistry. It was found the expression of integrin β4 (ITGb4) was HaCaT cell-specific and the mRNA level of ITGb4 in undetached HaCaT cells was significantly higher than that in detached ones. By modulating ITGb4 activity with specific functional blocking antibody ASC-8, the detachment ratio of HaCaT cells could be increased to be greater than 85%. Conversely, the addition of the ligand of ITGb4 laminin into the culture system decreased the medium pH-induced detachment ratio for HaCaT cells, but not for Hs68 cells. Further addition of ASC-8 could rescue the effect of laminin on preventing the detachment of HaCaT cells from pH-sensitive chitosan surface. Therefore, this study demonstrated the interaction of ITGb4 and laminin played an important role in controlling the detachment of HaCaT cells on pH-responsive chitosan. Copyright © 2014 Elsevier Ltd. All rights reserved.

Analysis of CD44-Hyaluronan Interactions in an Artificial Membrane System

PubMed Central

Wolny, Patricia M.; Banerji, Suneale; Gounou, Céline; Brisson, Alain R.; Day, Anthony J.; Jackson, David G.; Richter, Ralf P.

2010-01-01

CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ∼30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of ≤10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan. PMID:20663884

Metabolism, survival, and gene expression of Pseudomonas putida to hematite nanoparticles mediated by surface-bound humic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ouyang, Kai; Walker, Sharon L.; Yu, Xiao-Ying

Natural organic matter (NOM) is likely to coat naturally occurring nanoparticles (NNPs) in the soil environment and poses distinct effects on the interaction between NPs and soil microorganisms, however such topic has not been well investigated. This study explored the influence of nanoparticle surface-bound humic acid (HA, as a model NOM) on the toxicity of hematite NPs (i.e., nano-Fe2O3) to Pseudomonas putida (P. putida). Results showed that nano-Fe2O3 could inhibit the bacterial growth with an IC50 of 23.58 mg L-1, while nanoparticle surface-bound HA could significantly alleviate the P. putida toxicity of nano-Fe2O3. IC50 of nano-Fe2O3 increased to 4774.23 mgmore » L-1 as a result of surface-saturation by HA. Co-precipitation experiment and transmission electron microscopy observation revealed that nanoparticle surface-bound HA prevented the adhesion of nano-Fe2O3 to the cells as well as limited cell internalization of nanoparticles due to the increased electrostatic repulsion. The generation of intracellular reactive oxygen species (ROS) was significantly limited by the nanoparticle surface-bound HA. The prevention of adhesion and inhibition of ROS generation could account for the HA-mitigated nanotoxicity. Interfacial interactions between hematite NPs and cell membrane were also evaluated on the basis of the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory, and the magnitude of interaction energy barrier correlated well with the 48 h LC50 data of hematite NPs to P. putida. This result implies that metal oxide NPs with strong association with the cell surface might induce more severe cytotoxicity in microorganisms.« less

Biodegradable magnesium-hydroxyapatite metal matrix composites.

PubMed

Witte, Frank; Feyerabend, Frank; Maier, Petra; Fischer, Jens; Störmer, Michael; Blawert, Carsten; Dietzel, Wolfgang; Hort, Norbert

2007-04-01

Recent studies indicate that there is a high demand to design magnesium alloys with adjustable corrosion rates and suitable mechanical properties. An approach to this challenge might be the application of metal matrix composite (MMC) based on magnesium alloys. In this study, a MMC made of magnesium alloy AZ91D as a matrix and hydroxyapatite (HA) particles as reinforcements have been investigated in vitro for mechanical, corrosive and cytocompatible properties. The mechanical properties of the MMC-HA were adjustable by the choice of HA particle size and distribution. Corrosion tests revealed that HA particles stabilised the corrosion rate and exhibited more uniform corrosion attack in artificial sea water and cell solutions. The phase identification showed that all samples contained hcp-Mg, Mg(17)Al(12), and HA before and after immersion. After immersion in artificial sea water CaCO3 was found on MMC-HA surfaces, while no formation of CaCO3 was found after immersion in cell solutions with and without proteins. Co-cultivation of MMC-HA with human bone derived cells (HBDC), cells of an osteoblasts lineage (MG-63) and cells of a macrophage lineage (RAW264.7) revealed that RAW264.7, MG-63 and HBDC adhere, proliferate and survive on the corroding surfaces of MMC-HA. In summary, biodegradable MMC-HA are cytocompatible biomaterials with adjustable mechanical and corrosive properties.

Preparation, characterization, and in vitro osteoblast functions of a nano-hydroxyapatite/polyetheretherketone biocomposite as orthopedic implant material

PubMed Central

Ma, Rui; Tang, Songchao; Tan, Honglue; Lin, Wentao; Wang, Yugang; Wei, Jie; Zhao, Liming; Tang, Tingting

2014-01-01

A bioactive composite was prepared by incorporating 40 wt% nano-hydroxyapatite (nHA) into polyetheretherketone (PEEK) through a process of compounding, injection, and molding. The mechanical and surface properties of the nHA/PEEK composite were characterized, and the in vitro osteoblast functions in the composite were investigated. The mechanical properties (elastic modulus and compressive strength) of the nHA/PEEK composite increased significantly, while the tensile strength decreased slightly as compared with PEEK. Further, the addition of nHA into PEEK increased the surface roughness and hydrophilicity of the nHA/PEEK composite. In cell tests, compared with PEEK and ultra-high-molecular-weight polyethylene, it was found that the nHA/PEEK composite could promote the functions of MC3T3-E1 cells, including cell attachment, spreading, proliferation, alkaline phosphatase activity, calcium nodule formation, and expression of osteogenic differentiation-related genes. Incorporation of nHA into PEEK greatly improved the bioperformance of PEEK. The nHA/PEEK composite might be a promising orthopedic implant material. PMID:25170265

PLGA/nHA hybrid nanofiber scaffold as a nanocargo carrier of insulin for accelerating bone tissue regeneration

NASA Astrophysics Data System (ADS)

Haider, Adnan; Gupta, Kailash Chandra; Kang, Inn-Kyu

2014-06-01

The development of tissue engineering in the field of orthopedic surgery is booming. Two fields of research in particular have emerged: approaches for tailoring the surface properties of implantable materials with osteoinductive factors as well as evaluation of the response of osteogenic cells to these fabricated implanted materials (hybrid material). In the present study, we chemically grafted insulin onto the surface of hydroxyapatite nanorods (nHA). The insulin-grafted nHAs (nHA-I) were dispersed into poly(lactide-co-glycolide) (PLGA) polymer solution, which was electrospun to prepare PLGA/nHA-I composite nanofiber scaffolds. The morphology of the electrospun nanofiber scaffolds was assessed by field emission scanning electron microscopy (FESEM). After extensive characterization of the PLGA/nHA-I and PLGA/nHA composite nanofiber scaffolds by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction spectroscopy (XRD), X-ray photoelectron spectroscopy (XPS), energy-dispersive X-ray spectrometry (EDS), and transmission electron microscopy (TEM), the PLGA/nHA-I and PLGA/nHA (used as control) composite nanofiber scaffolds were subjected to cell studies. The results obtained from cell adhesion, alizarin red staining, and Von Kossa assay suggested that the PLGA/nHA-I composite nanofiber scaffold has enhanced osteoblastic cell growth, as more cells were proliferated and differentiated. The fact that insulin enhanced osteoblastic cell proliferation will open new possibilities for the development of artificial scaffolds for bone tissue regeneration.

Effect of micro-nano-hybrid structured hydroxyapatite bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway

PubMed Central

Mao, Lixia; Liu, Jiaqiang; Zhao, Jinglei; Chang, Jiang; Xia, Lunguo; Jiang, Lingyong; Wang, Xiuhui; Lin, Kaili; Fang, Bing

2015-01-01

The surface structure of bioceramic scaffolds is crucial for its bioactivity and osteoinductive ability, and in recent years, human periodontal ligament stem cells have been certified to possess high osteogenic and cementogenic differential ability. In the present study, hydroxyapatite (HA) bioceramics with micro-nano-hybrid surface (mnHA [the hybrid of nanorods and microrods]) were fabricated via hydrothermal reaction of the α-tricalcium phosphate granules as precursors in aqueous solution, and the effects of mnHA on the attachment, proliferation, osteogenic and cementogenic differentiations of human periodontal ligament stem cells as well as the related mechanisms were systematically investigated. The results showed that mnHA bioceramics could promote cell adhesion, proliferation, alkaline phosphatase (ALP) activity, and expression of osteogenic/cementogenic-related markers including runt-related transcription factor 2 (Runx2), ALP, osteocalcin (OCN), cementum attachment protein (CAP), and cementum protein (CEMP) as compared to the HA bioceramics with flat and dense surface. Moreover, mnHA bioceramics stimulated gene expression of low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin, which are the key genes of canonical Wnt signaling. Moreover, the stimulatory effect on ALP activity and osteogenic and cementogenic gene expression, including that of ALP, OCN, CAP, CEMP, and Runx2 of mnHA bioceramics could be repressed by canonical Wnt signaling inhibitor dickkopf1 (Dkk1). The results suggested that the HA bioceramics with mnHA could act as promising grafts for periodontal tissue regeneration. PMID:26648716

Biocompatibility of nano-hydroxyapatite/polyetheretherketone composite materials with osteoblasts cultured in vitro

NASA Astrophysics Data System (ADS)

Wang, Lin; Huang, Feijuan; Wu, Zhengzhi; Ma, Rui

2017-04-01

The biocompatibility of the Sprague Dawley (SD) rat osteoblasts, which were cultured on the surfaces of nano-hydroxyapatite/polyetheretherketone (n-HA/PEEK) composites were investigated in this work. The osteoblasts of 24- hour old SD rats were cultured and identified by modified enzymatic digestion in vitro. The morphology and proliferation of cells were observed in CCK-8 regent staining, inverted microscopes, and by scanning electron microscopy (SEM) respectively. The results show that n-HA/PEEK composites have good biocompatibility with SD osteoblasts and that they can promote the growth of the cells that were cultured on the surfaces of the composites. The content of HA in n- HA/PEEK composites plays an important role in cell proliferation.

Control of hydroxyapatite coating by self-assembled monolayers on titanium and improvement of osteoblast adhesion.

PubMed

Shen, Juan; Qi, Yongcheng; Jin, Bo; Wang, Xiaoyan; Hu, Yamin; Jiang, Qiying

2017-01-01

Self-assembly technique was applied to introduce functional groups and form hydroxyl-, amine-, and carboxyl-terminal self-assembled monolayers (SAMs). The SAMs were grafted onto titanium substrates to obtain a molecularly smooth functional surface. Subsequent hydrothermal crystal growth formed homogeneous and crack-free crystalline hydroxyapatite (HA) coatings on these substrates. AFM and XPS were used to characterize the SAM surfaces, and XRD, SEM, and TEM were used to characterize the HA coatings. Results show that highly crystalline, dense, and oriented HA coatings can be formed on the OH-, NH 2 -, and COOH-SAM surfaces. The SAM surface with -COOH exhibited stronger nucleating ability than that with -OH and -NH 2 . The nucleation and growth processes of HA coatings were effectively controlled by varying reaction time, pH, and temperature. By using this method, highly crystalline, dense, and adherent HA coatings were obtained. In addition, in vitro cell evaluation demonstrated that HA coatings improved cell adhesion as compared with pristine titanium substrate. The proposed method is considerably effective in introducing the HA coatings on titanium surfaces for various biomedical applications and further usage in other industries. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 124-135, 2017. © 2015 Wiley Periodicals, Inc.

[Construction of porous hydroxyapatite (HA) block loaded with cultured chondrocytes].

PubMed

Yan, M; Dang, G

1999-07-01

To construct a kind of bone healing enhancing implant with cultured chondrocytes bound to hydroxyapatite (HA). Chondrocytes were obtained from the costicartilage of rat and were cultured on the porous HA blocks, 3 mm x 3 mm x 4 mm size, for three and seven days. Scanning electron micrograph was taken to show whether the cells grew outside and inside the pore of HA block. The cells cultured on tiny glass sheet for 2 days were used to prove where the cells come from by in situ hybridization technique with alpha1 (II) cDNA probe. Scanning electron micrographs showed that the pores of the HA surface and inside of the blocks are filled with cultured cells, especially the longer cultured block. The cells were chondrocytes confirmed by in situ hybridization. The porous HA can be used as cell cultured substrate and chondrocyte can adhere and proliferate inside the porous HA block.

Cellular proliferation, cellular viability, and biocompatibility of HA-ZnO composites.

PubMed

Saha, Naresh; Dubey, Ashutosh K; Basu, Bikramjit

2012-01-01

One of the important issues in the development of hydroxyapatite (HA)-based biomaterials is the prosthetic infection, which limits wider use of monolithic HA despite superior cellular response. Recently, we reported that ZnO addition to HA can induce bactericidal property. It is therefore important to assess how ZnO addition influences the cytotoxicity property and cell adhesion/proliferation on HA-ZnO composite surfaces in vitro. In the above perspective, the objective of this study is to investigate the cell type and material composition dependent cellular proliferation and viability of pressureless sintered HA-ZnO composites. The combination of cell viability data as well as morphological observations of cultured human osteoblast-like SaOS2 cells and mouse fibroblast L929 cells suggests that HA-ZnO composites containing 10 Wt % or lower ZnO exhibit the ability to support cell adhesion and proliferation. Both SaOS2 and L929 cells exhibit extensive multidirectional network of actin cytoskeleton and cell flattening on the lower ZnO containing (≤10 Wt %) HA-ZnO composites. The in vitro results illustrate how variation in ZnO content can influence significantly the cell vitality, as evaluated using MTT biochemical assay. Also, the critical statistical analysis reveals that ZnO addition needs to be carefully tailored to ensure good in vitro cytocompatibility. The underlying reasons for difference in biological properties are analyzed. It is suggested that surface wettability as well as dissolution of ZnO, both contribute to the observed differences in cellular viability and proliferation. Copyright © 2011 Wiley Periodicals, Inc.

Activation of Osteoblastic Function on Titanium Surface with Titanium-Doped Hydroxyapatite Nanoparticle Coating: An In Vitro Study.

PubMed

Nakazawa, Masahiro; Yamada, Masahiro; Wakamura, Masato; Egusa, Hiroshi; Sakurai, Kaoru

Titanium-doped hydroxyapatite (TiHA) nanoparticles contain titanium atoms in the hydroxyapatite lattice, which can physicochemically functionalize the titanium surface without modification of the surface topography. This study aimed to evaluate the physicochemical properties of machined or microroughened titanium surfaces coated with TiHA nanoparticles and the functions of osteoblasts cultured on them. Titanium disks with commercially available surface topography, such as machined or sandblasted, large-grit, and acid-etched (SLA) surfaces, were coated with TiHA. The disks with original or TiHA-coated surfaces were evaluated in topography, wettability, and chemical composition. Osteoblastic cells from rat femurs were cultured on the disks and evaluated in proliferation and differentiation. TiHA coating changed from hydrophobicity to hydrophilicity on both machined and SLA surfaces. Calcium and phosphate atoms were detected all over the surface with TiHA coating regardless of the surface topography. However, the considerable change in the inherent surface topographies was not observed on both types of surfaces after TiHA coating. Osteoblastic proliferative activity at day 4 was increased by TiHA coating on both types of surfaces. TiHA coating did not enhance expressions of bone matrix-related genes such as osteocalcin, osteopontin, bone sialoprotein, alkaline phosphatase, and collagen I. However, depositions of collagen, osteocalcin, and calcium in the culture at days 7 and 20 were increased on both types of surface topographies with TiHA coating. TiHA coating enhanced extracellular matrix formation on smooth and microroughened titanium surfaces by increasing osteoblastic proliferative activity without the deterioration of differentiation through hydrophilic and chemical functionalization.

Superior in vitro biological response and mechanical properties of an implantable nanostructured biomaterial: Nanohydroxyapatite-silicone rubber composite.

PubMed

Thein-Han, W W; Shah, J; Misra, R D K

2009-09-01

A potential approach to achieving the objective of favorably modulating the biological response of implantable biopolymers combined with good mechanical properties is to consider compounding the biopolymer with a bioactive nanocrystalline ceramic biomimetic material with high surface area. The processing of silicone rubber (SR)-nanohydroxyapatite (nHA) composite involved uniform dispersion of nHA via shear mixing and ultrasonication, followed by compounding at sub-ambient temperature, and high-pressure solidification when the final curing reaction occurs. The high-pressure solidification approach enabled the elastomer to retain the high elongation of SR even in the presence of the reinforcement material, nHA. The biological response of the nanostructured composite in terms of initial cell attachment, cell viability and proliferation was consistently greater on SR-5wt.% nHA composite surface compared to pure SR. Furthermore, in the nanocomposite, cell spreading, morphology and density were distinctly different from that of pure SR. Pre-osteoblasts grown on SR-nHA were well spread, flat, large in size with a rough cell surface, and appeared as a group. In contrast, these features were less pronounced in SR (e.g. smooth cell surface, not well spread). Interestingly, an immunofluorescence study illustrated distinct fibronectin expression level, and stronger vinculin focal adhesion contacts associated with abundant actin stress fibers in pre-osteoblasts grown on the nanocomposite compared to SR, implying enhanced cell-substrate interaction. This finding was consistent with the total protein content and SDS-PAGE analysis. The study leads us to believe that further increase in nHA content in the SR matrix beyond 5wt.% will encourage even greater cellular response. The integration of cellular and molecular biology with materials science and engineering described herein provides a direction for the development of a new generation of nanostructured materials.

A novel route for synthesis of nanocrystalline hydroxyapatite from eggshell waste.

PubMed

Siva Rama Krishna, D; Siddharthan, A; Seshadri, S K; Sampath Kumar, T S

2007-09-01

The eggshell waste has been value engineered to a nanocrystalline hydroxyapatite (HA) by microwave processing. To highlight the advantages of eggshell as calcium precursor in the synthesis of HA (OHA), synthetic calcium hydroxide was also used to form HA (SHA) following similar procedure and were compared with a commercially available pure HA (CHA). All the HAs were characterized by X-ray powder diffraction (XRD) method, Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and specific surface area measurements. Nanocrystalline nature of OHA is revealed through characteristic broad peaks in XRD patterns, platelets of length 33-50 nm and width 8-14 nm in TEM micrograph and size calculations from specific surface area measurements. FT-IR spectra showed characteristic bands of HA and additionally peaks of carbonate ions. The cell parameter calculations suggest the formation of carbonated HA of B-type. The OHA exhibits superior sinterability in terms of hardness and density than both SHA and CHA may be due to larger surface area of its spherulite structure. The in vitro dissolution study shows longer stability in phosphate buffer and cell culture test using osteoblast cells establishes biocompatibility of OHA.

Nano-hydroxyapatite/β-CD/chitosan nanocomposite for potential applications in bone tissue engineering.

PubMed

Shakir, Mohammad; Jolly, Reshma; Khan, Mohd Shoeb; Rauf, Ahmar; Kazmi, Shadab

2016-12-01

Herein, we report the synthesis of a novel tri-component nanocomposite system incorporating β-cyclodextrin (β-CD) with nano-hydroxyapatite (n-HA) and chitosan (CS), (n-HA/β-CD/CS) at three different temperatures via co-precipitation method. The chemical interactions and surface morphology have been evaluated by TEM, SEM and AFM techniques revealing the agglomerated nanoparticles in CS/n-HA-HA binary system whereas the ternary systems produced needle shaped nanoparticles dispersed homogeneously at low temperature with more porous and rougher surface. The addition of β-CD in CS/n-HA at low temperature decreased the particle size and raised the thermal stability as compared to CS/n-HA. The comparative hemolytic, protein adsorption and platelet adhesion studies confirmed the better hemocompatibility of n-HA/β-CD/CS-(RT,HT,LT) nanocomposites relative to CS/n-HA. The cell viability has been evaluated in vitro using MG-63 cell line which revealed superior non toxicity of n-HA/β-CD/CS-LT nanocomposite in comparison to n-HA/β-CD/CS-(RT,HT) and CS/n-HA nanocomposites. Thus it may be concluded that the orchestrated organic/inorganic n-HA/β-CD/CS-(RT,HT,LT) nanocomposites exhibited relatively higher cell viability of human osteoblast cells, stimulated greater osteogenesis, controlled biodegradation, enhanced antibacterial activity with excellent in-vitro biomineralization and remarkable mechanical parameters as compared to CS/n-HA nanocomposite and thus may provide opportunities for potential use as an alternative biomaterial for Bone tissue engineering applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of drug loaded nanoparticles for tumor targeting. Part 1: Synthesis, characterization, and biological evaluation in 2D cell cultures.

PubMed

El-Dakdouki, Mohammad H; Puré, Ellen; Huang, Xuefei

2013-05-07

Nanoparticles (NPs) are being extensively studied as carriers for drug delivery, but they often have limited penetration inside tumors. We envision that by targeting an endocytic receptor on the cell surface, the uptake of NPs can be significantly enhanced through receptor mediated endocytosis. In addition, if the receptor is recycled to the cell surface, the NP cargo can be transported out of the cells, which is then taken up by neighboring cells thus enhancing solid tumor penetration. To validate our hypothesis, in the first of two articles, we report the synthesis of doxorubicin (DOX)-loaded, hyaluronan (HA) coated silica nanoparticles (SNPs) containing a highly fluorescent core to target CD44, a receptor expressed on the cancer cell surface. HA was conjugated onto amine-functionalized SNPs prepared through an oil-water microemulsion method. The immobilization of the cytotoxic drug DOX was achieved through an acid sensitive hydrazone linkage. The NPs were fully characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements, thermogravimetric analysis (TGA), UV-vis absorbance, and nuclear magnetic resonance (NMR). Initial biological evaluation experiments demonstrated that compared to ligand-free SNPs, the uptake of HA-SNPs by the CD44-expressing SKOV-3 ovarian cancer cells was significantly enhanced when evaluated in the 2D monolayer cell culture. Mechanistic studies suggested that cellular uptake of HA-SNPs was mainly through CD44 mediated endocytosis. HA-SNPs with immobilized DOX were endocytosed efficiently by the SKOV-3 cells as well. The enhanced tumor penetration and drug delivery properties of HA-SNPs will be evaluated in 3D tumor models in the subsequent paper.

Influence of nanoporosity on biological response of sol-gel-derived 70S30C bioactive glass monoliths

NASA Astrophysics Data System (ADS)

Thamma, Ukrit

In the field of bioactive glasses for hard tissue regeneration, the bioactivity of a material is measured by its ability to induce the formation of hydroxyapatite (HA), Ca10(PO4)6(OH)2, under physiological conditions. Due to its close chemical crystallographic resemblance to natural bones, the newly formed HA layer has been shown to be critical for the biological interaction and bonding between the surfaces of bioactive glasses and osteoblast (bone) cells. Since the formation mechanism of HA is dependent on the dissolution behavior of the bioactive glass substrate, the characteristics of HA layer are dominated by the glass composition and structure. By introducing nanoporosity into glass structure, the dissolution rate and HA growth rate on nanoporous sol-gel-derived glasses are drastically enhanced compared to that of non-porous melt-quench glasses with the same composition. While enhanced HA growth on nanoporous glass, compared to non-porous glass, was hypothesized to be associated with greater specific surface area (SSA), other studies argued that growth rate of HA layer on nanoporous glass is dominated by nanopore size distribution, and minimally affected by the bulk SSA of the underlying glass. In order to decouple the influence of nanopore size and SSA on HA formation, we have successfully fabricated homogeneous 70S30C bioactive glass monoliths with different nanopore sizes, yet similar SSA via sol-gel process. After 3-day PBS incubation of 70S30C nanoporous glass monoliths, the presence of hydroxyapatite and Type-B carbonated hydroxyapatite (HA/B-CHA) was confirmed by XPS and FTIR. Here, we report the influence of nanopore size on HA/CHA formation pathway, growth rate, and its microstructure. Due to pore-size limited diffusion of PO43-, two HA/CHA formation pathways were observed: HA/CHA surface deposition and/or HA/CHA incorporation into nanopores. HA/CHA growth rate on the surface of a nanoporous glass monolith is dominated by the pore-size limited transport of Ca2+ ions dissolved from nanoporous glass substrates. Furthermore, with rising overall growth rate controlled by nanopore size, HA/CHA microstructures evolved from needle-like, plate-like, and flower-like, respectively. Furthermore, the levels of initial cell attachment and protein adsorption on HA/CHA microstructures formed on different nanopore sizes were investigated. The initial cell attachment was quantified by measuring the density and average size of attached MC3T3-E1 cells after 2-hour seeding period. The amounts and conformation of adsorbed proteins after 2-hour incubation with HA/CHA were characterized by Western blot and FTIR, respectively. It was shown that the amounts of protein adsorption on various HA/CHA microstructures do not correlate with the initial MC3T3-E1 attachment, while the beta-sheet/alpha-helix ratios in Amide I of bovine albumin serum (BSA) adsorbed on HA/CHA microstructures do correlate to the level of initial cell attachment. This result suggests that the beta-sheet structure in BSA interacts with and activates the RGD sequence of adhesion proteins, such as fibronectin, upon adsorption, thus significantly enhancing the initial attachment of MC3T3-E1 cells. These findings provide new insights that can lead to a more detailed fundamental understanding of protein-surface and protein-protein interactions, which are crucial for the further development of bioactive material.

Nanostructured Ag+-substituted fluorhydroxyapatite-TiO2 coatings for enhanced bactericidal effects and osteoinductivity of Ti for biomedical applications

PubMed Central

Chang, Xiaotong; Wang, Zhenhui; Zhang, Xuejiao; Han, Shuguang; Su, Zhuobin; Yang, Hejie; Yang, Dongdong; Zhang, Xiaojun

2018-01-01

Background Poor mechanical properties, undesirable fast dissolution rate, and lack of antibacterial activity limit the application of hydroxyapatite (HA) as an implant coating material. To overcome these limitations, a hybrid coating of Ag+-substituted fluorhydroxyapatite and titania nanotube (TNT) was prepared. Methods The incorporation of silver into the HA-TiO2 hybrid coating improves its antimicrobial properties. The addition of F as a second binary element increases the structural stability of the coating. The TNT/F-and-Ag-substituted HA (FAgHA) bilayer coating on the Ti substrate was confirmed by X-ray diffraction, scanning electron microscope, energy-dispersive X-ray spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS). Results The results indicate that the FAgHA/TNT nanocomposite coating has a dense and uniform morphology with a nano-rod-like structure. The solubility measurement result shows that the substitution of F− ions into the AgHA structure has a positive effect on the dissolution resistance of HA. The adhesion strength of FAgHA/TNT has significantly increased because of the interlocking of the roughened surface with nano-rod-like particles that entered into the voids of the TiO2 nanotubes. Compared with that of the bare Ti, the corrosion current density of FAgHA/TNT-coated Ti substrate decreased from 3.71 to 0.18 μA, and its corrosion resistance increased by almost two orders of magnitude. Moreover, despite pure HA, the FAgHA killed all viable Staphylococcus aureus after 24 hours of incubation. Although the fabricated FAgHA/TNT coating is hydrophobic, it induced deposition of the typical spherical apatite when immersed in a simulated body fluid (SBF); the osteoblasts spread very well on the surface of the coating. In addition, in vitro cell culture tests demonstrated cell viability and alkaline phosphatase (ALP) similar to pure HA, which indicated good cytocompatibility. Interestingly, compared with bare Ti, FAgHA/TNT-coated Ti surface was innocent for cell vitality and even more beneficial for cell osteogenesis in vitro. Conclusion Enhancing the osseointegration and preventing infection in implants, the FAgHA/TNT-coated Ti makes implants more successful. PMID:29760549

In vitro evaluation of different heat-treated radio frequency magnetron sputtered calcium phosphate coatings.

PubMed

Yonggang, Yan; Wolke, Joop G C; Yubao, Li; Jansen, John A

2007-06-01

Surface chemical compositions, such as calcium/phosphorus ratio and phase content, have a strong influence on the bioactivity and biocompatibility of calcium phosphate (CaP) coatings as applied on orthopedic and dental implants. Hydroxylapatite (HA) and dicalcium pyrophosphate (DCPP) coatings were prepared on titanium substrates by RF magnetron sputter deposition. The surfaces were left as-prepared (amorphous HA coating; A-HA, amorphous DCPP coating; A-DCPP) or heat treated with: infrared (IR) at 550 degrees C (I-HA) or at 650 degrees C (I-DCPP), and a water steam at 140 degrees C (S-HA and S-DCPP). The surface changes of these coatings were determined after incubation in simulated body fluid (SBF). Also, the growth of rat bone marrow cells (RBM) was studied with scanning electron microscopy (SEM). Both IR and water steam heat treatment changed the sputter-deposited coatings from the amorphous into the crystalline phase. As-prepared amorphous coatings dissolved partially in SBF within 4 weeks of incubation, while heat-treated coatings supported the deposition of a precipitate, i.e., carbonated apatite on both I-HA and S-HA specimens, and tricalciumphosphate on the I-DCPP and S-DCPP specimens. The Ca/P ratio of the A-HA, I-HA, S-HA, A-DCPP, I-DCPP and S-DCPP coatings changed, respectively, from 1.98 to 1.12, 2.01 to 1.76, 1.91 to 1.68, 0.76 to 1.23, 0.76 to 1.26 and 1.62 to 1.55 after 4 weeks of incubation in SBF. Finally, the RBM cells grew well on all heat-treated coatings, but showed different mineralization morphology during cell culturing. The different heat-treatment procedures for the sputtered HA and DCPP coatings influenced the surface characteristics of these coatings, whereby a combination of crystallinity and specific phase composition (Ca/P ratio) strongly affected their in vitro bioactivity.

Measuring pKa of activation and pKi of inactivation for influenza hemagglutinin from kinetics of membrane fusion of virions and of HA expressing cells.

PubMed

Mittal, Aditya; Shangguan, Tong; Bentz, Joe

2002-11-01

The data for the pH dependence of lipid mixing between influenza virus (A/PR/8/34 strain) and fluorescently labeled liposomes containing gangliosides has been analyzed using a comprehensive mass action kinetic model for hemaglutinin (HA)-mediated fusion. Quantitative results obtained about the architecture of HA-mediated membrane fusion site from this analysis are in agreement with the previously reported results from analyses of data for HA-expressing cells fusing with various target membranes. Of the eight or more HAs forming a fusogenic aggregate, only two have to undergo the "essential" conformational change needed to initiate fusion. The mass action kinetic model has been extended to allow the analysis of the pKa for HA activation and pKi for HA inactivation. Inactivation and activation of HA following protonation were investigated for various experimental systems involving different strains of HA (A/PR/8/34, X:31, A/Japan). We find that the pKa for the final protonation site on each monomer of the trimer molecule is 5.6 to 5.7, irrespective of the strain. We also find that the pKi for the PR/8 strain is 4.8 to 4.9. The inactivation rate constants for HA, measured from experiments done with PR/8 virions fusing with liposomes and X:31 HA-expressing cells fusing with red blood cells, were both found to be of the order of 10(-4) s(-1). This number appears to be the minimal rate for HA's essential conformational change at low HA surface density. At high HA surface densities, we find evidence for cooperativity in the conformational change, as suggested by other studies.

Measuring pKa of activation and pKi of inactivation for influenza hemagglutinin from kinetics of membrane fusion of virions and of HA expressing cells.

PubMed Central

Mittal, Aditya; Shangguan, Tong; Bentz, Joe

2002-01-01

The data for the pH dependence of lipid mixing between influenza virus (A/PR/8/34 strain) and fluorescently labeled liposomes containing gangliosides has been analyzed using a comprehensive mass action kinetic model for hemaglutinin (HA)-mediated fusion. Quantitative results obtained about the architecture of HA-mediated membrane fusion site from this analysis are in agreement with the previously reported results from analyses of data for HA-expressing cells fusing with various target membranes. Of the eight or more HAs forming a fusogenic aggregate, only two have to undergo the "essential" conformational change needed to initiate fusion. The mass action kinetic model has been extended to allow the analysis of the pKa for HA activation and pKi for HA inactivation. Inactivation and activation of HA following protonation were investigated for various experimental systems involving different strains of HA (A/PR/8/34, X:31, A/Japan). We find that the pKa for the final protonation site on each monomer of the trimer molecule is 5.6 to 5.7, irrespective of the strain. We also find that the pKi for the PR/8 strain is 4.8 to 4.9. The inactivation rate constants for HA, measured from experiments done with PR/8 virions fusing with liposomes and X:31 HA-expressing cells fusing with red blood cells, were both found to be of the order of 10(-4) s(-1). This number appears to be the minimal rate for HA's essential conformational change at low HA surface density. At high HA surface densities, we find evidence for cooperativity in the conformational change, as suggested by other studies. PMID:12414698

Controlling the Biodegradation of Magnesium Implants Through Nanostructured Calcium-Phosphate Coating

NASA Astrophysics Data System (ADS)

Iskandar, Maria Emil

Magnesium (Mg) alloys, a novel class of degradable, metallic biomaterials, have attracted growing interest as a promising alternative for medical implant and device applications due to their advantageous mechanical and biological properties. Moreover, Mg is biodegradable in the physiological environments. However, the major obstacle for Mg to be used as medical implants is its rapid degradation in physiological fluids. Therefore, the present key challenge lies in controlling Mg degradation rate in the physiological environment. The objective of this study was to develop a nanostructured-hydroxyapatite (nHA) coating on polished Mg implants to control the degradation and bone tissue integration of the implants. The nHA coatings were deposited on Mg using the Spire's patented TPA process to moderate the aggressive degradation of Mg and to improve quick osteointegration between Mg and natural bone. Nanostructured-HA coatings mimic the nanostructure and chemistry of natural bone, which will provide a desirable environment for bone tissue regeneration. Surface morphology, element compositions, and crystal structures were characterized using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and x-ray diffractometry (XRD), respectively. SEM images of the deposited nHA-coating was analyzed using ImageJ's quantitative image analysis tool, to determine the nHA-coating particle size and thickness. The degradation of nHA-coated and non-coated Mg samples was investigated by incubating samples in phosphate buffered saline (PBS) and revised simulated body fluid (r-SBF), under standard cell culture conditions. To mimic the in vivo cell response in the physiological environment, rat bone marrow stromal cells (BMSC) were harvested and cultured with nHA-coated and non-coated polished Mg samples to determine cytocompatibilty. The degradation results suggested that the nanocoatings positively mediated Mg degradation. It can therefore be concluded that nHA-coatings show promise for controlling the biodegradation of Mg-based orthopedic implants and devices. Cell studies indicated significantly improved BMSC adhesion on the surfaces of the nHA-coated and non-coated Mg samples, in comparison to the cells surrounding the Mg samples. These results indicated that the nHA-coated and non-coated Mg samples promote cell activity on the surface. However, cell experiments must be repeated on a larger number of samples with extensive short and long term cell studies, to achieve more verifiable results.

Three-dimensional polycaprolactone-hydroxyapatite scaffolds combined with bone marrow cells for cartilage tissue engineering.

PubMed

Wei, Bo; Yao, Qingqiang; Guo, Yang; Mao, Fengyong; Liu, Shuai; Xu, Yan; Wang, Liming

2015-08-01

The goal of this study was to investigate the chondrogenic potential of three-dimensional polycaprolactone-hydroxyapatite (PCL-HA) scaffolds loaded with bone marrow cells in vitro and the effect of PCL-HA scaffolds on osteochondral repair in vivo. Here, bone marrow was added to the prepared PCL-HA scaffolds and cultured in chondrogenic medium for 10 weeks. Osteochondral defects were created in the trochlear groove of 29 knees in 17 New Zealand white rabbits, which were then divided into four groups that underwent: implantation of PCL-HA scaffolds (left knee, n = 17; Group 1), microfracture (right knee, n = 6; Group 2), autologous osteochondral transplantation (right knee, n = 6; Group 3), and no treatment (right knee, n = 5; Control). Extracellular matrix produced by bone marrow cells covered the surface and filled the pores of PCL-HA scaffolds after 10 weeks in culture. Moreover, many cell-laden cartilage lacunae were observed, and cartilage matrix was concentrated in the PCL-HA scaffolds. After a 12-week repair period, Group 1 showed excellent vertical and lateral integration with host bone, but incomplete cartilage regeneration and matrix accumulation. An uneven surface of regenerated cartilage and reduced distribution of cartilage matrix were observed in Group 2. In addition, abnormal bone growth and unstable integration between repaired and host tissues were detected. For Group 3, the integration between transplanted and host cartilage was interrupted. Our findings indicate that the PCL-HA scaffolds loaded with bone marrow cells improved chondrogenesis in vitro and implantation of PCL-HA scaffolds for osteochondral repairenhanced integration with host bone. However, cartilage regeneration remained unsatisfactory. The addition of trophic factors or the use of precultured cell-PCL-HA constructs for accelerated osteochondral repair requires further investigation. © The Author(s) 2015.

Influence of nanohydroxyapatite surface properties on Staphylococcus epidermidis biofilm formation.

PubMed

Barros, J; Grenho, L; Manuel, C M; Ferreira, C; Melo, L; Nunes, O C; Monteiro, F J; Ferraz, M P

2014-05-01

Nanohydroxyapatite (nanoHA), due to its chemical properties, has appeared as an exceptionally promising bioceramic to be used as bone regeneration material. Staphylococcus epidermidis have emerged as major nosocomial pathogens associated with infections of implanted medical devices. In this work, the purpose was to study the influence of the nanoHA surface characteristics on S. epidermidis RP62A biofilm formation. Therefore, two different initial inoculum concentrations (Ci) were used in order to check if these would affect the biofilm formed on the nanoHA surfaces. Biofilm formation was followed by the enumeration of cultivable cells and by scanning electron microscopy. Surface topography, contact angle, total surface area and porosimetry of the biomaterials were studied and correlated with the biofilm data. The surface of nanoHA sintered at 830 (nanoHA830) showed to be more resistant to S. epidermidis attachment and accumulation than that of nanoHA sintered at 1000 (nanoHA1000). The biofilm formed on nanoHA830 presented differences in terms of structure, surface coverage and EPS production when compared to the one formed on nanoHA1000 surface. It was observed that topography and surface area of nanoHA surfaces had influence on the bacterial attachment and accumulation. Ci influenced bacteria attachment and accumulation on nanoHA surfaces over time. The choice of the initial inoculum concentration was relevant proving to have an effect on the extent of adherence thus being a critical point for human health if these materials are used in implantable devices. This study showed that the initial inoculum concentration and surface material properties determine the rate of microbial attachment to substrata and consequently are related to biofilm-associated infections in biomaterials.

Influence of processing parameters on microstructure and biocompatibility of surface laser sintered hydroxyapatite-SiO2 composites.

PubMed

Kivitz, E; Görke, R; Schilling, A F; Zhang, J; Heinrich, J G

2013-05-01

Silica-doped hydroxyapatite (HA) is a promising material concerning biocompatibility to natural bone, bioactivity and osteoconductive characteristics. HA exhibits phase transformations during sintering which are attendant to the change in volume and thermal strain. To avoid cracks during sintering, the exact knowledge of the phase transition temperatures is necessary. The sintering behavior of HA can be improved by adding amorphous silica with a low coefficient of thermal expansion. Therefore, the phase transformations in the system HA-SiO2 were analyzed by using differential scanning calorimetry followed by quantitative phase analysis by X-ray diffraction with the Riedveld method. The maximum sintering temperature without reversible phase transformation was defined as 1265°C. In laser surface sintered (LSS) samples, amorphous SiO2 , HA, and Si-α-TCP (or α-TCP) were detected. By comparison, only crystalline phases, such as cristobalite, HA, β-TCP, and Si-α-TCP (or α-TCP), were determined after furnace sintering. Scanning electron microscopy micrographs of furnace sintered and LSS samples show the differences in the resulting microstructures. Biocompatibility was determined by measuring cell activity of osteoblasts cultivated on four laser-sintered materials in the HA-SiO2 system in comparison to normal cell culture plastic. Cell proliferation was similar on all surfaces. The level of the cell activity on day 8 varied depending on the composition of the material and increased linearly as the amorphous SiO2 content rose. Taken together a laser-based method to develop novel biocompatible HA-SiO2 ceramics with adjustable properties and possible applications as orthopedic bioceramics are discussed. Copyright © 2012 Wiley Periodicals, Inc.

Carbon fibers with a nano-hydroxyapatite coating as an excellent biofilm support for bioreactors

NASA Astrophysics Data System (ADS)

Liu, Qijie; Zhang, Chao; Bao, Yanling; Dai, Guangze

2018-06-01

A biofilm support with high biocompatibility is needed for bioreactors. A nano-hydroxyapatite (HA) coating on carbon fibers (CFs) was prepared by electrochemical deposition (ECD). The sludge immobilization assays, bacterial cells adhesion assays and Derjaguin-Landau-Verwey-Overbeek (DLVO) theory were used to evaluate the capacity of CF supports to immobilize activated sludge and bacterial cells. The sludge immobilization and bacterial cells adhesion assays illustrated that HA coating could enhance the capacity of CFs to immobilize microorganisms. SEM images showed that HA and bacterial cells formed a dense film on CFs surface. In addition, HA, acting as a glue, could combine CFs with bacterial cells or between cells, which helped CFs capture more bacterial cells. DLVO theory illustrated that CFs with HA coating had a lower total interaction energy than CFs without handling, explaining the higher capacity of CFs with HA coating to immobilize bacterial cells. This result was owning to the less negative zeta potential and higher hydrophilicity of CFs with HA coating, and the hydrophilicity made a greater contribution to the lower total interaction energy. Experiments and theory reveal that HA coating could enhance the biocompatibility of CFs, and CFs with HA coating could be used as an excellent biofilm support for bioreactors.

Sr-containing hydroxyapatite: morphologies of HA crystals and bioactivity on osteoblast cells.

PubMed

Aina, Valentina; Bergandi, Loredana; Lusvardi, Gigliola; Malavasi, Gianluca; Imrie, Flora E; Gibson, Iain R; Cerrato, Giuseppina; Ghigo, Dario

2013-04-01

A series of Sr-substituted hydroxyapatites (HA), of general formula Ca(10-x)Srx(PO4)6(OH)2, where x=2 and 4, were synthesized by solid state methods and characterized extensively. The reactivity of these materials in cell culture medium was evaluated, and the behavior towards MG-63 osteoblast cells (in terms of cytotoxicity and proliferation assays) was studied. Future in vivo studies will give further insights into the behavior of the materials. A paper by Lagergren et al. (1975), concerning Sr-substituted HA prepared by a solid state method, reports that the presence of Sr in the apatite composition strongly influences the apatite diffraction patterns. Zeglinsky et al. (2012) investigated Sr-substituted HA by ab initio methods and Rietveld analyses and reported changes in the HA unit cell volume and shape due to the Sr addition. To further clarify the role played by the addition of Sr on the physico-chemical properties of these materials we prepared Sr-substituted HA compositions by a solid state method, using different reagents, thermal treatments and a multi-technique approach. Our results indicated that the introduction of Sr at the levels considered here does influence the structure of HA. There is also evidence of a decrease in the crystallinity degree of the materials upon Sr addition. The introduction of increasing amounts of Sr into the HA composition causes a decrease in the specific surface area and an enrichment of Sr-apatite phase at the surface of the samples. Bioactivity tests show that the presence of Sr causes changes in particle size and/or morphology during soaking in MEM solution; on the contrary the morphology of pure HA does not change after 14 days of reaction. The presence of Sr, as Sr-substituted HA and SrCl2, in cultures of human MG-63 osteoblasts did not produce any cytotoxic effect. In fact, Sr-substituted HA increased the proliferation of osteoblast cells and enhanced cell differentiation: Sr in HA has a positive effect on MG-63 cells. In contrast, Sr ions alone, at the concentrations released by Sr-HA (1.21-3.24 ppm), influenced neither cell proliferation nor differentiation. Thus the positive effects of Sr in Sr-HA materials are probably due to the co-action of other ions such as Ca and P. Copyright © 2012 Elsevier B.V. All rights reserved.

Sol-gel-derived hydroxyapatite-carbon nanotube/titania coatings on titanium substrates.

PubMed

Ji, Xiaoli; Lou, Weiwei; Wang, Qi; Ma, Jianfeng; Xu, Haihong; Bai, Qing; Liu, Chuantong; Liu, Jinsong

2012-01-01

In this paper, hydroxyapatite-carbon nanotube/titania (HA-CNT/TiO(2)) double layer coatings were successfully developed on titanium (Ti) substrates intended for biomedical applications. A TiO(2) coating was firstly developed by anodization to improve bonding between HA and Ti, and then the layer of HA and CNTs was coated on the surface by the sol-gel process to improve the biocompatibility and mechanical properties of Ti. The surfaces of double layer coatings were uniform and crack-free with a thickness of about 7 μm. The bonding strength of the HA-CNT/TiO(2) coating was higher than that of the pure HA and HA-CNT coatings. Additionally, in vitro cell experiments showed that CNTs promoted the adhesion of preosteoblasts on the HA-CNT/TiO(2) double layer coatings. These unique surfaces combined with the osteoconductive properties of HA exhibited the excellent mechanical properties of CNTs. Therefore, the developed HA-CNT/TiO(2) coatings on Ti substrates might be a promising material for bone replacement.

Effect of Magnesium and Osteoblast Cell Presence on Hydroxyapatite Formation on (Ti,Mg)N Thin Film Coatings

NASA Astrophysics Data System (ADS)

Onder, Sakip; Calikoglu-Koyuncu, Ayse Ceren; Torun Kose, Gamze; Kazmanli, Kursat; Kok, Fatma Nese; Urgen, Mustafa

2017-07-01

TiN and (Ti,Mg)N thin film coatings were deposited on Ti substrates by an arc-physical vapor deposition technique. The effect of cell presence on hydroxyapatite (HA) formation was investigated using surfaces with four different Mg contents (0, 8.1, 11.31, and 28.49 at.%). Accelerated corrosion above 10 at.% Mg had a negative effect on the performance in terms of both cell proliferation and mineralization. In the absence of cells, Mg-free TiN coatings and low-Mg (8.1 at.%)-doped (Ti,Mg)N surfaces led to an early HA deposition (after 7 days and 14 days, respectively) in cell culture medium (DMEM), but the crystallinity was low. More crystalline HA structures were obtained in the presence of the cells. HA deposits with an ideal Ca/P ratio were obtained at least a week earlier, at day 14, in TiN and low-Mg (8.1 at.%)-doped (Ti,Mg)N compared with that of high-Mg-containing surfaces (>10 at.%). A thicker mineralized matrix was formed on low-Mg (8.1 at.%)-doped (Ti,Mg)N relative to that of the TiN sample. Low-Mg doping (<10 at.%) into TiN coatings resulted in better cell proliferation and thicker mineralized matrix formation, so it could be a promising alternative for hard tissue applications.

Physical and biological evaluations of sintered hydroxyapatite/silicone composite with covalent bonding for a percutaneous implant material.

PubMed

Furuzono, Tsutomu; Wang, Pao-Li; Korematsu, Arata; Miyazaki, Kozo; Oido-Mori, Mari; Kowashi, Yusuke; Ohura, Kiyoshi; Tanaka, Junzo; Kishida, Akio

2003-05-15

A composite (HA/silicone) of hydroxyapatite (HA) microparticles with an average diameter of 2.0 micro m covalently linked to a silicone substrate has been developed, and its physical and biological properties as a percutaneous soft-tissue-compatible material have been evaluated. In tensile property measurement, samples of HA/silicone and the original silicone were similar in tensile strength, ca. 7.8 MPa, and elongation at break, ca. 570%. It was found that chemical surface modification with HA particles presented no mechanical disadvantage. In an adhesive-tape peeling test, scanning electron microscopic (SEM) observation showed that HA particles coupled directly to the substrate were not removed. HA particles may bond strongly with the substrate. In human periodontal ligament fibroblast attachment and proliferation experiments, the number of cells attached to HA/silicone was 14 times greater than that attached to the original silicone after 24 h of incubation. The value on HA/silicone was ca. 80% versus that on a tissue-culture plastic used as a positive control. After 72 h of incubation, the number of cells grown on HA/silicone increased to the level of the positive control. In observation of fluorescence microscopy stained by Hoechst 33342, cells appeared to tightly adhere to HA particles coupled to the silicone sheet due to intact nuclear morphology. Observation of cells by fluorescence dye with rhodamin phalloidin showed an extensive F-actin cytoskeleton on HA/silicone. In a 4-week animal implant test, force required to pull out the HA/silicone sheet was 15 times that of the original silicone. HA-particle coating on silicone with covalent linkage gave the inert surface bioactivity. The HA composite thus effectively prevents germ infection percutaneously. Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 65B: 217-226, 2003

Interface activation and surface characteristics of Ti/TiN/HA coated sintered stainless steels

NASA Astrophysics Data System (ADS)

Choe, Han-Cheol; Ko, Yeong-Mu

2006-02-01

Interface activation and surface characteristics of Ti/TiN/HA film coated sintered stainless steels (SSS) have been investigated by electrochemical and biocompatibility tests. HA (hydroxyapatite), Ti, and Ti/TiN film coatings were applied using electron-beam deposition method (EB-PVD). Ti, Ti/TiN, and Ti/TiN/HA film coated surfaces and layers were investigated by SEM and XPS. The coated films showed micro-columnar structure, and Ti/TiN/HA films were denser than Ti or HA-only film. The corrosion resistance of the HA coating was similar to that of Ti/TiN/HA film coating when Cu content reached 4 wt.%, but the corrosion resistance of the HA coating decreased when Cu content increased from 4 wt.% in 0.9% NaCl solution. Therefore, HA-only coating could ensure corrosion resistance when Cu content does not exceed 4 wt.%. The results of biocompatibility tests of SSS on dogs showed that bone formation and biocompatibility were favorable when Cu content did not exceed 4 wt.%. The biocompatibility with bone was generally favorable in Ti/TiN/HA film coating and HA-only coating, while bone formation was somewhat faster for the HA film coated surface than for the Ti/TiN/HA film coating. Also, good cell growth and osseointegration without toxicity were observed.

Influence of three-dimensional hyaluronic acid microenvironments on mesenchymal stem cell chondrogenesis.

PubMed

Chung, Cindy; Burdick, Jason A

2009-02-01

Mesenchymal stem cells (MSCs) are multipotent progenitor cells whose plasticity and self-renewal capacity have generated significant interest for applications in tissue engineering. The objective of this study was to investigate MSC chondrogenesis in photo-cross-linked hyaluronic acid (HA) hydrogels. Because HA is a native component of cartilage, and MSCs may interact with HA via cell surface receptors, these hydrogels could influence stem cell differentiation. In vitro and in vivo cultures of MSC-laden HA hydrogels permitted chondrogenesis, measured by the early gene expression and production of cartilage-specific matrix proteins. For in vivo culture, MSCs were encapsulated with and without transforming growth factor beta-3 (TGF-beta3) or pre-cultured for 2 weeks in chondrogenic medium before implantation. Up-regulation of type II collagen, aggrecan, and sox 9 was observed for all groups over MSCs at the time of encapsulation, and the addition of TGF-beta3 further enhanced the expression of these genes. To assess the influence of scaffold chemistry on chondrogenesis, HA hydrogels were compared with relatively inert poly(ethylene glycol) (PEG) hydrogels and showed enhanced expression of cartilage-specific markers. Differences between HA and PEG hydrogels in vivo were most noticeable for MSCs and polymer alone, indicating that hydrogel chemistry influences the commitment of MSCs to undergo chondrogenesis (e.g., approximately 43-fold up-regulation of type II collagen of MSCs in HA over PEG hydrogels). Although this study investigated only early markers of tissue regeneration, these results emphasize the importance of material cues in MSC differentiation microenvironments, potentially through interactions between scaffold materials and cell surface receptors.

Increase in gap junctional intercellular communication by high molecular weight hyaluronic acid associated with fibroblast growth factor 2 and keratinocyte growth factor production in normal human dermal fibroblasts.

PubMed

Park, Jeong Ung; Tsuchiya, Toshie

2002-07-01

The effects of different molecular weights of hyaluronic acid (HA), a major component of extracellular matrix, on gap junctional intercellular communication (GJIC) in normal human dermal fibroblasts (NHDF cells) were investigated. NHDF cells were cultured for 4 days with different molecular weights of HA and then the extent of GJIC was assessed by the scrape-loading dye transfer method, using Lucifer yellow. The area of dye transfer was greater in the dishes coated with HA than in those to which HA was added. Thus, NHDF cells cultured on surfaces coated with high molecular weight (HMW) HA (MW, 800 kDa) showed greatly enhanced GJIC. Furthermore, another aim of this study was to evaluate the effects of different molecular weights of HA on the production of FGF-2 and KGF, because both are important cytokines produced by NHDF cells. When FGF-2 and KGF cultured levels of cell extracts and media were determined by ELISA, both levels were significantly enhanced when cells were grown on plates coated with HMW HA. This finding indicated that the function of gap junction channels in NHDF cells grown on plates coated with HMW HA may promote the biosynthesis of growth factors such as FGF-2 and KGF.

Real-Time Protein and Cell Binding Measurements on Hydroxyapatite Coatings

PubMed Central

Vilardell, A. M.; Cinca, N.; Jokinen, A.; Garcia-Giralt, N.; Dosta, S.; Cano, I. G.; Guilemany, J. M.

2016-01-01

Although a lot of in vitro and in vivo assays have been performed during the last few decades years for hydroxyapatite bioactive coatings, there is a lack of exploitation of real-time in vitro interaction measurements. In the present work, real-time interactions for a plasma sprayed hydroxyapatite coating were measured by a Multi-Parametric Surface Plasmon Resonance (MP-SPR), and the results were compared with standard traditional cell viability in vitro assays. MP-SPR is proven to be suitable not only for measurement of molecule–molecule interactions but also molecule–material interaction measurements and cell interaction. Although SPR is extensively utilized in interaction studies, recent research of protein or cell adsorption on hydroxyapatite coatings for prostheses applications was not found. The as-sprayed hydroxyapatite coating resulted in 62.4% of crystalline phase and an average thickness of 24 ± 6 μm. The MP-SPR was used to measure lysozyme protein and human mesenchymal stem cells interaction to the hydroxyapatite coating. A comparison between the standard gold sensor and Hydroxyapatite (HA)-plasma coated sensor denoted a clearly favourable cell attachment on HA coated sensor as a significantly higher signal of cell binding was detected. Moreover, traditional cell viability and proliferation tests showed increased activity with culture time indicating that cells were proliferating on HA coating. Cells show homogeneous distribution and proliferation along the HA surface between one and seven days with no significant mortality. Cells were flattened and spread on rough surfaces from the first day, with increasing cytoplasmatic extensions during the culture time. PMID:27618911

Expression and purification of RHC-EGFP fusion protein and its application in hyaluronic acid assay.

PubMed

Duan, Ningjun; Lv, Wansheng; Zhu, Lingli; Zheng, Weijuan; Hua, Zichun

2017-03-16

Hyaluronan is a widely distributed glycosaminoglycan which has multiple functions. Hyaluronic acid (HA) accumulation has been reported in many human diseases. Understanding the role of hyaluronan and its binding proteins in the pathobiology of disease will facilitate the development of novel therapeutics for many critical diseases. Current techniques described for the analysis of HA are mainly for HA quantification in solutions, not for the direct detection of HA in tissues or on cell surfaces. In our study, a fusion protein, named C-terminal domain of RHAMM-enhanced green fluorescence protein (RHC-EGFP), combined the HA-binding domain, C-terminal of receptor for hyaluronan-mediated motility, with EGFP, a widely used enhanced green fluorescence protein, was expressed and purified from Escherichia coli with high purity. Based on the sensitivity and convenience of fluorescence detection, methods for direct assay of HA in solutions, on cell surface or in tissues were established using RHC-EGFP. The binding specificity was also confirmed by competitive binding experiment and hyaluronidase degradation experiment. Our results provide an alternative choice for the specific and convenient assay of HA in various samples, and maybe helpful for further understanding of the fundamental and comprehensive functions of HA.

Replacement of the V3 domain in the surface subunit of the feline immunodeficiency virus envelope glycoprotein with the equivalent region of a T cell-tropic human immunodeficiency virus type 1 results in a chimeric surface protein that efficiently binds to CXCR4.

PubMed

González, Silvia A; Falcón, Juan I; Affranchino, José L

2014-03-01

Feline immunodeficiency virus (FIV) and the T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1) share the use of the chemokine receptor CXCR4 for cell entry. To study this process further we developed a cell surface binding assay based on the expression of a soluble version of the FIV SU C-terminally tagged with the influenza virus hemagglutinin epitope (HA). The specificity of the assay was demonstrated by the following evidence: (1) the SU-HA protein bound to HeLa cells that express CXCR4 but not to MDCK cells that lack this chemokine receptor; and (2) binding of the SU-HA to HeLa cells was blocked by incubation with the CXCR4 antagonist AMD3100 as well as with the anti-CXCR4 monoclonal antibody (MAb) 12G5. Deletion of the V3 region from the FIV SU glycoprotein abolished its ability to bind CXCR4-expressing cells. Remarkably, substitution of the V3 domain of the FIV SU by the equivalent region of the HIV-1 NL4-3 isolate resulted in efficient cell surface binding of the chimeric SU protein to CXCR4. Moreover, transfection of MDCK cells with a plasmid encoding human CXCR4 allowed the association of the chimeric SU-HA glycoprotein to the transfected cells. Interestingly, while cell binding of the chimeric FIV-HIV SU was inhibited by an anti-HIV-1 V3 MAb, its association with CXCR4 was found to be resistant to AMD3100. Of note, the chimeric FIV-HIV Env glycoprotein was capable of promoting CXCR4-dependent cell-to-cell fusion.

Fortifying the Bone-Implant Interface Part 1: An In Vitro Evaluation of 3D-Printed and TPS Porous Surfaces.

PubMed

MacBarb, Regina F; Lindsey, Derek P; Bahney, Chelsea S; Woods, Shane A; Wolfe, Mark L; Yerby, Scott A

2017-01-01

An aging society and concomitant rise in the incidence of impaired bone health have led to the need for advanced osteoconductive spinal implant surfaces that promote greater biological fixation ( e.g. for interbody fusion cages, sacroiliac joint fusion implants, and artificial disc replacements). Additive manufacturing, i.e. 3D-printing, may improve bone integration by generating biomimetic spinal implant surfaces that mimic bone morphology. Such surfaces may foster an enhanced cellular response compared to traditional implant surfacing processes. This study investigated the response of human osteoblasts to additive manufactured (AM) trabecular-like titanium implant surfaces compared to traditionally machined base material with titanium plasma spray (TPS) coated surfaces, with and without a nanocrystalline hydroxyapatite (HA) coating. For TPS-coated discs, wrought Ti6Al4V ELI was machined and TPS-coating was applied. For AM discs, Ti6Al4V ELI powder was 3D-printed to form a solid base and trabecular-like porous surface. The HA-coating was applied via a precipitation dip-spin method. Surface porosity, pore size, thickness, and hydrophilicity were characterized. Initial cell attachment, proliferation, alkaline phosphatase (ALP) activity, and calcium production of hFOB cells ( n =5 per group) were measured. Cells on AM discs exhibited expedited proliferative activity. While there were no differences in mean ALP expression and calcium production between TPS and AM discs, calcium production on the AM discs trended 48% higher than on TPS discs ( p =0.07). Overall, HA-coating did not further enhance results compared to uncoated TPS and AM discs. Results demonstrate that additive manufacturing allows for controlled trabecular-like surfaces that promote earlier cell proliferation and trends toward higher calcium production than TPS coating. Results further showed that nanocrystalline HA may not provide an advantage on porous titanium surfaces. Additive manufactured porous titanium surfaces may induce a more osteogenic environment compared to traditional TPS, and thus present as an attractive alternative to TPS-coating for orthopedic spinal implants.

Osteogenic Responses to Zirconia with Hydroxyapatite Coating by Aerosol Deposition

PubMed Central

Cho, Y.; Hong, J.; Ryoo, H.; Kim, D.; Park, J.

2015-01-01

Previously, we found that osteogenic responses to zirconia co-doped with niobium oxide (Nb2O5) or tantalum oxide (Ta2O5) are comparable with responses to titanium, which is widely used as a dental implant material. The present study aimed to evaluate the in vitro osteogenic potential of hydroxyapatite (HA)-coated zirconia by an aerosol deposition method for improved osseointegration. Surface analysis by scanning electron microscopy and x-ray diffraction proved that a thin as-deposited HA film on zirconia showed a shallow, regular, crater-like surface. Deposition of dense and uniform HA films was measured by SEM, and the contact angle test demonstrated improved wettability of the HA-coated surface. Confocal laser scanning microscopy indicated that MC3T3-E1 pre-osteoblast attachment did not differ notably between the titanium and zirconia surfaces; however, cells on the HA-coated zirconia exhibited a lower proliferation than those on the uncoated zirconia late in the culture. Nevertheless, ALP, alizarin red S staining, and bone marker gene expression analysis indicated good osteogenic responses on HA-coated zirconia. Our results suggest that HA-coating by aerosol deposition improves the quality of surface modification and is favorable to osteogenesis. PMID:25586588

Antibacterial properties of Ag-doped hydroxyapatite layers prepared by PLD method

NASA Astrophysics Data System (ADS)

Jelínek, Miroslav; Kocourek, Tomáš; Jurek, Karel; Remsa, Jan; Mikšovský, Jan; Weiserová, Marie; Strnad, Jakub; Luxbacher, Thomas

2010-12-01

Thin hydroxyapatite (HA), silver-doped HA and silver layers were prepared using a pulsed laser deposition method. Doped layers were ablated from silver/HA targets. Amorphous and crystalline films of silver concentrations of 0.06 at.%, 1.2 at.%, 4.4 at.%, 8.3 at.% and 13.7 at.% were synthesized. Topology was studied using scanning electron microscopy and atomic force microscopy. Contact angle and zeta potential measurements were conducted to determine the wettability, surface free energy and electric surface properties. In vivo measurement (using Escherichia coli cells) of antibacterial properties of the HA, silver-doped HA and silver layers was carried out. The best antibacterial results were achieved for silver-doped HA layers of silver concentration higher than 1.2 at.%.

Fabrication and Characterization of Carbon Fiber-Reinforced Nano-Hydroxyapatite/Polyamide46 Biocomposite for Bone Substitute.

PubMed

Deng, Zhennan; Han, Hongjuan; Yang, Jingyuan; Li, Yuanyuan; Du, Shengnan; Ma, Jianfeng

2017-05-24

BACKGROUND Ideal bone repair material should be of good biocompatibility and high bioactivity. Besides, their mechanical properties should be equivalent to those of natural bone. The objective of this study was to fabricate a novel biocomposite suitable for load-bearing bone defect repair. MATERIAL AND METHODS A novel biocomposite composed of carbon fiber, hydroxyapatite and polyamide46 (CF/HA/PA46) was fabricated, and its mechanical performances and preliminary cell responses were evaluated to explore its feasibility for load-bearing bone defect repair. RESULTS The resultant CF/HA/PA46 biocomposite showed a bending strength of 159-223 MPa, a tensile strength of 127-199 MPa and a tensile modulus of 7.7-10.8 GPa, when the CF content was 5-20% (mass fraction) in biocomposite. The MG63 cells, showing an osteogenic phenotype, were well adhered and spread on the surface of the CF/HA/PA46 biocomposite. Moreover, the cells vitality and differentiation on the CF/HA/PA46 biocomposite surface were obviously increased during the culture time and there was no significant difference between the CF/HA/PA46 biocomposite and HA/PA (as control) at all the experimental time (P>0.05). CONCLUSIONS The addition of CF into HA/PA46 composite manifest improved the mechanical performances and showed favorable effects on biocompatibility of MG63 cells. The obtained biocomposite has high potential for bone repair in load-bearing sites.

Fabrication and Characterization of Carbon Fiber-Reinforced Nano-Hydroxyapatite/Polyamide46 Biocomposite for Bone Substitute

PubMed Central

Deng, Zhennan; Han, Hongjuan; Yang, Jingyuan; Li, Yuanyuan; Du, Shengnan; Ma, Jianfeng

2017-01-01

Background Ideal bone repair material should be of good biocompatibility and high bioactivity. Besides, their mechanical properties should be equivalent to those of natural bone. The objective of this study was to fabricate a novel biocomposite suitable for load-bearing bone defect repair. Material/Methods A novel biocomposite composed of carbon fiber, hydroxyapatite and polyamide46 (CF/HA/PA46) was fabricated, and its mechanical performances and preliminary cell responses were evaluated to explore its feasibility for load-bearing bone defect repair. Results The resultant CF/HA/PA46 biocomposite showed a bending strength of 159–223 MPa, a tensile strength of 127–199 MPa and a tensile modulus of 7.7–10.8 GPa, when the CF content was 5–20% (mass fraction) in biocomposite. The MG63 cells, showing an osteogenic phenotype, were well adhered and spread on the surface of the CF/HA/PA46 biocomposite. Moreover, the cells vitality and differentiation on the CF/HA/PA46 biocomposite surface were obviously increased during the culture time and there was no significant difference between the CF/HA/PA46 biocomposite and HA/PA (as control) at all the experimental time (P>0.05). Conclusions The addition of CF into HA/PA46 composite manifest improved the mechanical performances and showed favorable effects on biocompatibility of MG63 cells. The obtained biocomposite has high potential for bone repair in load-bearing sites. PMID:28536416

Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.

PubMed

Amatsu, Sho; Sugawara, Yo; Matsumura, Takuhiro; Kitadokoro, Kengo; Fujinaga, Yukako

2013-12-06

Clostridium botulinum HA is a component of the large botulinum neurotoxin complex and is critical for its oral toxicity. HA plays multiple roles in toxin penetration in the gastrointestinal tract, including protection from the digestive environment, binding to the intestinal mucosal surface, and disruption of the epithelial barrier. At least two properties of HA contribute to these roles: the sugar-binding activity and the barrier-disrupting activity that depends on E-cadherin binding of HA. HA consists of three different proteins, HA1, HA2, and HA3, whose structures have been partially solved and are made up mainly of β-strands. Here, we demonstrate structural and functional reconstitution of whole HA and present the complete structure of HA of serotype B determined by x-ray crystallography at 3.5 Å resolution. This structure reveals whole HA to be a huge triskelion-shaped molecule. Our results suggest that whole HA is functionally and structurally separable into two parts: HA1, involved in recognition of cell-surface carbohydrates, and HA2-HA3, involved in paracellular barrier disruption by E-cadherin binding.

Hyaluronic acid-coated, prodrug-based nanostructured lipid carriers for enhanced pancreatic cancer therapy.

PubMed

Lu, Zhihe; Su, Jingrong; Li, Zhengrong; Zhan, Yuzhu; Ye, Decai

2017-01-01

Gemcitabine (GEM) and Baicalein (BCL) are reported to have anti-tumor effects including pancreatic cancer. Hyaluronic acid (HA) can bind to over-expressed receptors in various kinds of cancer cells. The aim of this study is to develop prodrugs containing HA, BCL and GEM, and construct nanomedicine incorporate GEM and BCL in the core and HA on the surface. This system could target the cancer cells and co-deliver the drugs. GEM-stearic acid lipid prodrug (GEM-SA) and hyaluronic acid-amino acid-baicalein prodrug (HA-AA-BCL) were synthesized. Then, GEM and BCL prodrug-based targeted nanostructured lipid carriers (HA-GEM-BCL NLCs) were prepared by the nanoprecipitation technique. The in vitro cytotoxicity studies of the NLCs were evaluated on AsPC1 pancreatic cancer cell line. In vivo anti-tumor effects were observed on the murine-bearing pancreatic cancer model. HA-GEM-BCL NLCs were effective in entering pancreatic cancer cells over-expressing HA receptors, and showed cytotoxicity of tumor cells in vitro. In vivo study revealed significant tumor growth inhibition ability of HA-GEM-BCL NLCs in murine pancreatic cancer model. It could be concluded that HA-GEM-BCL NLCs could be featured as promising co-delivery, tumor-targeted nanomedicine for the treatment of cancers.

Effect of negatively charged cellulose nanofibers on the dispersion of hydroxyapatite nanoparticles for scaffolds in bone tissue engineering.

PubMed

Park, Minsung; Lee, Dajung; Shin, Sungchul; Hyun, Jinho

2015-06-01

Nanofibrous 2,2,6,6-tetramethylpiperidine-1-oxyl(TEMPO)-oxidized bacterial cellulose (TOBC) was used as a dispersant of hydroxyapatite (HA) nanoparticles in aqueous solution. The surfaces of TOBC nanofibers were negatively charged after the reaction with the TEMPO/NaBr/NaClO system at pH 10 and room temperature. HA nanoparticles were simply adsorbed on the TOBC nanofibers (HA-TOBC) and dispersed well in DI water. The well-dispersed HA-TOBC colloidal solution formed a hydrogel after the addition of gelatin, followed by crosslinking with glutaraldehyde (HA-TOBC-Gel). The chemical modification of the fiber surfaces and the colloidal stability of the dispersion solution confirmed TOBC as a promising HA dispersant. Both the Young's modulus and maximum tensile stress increased as the amount of gelatin increased due to the increased crosslinking of gelatin. In addition, the well-dispersed HA produced a denser scaffold structure resulting in the increase of the Young's modulus and maximum tensile stress. The well-developed porous structures of the HA-TOBC-Gel composites were incubated with Calvarial osteoblasts. The HA-TOBC-Gel significantly improved cell proliferation as well as cell differentiation confirming the material as a potential candidate for use in bone tissue engineering scaffolds. Copyright © 2015 Elsevier B.V. All rights reserved.

The differential regulation of osteoblast and osteoclast activity by surface topography of hydroxyapatite coatings.

PubMed

Costa, Daniel O; Prowse, Paul D H; Chrones, Tom; Sims, Stephen M; Hamilton, Douglas W; Rizkalla, Amin S; Dixon, S Jeffrey

2013-10-01

The behavior of bone cells is influenced by the surface chemistry and topography of implants and scaffolds. Our purpose was to investigate how the topography of biomimetic hydroxyapatite (HA) coatings influences the attachment and differentiation of osteoblasts, and the resorptive activity of osteoclasts. Using strategies reported previously, we directly controlled the surface topography of HA coatings on polycaprolactone discs. Osteoblasts and osteoclasts were incubated on HA coatings having distinct isotropic topographies with submicrometer and micro-scale features. Osteoblast attachment and differentiation were greater on more complex, micro-rough HA surfaces (Ra ~2 μm) than on smoother topographies (Ra ~1 μm). In contrast, activity of the osteoclast marker tartrate-resistant acid phosphatase was greater on smoother than on micro-rough surfaces. Furthermore, scanning electron microscopy revealed the presence of resorption lacunae exclusively on smoother HA coatings. Inhibition of resorption on micro-rough surfaces was associated with disruption of filamentous actin sealing zones. In conclusion, HA coatings can be prepared with distinct topographies, which differentially regulate responses of osteoblasts, as well as osteoclastic activity and hence susceptibility to resorption. Thus, it may be possible to design HA coatings that induce optimal rates of bone formation and degradation specifically tailored for different applications in orthopedics and dentistry. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

PubMed

Formosa, C; Lachaize, V; Galés, C; Rols, M P; Martin-Yken, H; François, J M; Duval, R E; Dague, E

2015-01-01

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.

Photo-crosslinked hyaluronic acid coated upconverting nanoparticles

NASA Astrophysics Data System (ADS)

Mrazek, Jiri; Kettou, Sofiane; Matuska, Vit; Svozil, Vit; Huerta-Angeles, Gloria; Pospisilova, Martina; Nesporova, Kristina; Velebny, Vladimir

2017-02-01

Hyaluronic acid (HA)-coated inorganic nanoparticles display enhanced interaction with the CD44 receptors which are overexpressed in many types of cancer cells. Here, we describe a modification of core-shell β-NaY0.80Yb0.18Er0.02F4@NaYF4 nanoparticles (UCNP) by HA derivative bearing photo-reactive groups. UCNP capped with oleic acid were firstly transferred to aqueous phase by an improved protocol using hydrochloric acid or lactic acid treatment. Subsequently, HA bearing furanacryloyl moieties (HA-FU) was adsorbed on the nanoparticle surface and crosslinked by UV irradiation. The crosslinking resulted in stable HA coating, and no polymer desorption was observed. As-prepared UCNP@HA-FU show a hydrodynamic diameter of about 180 nm and are colloidally stable in water and cell culture media. The cellular uptake by normal human fibroblasts and MDA MB-231 cancer cell line was investigated by upconversion luminescence imaging.

Evaluate the growth and adhesion of osteoblast cells on nanocomposite scaffold of hydroxyapatite/titania coated with poly hydroxybutyrate

PubMed Central

Pourmollaabbassi, Babak; Karbasi, Saeed; Hashemibeni, Batool

2016-01-01

Background: The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. Materials and Methods: In this paper, hydroxyapatite (HA) powder was made out of bovine bone by thermal analysis at 900°C and first, and then, porous HA (50 weight percentage) was produced by polyurethane sponge replication method. In order to improve the scaffold mechanical properties, they have been coated with poly hydroxybutyrate. In terms of phase studies, morphology, and specifying agent groups, the specific characterization devices such as X-ray diffraction and Fourier transform infrared, were employed. To compare the behavior of cellular scaffolds, they were divided into four groups of scaffolds. The osteoblast cells were cultured. To perform phase studies, analysis of Methylthiazole tetrazolium (MTT) and Trypan blue were carried out for the viability and attachment on the surface of the scaffold, and the specification of Scanning electron microscopy was employed for the morphology of the cells. Results: The results of MTT analysis performed on four groups of scaffolds have shown that Titanium oxide (Tio2) had no effect on cell growth alone and HA was the main factor of growth and cell osteoblast adhesion on the scaffold. Moreover, the results showed that the use of coating with poly-3-hydroxybutyrate saved the factors and placed the osteoblasts within the pore. Since the main part of bone consists of HA, the TiO2 accelerates the formation of apatite crystals at the scaffold surface which is the evidence for bone tissue regeneration. Conclusions: It is likely that the relation between HA and TiO2 leads to an increase in osteoblast adhesion and growth of cells on the scaffold surface. PMID:27761431

Evaluate the growth and adhesion of osteoblast cells on nanocomposite scaffold of hydroxyapatite/titania coated with poly hydroxybutyrate.

PubMed

Pourmollaabbassi, Babak; Karbasi, Saeed; Hashemibeni, Batool

2016-01-01

The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. In this paper, hydroxyapatite (HA) powder was made out of bovine bone by thermal analysis at 900°C and first, and then, porous HA (50 weight percentage) was produced by polyurethane sponge replication method. In order to improve the scaffold mechanical properties, they have been coated with poly hydroxybutyrate. In terms of phase studies, morphology, and specifying agent groups, the specific characterization devices such as X-ray diffraction and Fourier transform infrared, were employed. To compare the behavior of cellular scaffolds, they were divided into four groups of scaffolds. The osteoblast cells were cultured. To perform phase studies, analysis of Methylthiazole tetrazolium (MTT) and Trypan blue were carried out for the viability and attachment on the surface of the scaffold, and the specification of Scanning electron microscopy was employed for the morphology of the cells. The results of MTT analysis performed on four groups of scaffolds have shown that Titanium oxide (Tio 2 ) had no effect on cell growth alone and HA was the main factor of growth and cell osteoblast adhesion on the scaffold. Moreover, the results showed that the use of coating with poly-3-hydroxybutyrate saved the factors and placed the osteoblasts within the pore. Since the main part of bone consists of HA, the TiO 2 accelerates the formation of apatite crystals at the scaffold surface which is the evidence for bone tissue regeneration. It is likely that the relation between HA and TiO 2 leads to an increase in osteoblast adhesion and growth of cells on the scaffold surface.

The influence of dissolved and surface-bound humic acid on the toxicity of TiO₂ nanoparticles to Chlorella sp.

PubMed

Lin, Daohui; Ji, Jing; Long, Zhifeng; Yang, Kun; Wu, Fengchang

2012-09-15

NOM is likely to coat TiO₂ nanoparticles (nano-TiO₂) discharged into the aquatic environment and influence the nanotoxicity to aquatic organisms, which however has not been well investigated. This study explored the influence of nanoparticle surface-bound humic acid (HA, as a model NOM) as well as dissolved HA on the toxicity of nano-TiO₂ to Chlorella sp., with a specific focus on adhesion of the nanoparticles to the algae. Results showed that nano-TiO₂ and the dissolved HA could inhibit the algal growth with an IC₅₀ of 4.9 and 8.4 mg L⁻¹, respectively, while both dissolved and nanoparticle surface-bound HA could significantly alleviate the algal toxicity of nano-TiO₂. IC₅₀ of nano-TiO₂ increased to 18 mg L⁻¹ in the presence of 5 mg L⁻¹ of the dissolved HA and to 48 mg L⁻¹ as the result of surface-saturation by HA. Co-precipitation experiment and transmission electron microscopy observation revealed that both dissolved and nanoparticle surface-bound HA prevented the adhesion of nano-TiO₂ to the algal cells due to the increased electrosteric repulsion. The generation of intracellular reactive oxygen species (ROS) was significantly limited by the dissolved and nanoparticle surface-bound HA. The prevention of adhesion and inhibition of ROS generation could account for the HA-mitigated nanotoxicity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Effect of surface roughness on osteogenesis in vitro and osseointegration in vivo of carbon fiber-reinforced polyetheretherketone–nanohydroxyapatite composite

PubMed Central

Deng, Yi; Liu, Xiaochen; Xu, Anxiu; Wang, Lixin; Luo, Zuyuan; Zheng, Yunfei; Deng, Feng; Wei, Jie; Tang, Zhihui; Wei, Shicheng

2015-01-01

As United States Food and Drug Administration-approved implantable material, carbon fiber-reinforced polyetheretherketone (CFRPEEK) possesses an adjustable elastic modulus similar to cortical bone and is a prime candidate to replace surgical metallic implants. The bioinertness and inferior osteogenic properties of CFRPEEK, however, limit its clinical application as orthopedic/dental implants. In this study, CFRPEEK–nanohydroxyapatite ternary composites (PEEK/n-HA/CF) with variable surface roughness have been successfully fabricated. The effect of surface roughness on their in vitro cellular responses of osteoblast-like MG-63 cells (attachment, proliferation, apoptosis, and differentiation) and in vivo osseointegration is evaluated. The results show that the hydrophilicity and the amount of Ca ions on the surface are significantly improved as the surface roughness of composite increases. In cell culture tests, the results reveal that the cell proliferation rate and the extent of osteogenic differentiation of cells are a function of the size of surface roughness. The composite with moderate surface roughness significantly increases cell attachment/proliferation and promotes the production of alkaline phosphatase (ALP) activity and calcium nodule formation compared with the other groups. More importantly, the PEEK/n-HA/CF implant with appropriate surface roughness exhibits remarkably enhanced bioactivity and osseointegration in vivo in the animal experiment. These findings will provide critical guidance for the design of CFRPEEK-based implants with optimal roughness to regulate cellular behaviors, and to enhance biocompability and osseointegration. Meanwhile, the PEEK/n-HA/CF ternary composite with optimal surface roughness might hold great potential as bioactive biomaterial for bone grafting and tissue engineering applications. PMID:25733834

Precision Extruding Deposition for Freeform Fabrication of PCL and PCL-HA Tissue Scaffolds

NASA Astrophysics Data System (ADS)

Shor, L.; Yildirim, E. D.; Güçeri, S.; Sun, W.

Computer-aided tissue engineering approach was used to develop a novel Precision Extrusion Deposition (PED) process to directly fabricate Polycaprolactone (PCL) and composite PCL/Hydroxyapatite (PCL-HA) tissue scaffolds. The process optimization was carried out to fabricate both PCL and PCL-HA (25% concentration by weight of HA) with a controlled pore size and internal pore structure of the 0°/90° pattern. Two groups of scaffolds having 60 and 70% porosity and with pore sizes of 450 and 750 microns, respectively, were evaluated for their morphology and compressive properties using Scanning Electron Microscopy (SEM) and mechanical testing. The surface modification with plasma was conducted on PCL scaffold to increase the cellular attachment and proliferation. Our results suggested that inclusion of HA significantly increased the compressive modulus from 59 to 84 MPa for 60% porous scaffolds and from 30 to 76 MPa for 70% porous scaffolds. In vitro cell-scaffolds interaction study was carried out using primary fetal bovine osteoblasts to assess the feasibility of scaffolds for bone tissue engineering application. In addition, the results in surface hydrophilicity and roughness show that plasma surface modification can increase the hydrophilicity while introducing the nano-scale surface roughness on PCL surface. The cell proliferation and differentiation were calculated by Alamar Blue assay and by determining alkaline phosphatase activity. The osteoblasts were able to migrate and proliferate over the cultured time for both PCL as well as PCL-HA scaffolds. Our study demonstrated the viability of the PED process to the fabricate PCL and PCL-HA composite scaffolds having necessary mechanical property, structural integrity, controlled pore size and pore interconnectivity desired for bone tissue engineering.

Enhanced and Selective Antiproliferative Activity of Methotrexate-Functionalized-Nanocapsules to Human Breast Cancer Cells (MCF-7).

PubMed

de Oliveira, Catiúscia P; Büttenbender, Sabrina L; Prado, Willian A; Beckenkamp, Aline; Asbahr, Ana C; Buffon, Andréia; Guterres, Silvia S; Pohlmann, Adriana R

2018-01-04

Methotrexate is a folic acid antagonist and its incorporation into nanoformulations is a promising strategy to increase the drug antiproliferative effect on human breast cancer cells by overexpressing folate receptors. To evaluate the efficiency and selectivity of nanoformulations containing methotrexate and its diethyl ester derivative, using two mechanisms of drug incorporation (encapsulation and surface functionalization) in the in vitro cellular uptake and antiproliferative activity in non-tumoral immortalized human keratinocytes (HaCaT) and in human breast carcinoma cells (MCF-7). Methotrexate and its diethyl ester derivative were incorporated into multiwall lipid-core nanocapsules with hydrodynamic diameters lower than 160 nm and higher drug incorporation efficiency. The nanoformulations were applied to semiconfluent HaCaT or MCF-7 cells. After 24 h, the nanocapsules were internalized into HaCaT and MCF-7 cells; however, no significant difference was observed between the nanoformulations in HaCaT (low expression of folate receptors), while they showed significantly higher cellular uptakes than the blank-nanoformulation in MCF-7, which was the highest uptakes observed for the drug functionalized-nanocapsules. No antiproliferative activity was observed in HaCaT culture, whereas drug-containing nanoformulations showed antiproliferative activity against MCF-7 cells. The effect was higher for drug-surface functionalized nanocapsules. In conclusion, methotrexate-functionalized-nanocapsules showed enhanced and selective antiproliferative activity to human breast cancer cells (MCF-7) being promising products for further in vivo pre-clinical evaluations.

Hyaluronic Acid-Based pH-Sensitive Polymer-Modified Liposomes for Cell-Specific Intracellular Drug Delivery Systems.

PubMed

Miyazaki, Maiko; Yuba, Eiji; Hayashi, Hiroshi; Harada, Atsushi; Kono, Kenji

2018-01-17

For the enhancement of therapeutic effects and reduction of side effects derived from anticancer drugs in cancer chemotherapy, it is imperative to develop drug delivery systems with cancer-specificity and controlled release function inside cancer cells. pH-sensitive liposomes are useful as an intracellular drug delivery system because of their abilities to transfer their contents into the cell interior through fusion or destabilization of endosome, which has weakly acidic environment. We earlier reported liposomes modified with various types of pH-sensitive polymers based on synthetic polymers and biopolymers as vehicles for intracellular drug delivery systems. In this study, hyaluronic acid (HA)-based pH-sensitive polymers were designed as multifunctional polymers having not only pH-sensitivity but also targeting properties to cells expressing CD44, which is known as a cancer cell surface marker. Carboxyl group-introduced HA derivatives of two types, MGlu-HA and CHex-HA, which have a more hydrophobic side chain structure than that of MGlu-HA, were synthesized by reaction with various dicarboxylic anhydrides. These polymer-modified liposomes were stable at neutral pH, but showed content release under weakly acidic conditions. CHex-HA-modified liposomes delivered their contents into CD44-expressing cells more efficiently than HA-modified or MGlu-HA-modified liposomes or unmodified liposomes, whereas the same liposomes were taken up only slightly by cells expressing CD44 proteins less. Competition assay using free HA or other polymers revealed that HA derivative-modified liposomes might be recognized by CD44. Therefore, HA-derivative-modified liposomes are useful as cell-specific intracellular drug delivery systems.

Tailoring of the titanium surface by preparing cardiovascular endothelial extracellular matrix layer on the hyaluronic acid micro-pattern for improving biocompatibility.

PubMed

Li, Jingan; Zhang, Kun; Wu, Juejue; Zhang, Lijuan; Yang, Ping; Tu, Qiufen; Huang, Nan

2015-04-01

It has been proved that high molecular weight hyaluronic acid (HMW-HA, 1×10(6) Da) micro-strips on titanium (Ti) surface can elongate the human vascular endothelial cell (EC) morphology, subsequently enhance endothelial extracellular matrix (ECM) deposition in our previous work. The HMW-HA micro-strips were anticipated to possess good hemocompatibility and EC compatibility simultaneously. However, the single HMW-HA micro-strips on Ti substrate showed bad hemocompatibility. To solve this problem, a method combining HA micro-pattern and EC decellularization was developed, and the endothelial extracellular matrix layer on the HA micro-pattern (ECM/HAP) showed excellent hemocompatibility and endothelial progenitor cells (EPCs) compatibility (cell number: 14.3±0.5×10(5) cells/cm2>2.2±0.7×10(5) cells/cm2 on ECM/TiOH, 7.5±1.3×10(5) cells/cm2 on TiOH, 3.4±0.9×10(5) cells/cm2 on TiOH/HAP and 3.6±1.2×10(5) cells/cm2 on Ti). We also found that the ECM/HAP coating could significantly inhibit the excessive proliferation of smooth muscle cells (SMCs) (cck-8 absorption: 0.25±0.06<1.18±0.09 A.U. on ECM/TiOH, 0.87±0.15 A.U. on TiOH and 1.55±0.11 A.U. on Ti) and the attachment of macrophages (cell number: 1.3±0.1×10(3)<9.2±1.5×10(3) cells/cm2 on ECM/TiOH, 8.8±0.3×10(3) cells/cm2 on TiOH, 7.3±0.7×10(3) cells/cm2 on TiOH/HAP and 9.6±0.9×10(3) cells/cm2 on Ti in 12 h). These data suggest that the multifunctional ECM/HAP coating can be used to build the bionic human endothelial ECM on the biomaterials surface, which might provide a potential and effective method for surface modification of cardiovascular devices. Copyright © 2015. Published by Elsevier B.V.

Pyrrole-hyaluronic acid conjugates for decreasing cell binding to metals and conducting polymers

PubMed Central

Lee, Jae Young; Schmidt, Christine E.

2010-01-01

Surface modification of electrically conductive biomaterials has been studied to improve biocompatibility for a number of applications, such as implantable sensors and microelectrode arrays. In this study, we electrochemically coated electrodes with biocompatible and non-cell adhesive hyaluronic acid (HA) to reduce cellular adhesion for potential use in neural prostheses. To this end, pyrrole-conjugated hyaluronic acid (PyHA) was synthesized and employed for electrochemical coating of platinum, indium-tin-oxide, and polystyrene sulfonate-doped polypyrrole electrodes. This PyHA conjugate consists of (1) a pyrrole moiety that allows the compound to be electrochemically deposited onto a conductive substrate and (2) non-adhesive HA to minimize cell adhesion and to potentially decrease inflammatory tissue responses. Our characterization results showed the presence of a hydrophilic p(PyHA) layer on the modified electrode, and impedance measurements revealed impedance that was statistically the same as the unmodified electrode. We found that the p(PyHA)-coated electrodes minimized adhesion and migration of fibroblasts and astrocytes for a minimum of up to 3 months. Also, the coating was stable in physiological solution for 3 months and also stable against enzymatic degradation by hyaluronidase. These studies suggest that this p(PyHA)-coating has the potential to be used to mask conducting electrodes from adverse glial responses that occur upon implantation. In addition, electrochemical coating with PyHA can be potentially extended for the surface modification of other metallic and conducting substances such as stents and biosensors. PMID:20558330

Characteristics and in vitro response of thin hydroxyapatite–titania films produced by plasma electrolytic oxidation of Ti alloys in electrolytes with particle additions

PubMed Central

Yeung, W. K.; Sukhorukova, I. V.; Shtansky, D. V.; Levashov, E. A.; Zhitnyak, I. Y.; Gloushankova, N. A.; Kiryukhantsev-Korneev, P. V.; Petrzhik, M. I.; Matthews, A.

2016-01-01

The enhancement of the biological properties of Ti by surface doping with hydroxyapatite (HA) is of great significance, especially for orthodontic applications. This study addressed the effects of HA particle size in the electrolyte suspension on the characteristics and biological properties of thin titania-based coatings produced on Ti–6Al–4V alloy by plasma electrolytic oxidation (PEO). Detailed morphological investigation of the coatings formed by a single-stage PEO process with two-step control of the electrical parameters was performed using the Minkowski functionals approach. The surface chemistry was studied by glow discharge optical emission spectroscopy and Fourier transform infrared spectroscopy, whereas mechanical properties were evaluated using scratch tests. The biological assessment included in vitro evaluation of the coating bioactivity in simulated body fluid (SBF) as well as studies of spreading, proliferation and osteoblastic differentiation of MC3T3-E1 cells. The results demonstrated that both HA micro- and nanoparticles were successfully incorporated in the coatings but had different effects on their surface morphology and elemental distributions. The micro-particles formed an irregular surface morphology featuring interpenetrated networks of fine pores and coating material, whereas the nanoparticles penetrated deeper into the coating matrix which retained major morphological features of the porous TiO2 coating. All coatings suffered cohesive failure in scratch tests, but no adhesive failure was observed; moreover doping with HA increased the coating scratch resistance. In vitro tests in SBF revealed enhanced bioactivity of both HA-doped PEO coatings; furthermore, the cell proliferation/morphometric tests showed their good biocompatibility. Fluorescence microscopy revealed a well-organised actin cytoskeleton and focal adhesions in MC3T3-E1 cells cultivated on these substrates. The cell alkaline phosphatase activity in the presence of ascorbic acid and β-glycerophosphate was significantly increased, especially in HA nanoparticle-doped coatings. PMID:27019704

Atomic force microscopy evaluation of aqueous interfaces of immobilized hyaluronan.

PubMed

Morra, Marco; Cassinelli, Clara; Pavesio, Alessandra; Renier, Davide

2003-03-15

Hyaluronan (HA) was immobilized on aminated glass surfaces in three different ways: by simple ionic interaction and by covalent linking at low density and at full density. In agreement with previous reports, in vitro experiments show that the outcome of fibroblast adhesion tests is markedly affected by the details of the coupling procedure, suggesting that different interfacial forces are operating at the aqueous/HA interface in the three cases investigated. The interfacial properties of the HA-coated surfaces were probed by force-distance curves obtained with the atomic force microscope (AFM). This approach readily shows significant differences among the tested samples, which are directly related to the coupling strategy and to results of cell adhesion tests. In particular, the range of interaction between the tip and the surface is much lower when HA is covalently linked than when it is ionically coupled, suggesting a more compact surface structure in the former case. Increasing HA surface density minimizes the interaction force between the surface and the AFM tip, likely reflecting more complete shielding by the HA chains of the underlying substrate. In summary, these measurements clearly show the different nature of the aqueous interfaces tested, and underline the role of this analytical approach in the development and control of finely tuned biomaterial surfaces.

Comparison of candidate materials for a synthetic osteo-odonto keratoprosthesis device.

PubMed

Tan, Xiao Wei; Perera, A Promoda P; Tan, Anna; Tan, Donald; Khor, K A; Beuerman, Roger W; Mehta, Jodhbir S

2011-01-05

Osteo-odonto keratoprosthesis is one of the most successful forms of keratoprosthesis surgery for end-stage corneal and ocular surface disease. There is a lack of detailed comparison studies on the biocompatibilities of different materials used in keratoprosthesis. The aim of this investigation was to compare synthetic bioinert materials used for keratoprosthesis surgery with hydroxyapatite (HA) as a reference. Test materials were sintered titanium oxide (TiO(2)), aluminum oxide (Al(2)O(3)), and yttria-stabilized zirconia (YSZ) with density >95%. Bacterial adhesion on the substrates was evaluated using scanning electron microscopy and the spread plate method. Surface properties of the implant discs were scanned using optical microscopy. Human keratocyte attachment and proliferation rates were assessed by cell counting and MTT assay at different time points. Morphologic analysis and immunoblotting were used to evaluate focal adhesion formation, whereas cell adhesion force was measured with a multimode atomic force microscope. The authors found that bacterial adhesion on the TiO(2), Al(2)O(3), and YSZ surfaces were lower than that on HA substrates. TiO(2) significantly promoted keratocyte proliferation and viability compared with HA, Al(2)O(3,) and YSZ. Immunofluorescent imaging analyses, immunoblotting, and atomic force microscope measurement revealed that TiO(2) surfaces enhanced cell spreading and cell adhesion compared with HA and Al(2)O(3). TiO(2) is the most suitable replacement candidate for use as skirt material because it enhanced cell functions and reduced bacterial adhesion. This would, in turn, enhance tissue integration and reduce device failure rates during keratoprosthesis surgery.

Design of magnetic polyplexes taken up efficiently by dendritic cell for enhanced DNA vaccine delivery.

PubMed

Nawwab Al-Deen, F M; Selomulya, C; Kong, Y Y; Xiang, S D; Ma, C; Coppel, R L; Plebanski, M

2014-02-01

Dendritic cells (DC) targeting vaccines require high efficiency for uptake, followed by DC activation and maturation. We used magnetic vectors comprising polyethylenimine (PEI)-coated superparamagnetic iron oxide nanoparticles, with hyaluronic acid (HA) of different molecular weights (<10 and 900 kDa) to reduce cytotoxicity and to facilitate endocytosis of particles into DCs via specific surface receptors. DNA encoding Plasmodium yoelii merozoite surface protein 1-19 and a plasmid encoding yellow fluorescent gene were added to the magnetic complexes with various % charge ratios of HA: PEI. The presence of magnetic fields significantly enhanced DC transfection and maturation. Vectors containing a high-molecular-weight HA with 100% charge ratio of HA: PEI yielded a better transfection efficiency than others. This phenomenon was attributed to their longer molecular chains and higher mucoadhesive properties aiding DNA condensation and stability. Insights gained should improve the design of more effective DNA vaccine delivery systems.

Optimization of implant/bone attachment: The effects of implant surface porosity, bioactive ceramic coatings, and delivery of adsorbed growth factors

NASA Astrophysics Data System (ADS)

Melican, Mora Carolynne

Various surface treatments and coating materials have been tested for use on metal alloy orthopaedic implants. Their purpose has been to enhance the bioactivity of the implant surfaces, and thus to increase the rate and degree of bony attachment in vivo in an attempt to hasten recovery time, increase implant service lifetime, and lessen pain associated with loosened orthopaedic implants. A series of in vivo and in vitro studies were performed to determine the influence of different implant surfaces including porous metal surfaces with varied porosity with depth, resorbable and non-resorbable plasma-sprayed hydroxyapatite (HA) coatings, and finally HA coatings with an adsorbed layer of human recombinant bone morphogenetic protein (rhBMP-2), an osteoinductive protein. Textured as-cast metal surfaces produced by investment casting in three dimensionally printed ceramic molds have exhibited superior bony ingrowth and attachment. Plasma-sprayed HA coatings have been shown to be appropriate substrates for osteoblast proliferation (particularly on highly crystalline HA) and stem cell proliferation (particularly on less crystalline HA). Less crystalline HA coatings have shown promise as delivery systems for different levels of rhBMP-2. The osteoinductive protein has been shown to remain active after delivery to the system, and was most effective when delivered in concentrations ranging from 30 to 50 ng/ml. Combinations of these surface treatments for metal implant surfaces warrant further investigation.

Hyaluronic acid as a potential boron carrier for BNCT: Preliminary evaluation.

PubMed

Zaboronok, A; Yamamoto, T; Nakai, K; Yoshida, F; Uspenskii, S; Selyanin, M; Zelenetskii, A; Matsumura, Akira

2015-12-01

Hyaluronic acid (HA), a nonimmunogenic, biocompatible polymer found in different biological tissues, has the potential to attach to CD44 receptors on the surface of certain cancer cells, where the receptor is overexpressed compared with normal cells. Boron-hyaluronic acid (BHA) was tested for its feasibility as a potential agent for BNCT. BHA with low-viscosity 30 kDa HA could be administered by intravenous injection. The compound showed a certain degree of cytotoxicity and accumulation in C6 rat glioma cells in vitro. Instability of the chelate bonds between boron and HA and/or insufficient specificity of CD44 receptors on C6 cells to BHA could account for the insufficient in vitro accumulation. To ensure the future eligibility of BHA for BNCT experiments, using alternative tumor cell lines and chemically securing the chelate bonds or synthesizing BHA with boron covalently attached to HA might be required. Copyright © 2015 Elsevier Ltd. All rights reserved.

Endocytosis of hyaluronan in rat Kupffer cells.

PubMed

Alston-Smith, J; Pertoft, H; Laurent, T C

1992-09-01

The binding, uptake and degradation of hyaluronan (HA) labelled with 3H in its acetyl group were studied in cultured rat Kupffer cells (KC). At 4 degrees C the binding increased with increasing concentrations of HA in the culture medium up to at least 1 microgram/ml, when saturation occurred. Binding could be prevented efficiently by the addition of an excess of unlabelled HA, and to a lesser extent by chondroitin sulphate and oligosaccharide fragments of HA, consisting of four sugars or more. The labelled HA bound to the cells could be removed by incubating the cells with Streptomyces hyaluronidase, or trypsin, indicating that the HA-binding sites are located on the cell surface. At 37 degrees C HA was internalized in a concentration-dependent manner, and degradation products appeared in the supernatant after 1-5 h, depending on the concentration applied. At 50 ng of free HA/ml, each KC accumulated 60 ag of the polysaccharide/min in the first 1 h, and degraded a total amount of 10 fg of HA during an 8 h period. Addition of the negatively charged polysaccharide dextran sulphate reduced binding, and to an even greater extent internalization, of HA in KC, while no effect was observed with dextran. Depletion of intracellular potassium caused a marked reduction in the rate of endocytosis of cell-membrane-associated HA into KC, without affecting binding. Addition of KCl to the culture medium returned endocytosis of [3H]HA to normal levels. There was no effect on binding and a partial effect on internalization by depletion of bivalent cations or in the presence of EDTA. The degradation of [3H]HA by KC cultures was abolished in the presence of weak bases, NH4Cl and chloroquine, supporting the idea that HA is endocytosed into lysosomes prior to degradation. The fluid-phase marker [14C]sucrose was internalized in the cells at much lower rate than was HA. Rates of binding, internalization and degradation of HA in KC point therefore to a specific endocytosis followed by an intracellular degradation to low-M(r) compounds. It was estimated that, under physiological conditions, KC only clear a minor proportion of circulating HA.

Zero-order controlled release of BMP2-derived peptide P24 from the chitosan scaffold by chemical grafting modification technique for promotion of osteogenesis in vitro and enhancement of bone repair in vivo.

PubMed

Chen, Yan; Liu, Xujie; Liu, Rui; Gong, Yong; Wang, Mingbo; Huang, Qianli; Feng, Qingling; Yu, Bo

2017-01-01

Combination of tissue-engineered bone scaffolds with cell-adhesive, osteoconductive, or osteoinductive biomolecules is a critical strategy to improve their properties that significantly influence cellular behaviors, such as adhesion, proliferation, and differentiation, which is beneficial for critical-sized bone defects repairing. However, the traditional surface modification techniques, such as physical adsorption, coating, and plasma treatment, et al, have great limitations for immobilization of bioactive molecules due to undesirable controlled delivery performance or overly complex multistep procedures. In this study, we functionalized the chitosan/hydroxyapatite (CS/HA) biomimetic composite scaffold for controlled delivery of BMP2-derived peptide (P24) by the chemical grafting modification technique: firstly, P24 was conjugated with a thiolated chitosan, chitosan-4-thiobutylamidine (CS-TBA); secondly, the resultant CS-P24 was then combined with HA to prepare CS-P24/HA scaffolds. The effect of CS-P24/HA scaffolds on bone regeneration was evaluated, along with the underlying biological mechanisms responsible in vitro and in vivo . In vitro , the controlled and sustained release of bioactive P24 could last up to 90 days, furthermore, the release profiles of CS-5%P24/HA and CS-10%P24/HA were linear and could be fitted according to zero-order kinetic model (R 2 =0.9929; R 2 =0.9757); P24 on the scaffold significantly promoted cell adhesion, proliferation, osteodifferentiation, and mineralization with synergistic effects. Bone marrow stromal cells (BMSCs) revealed spindle-shaped surface morphology, indicating the CS-P24/HA scaffolds supported cell adhesion and possessed a high proliferation rate that varied according to the P24 concentration levels. Furthermore, mRNA levels for OCN, Runx2, and collagen I were significantly up-regulated on CS-P24/HA scaffolds compared with cells grown on CS/HA scaffolds in vitro ( p < 0.05). Similarly, the BMSCs exhibited a higher ALP expression and calcium deposition level on CS-P24/HA scaffolds compared with CS/HA scaffolds ( p < 0.05). In vivo , osteoinductive studies revealed a significantly higher ectopic osteogenesis level of CS-10%P24/HA scaffolds in rat dorsal muscle pockets compared with that of CS/HA scaffolds. Finally, CS-P24/HA scaffolds showed superior performance in the reconstruction of rat calvarial bone defects. This novel CS-P24/HA scaffold is deemed a strong potential candidate for the repair of bone defects in human bone tissue engineering.

Zero-order controlled release of BMP2-derived peptide P24 from the chitosan scaffold by chemical grafting modification technique for promotion of osteogenesis in vitro and enhancement of bone repair in vivo

PubMed Central

Chen, Yan; Liu, Xujie; Liu, Rui; Gong, Yong; Wang, Mingbo; Huang, Qianli; Feng, Qingling; Yu, Bo

2017-01-01

Combination of tissue-engineered bone scaffolds with cell-adhesive, osteoconductive, or osteoinductive biomolecules is a critical strategy to improve their properties that significantly influence cellular behaviors, such as adhesion, proliferation, and differentiation, which is beneficial for critical-sized bone defects repairing. However, the traditional surface modification techniques, such as physical adsorption, coating, and plasma treatment, et al, have great limitations for immobilization of bioactive molecules due to undesirable controlled delivery performance or overly complex multistep procedures. In this study, we functionalized the chitosan/hydroxyapatite (CS/HA) biomimetic composite scaffold for controlled delivery of BMP2-derived peptide (P24) by the chemical grafting modification technique: firstly, P24 was conjugated with a thiolated chitosan, chitosan-4-thiobutylamidine (CS-TBA); secondly, the resultant CS-P24 was then combined with HA to prepare CS-P24/HA scaffolds. The effect of CS-P24/HA scaffolds on bone regeneration was evaluated, along with the underlying biological mechanisms responsible in vitro and in vivo. In vitro, the controlled and sustained release of bioactive P24 could last up to 90 days, furthermore, the release profiles of CS-5%P24/HA and CS-10%P24/HA were linear and could be fitted according to zero-order kinetic model (R2=0.9929; R2=0.9757); P24 on the scaffold significantly promoted cell adhesion, proliferation, osteodifferentiation, and mineralization with synergistic effects. Bone marrow stromal cells (BMSCs) revealed spindle-shaped surface morphology, indicating the CS-P24/HA scaffolds supported cell adhesion and possessed a high proliferation rate that varied according to the P24 concentration levels. Furthermore, mRNA levels for OCN, Runx2, and collagen I were significantly up-regulated on CS-P24/HA scaffolds compared with cells grown on CS/HA scaffolds in vitro (p < 0.05). Similarly, the BMSCs exhibited a higher ALP expression and calcium deposition level on CS-P24/HA scaffolds compared with CS/HA scaffolds (p < 0.05). In vivo, osteoinductive studies revealed a significantly higher ectopic osteogenesis level of CS-10%P24/HA scaffolds in rat dorsal muscle pockets compared with that of CS/HA scaffolds. Finally, CS-P24/HA scaffolds showed superior performance in the reconstruction of rat calvarial bone defects. This novel CS-P24/HA scaffold is deemed a strong potential candidate for the repair of bone defects in human bone tissue engineering. PMID:28435449

Investigation of Overrun-Processed Porous Hyaluronic Acid Carriers in Corneal Endothelial Tissue Engineering

PubMed Central

Lai, Jui-Yang; Cheng, Hsiao-Yun; Ma, David Hui-Kang

2015-01-01

Hyaluronic acid (HA) is a linear polysaccharide naturally found in the eye and therefore is one of the most promising biomaterials for corneal endothelial regenerative medicine. This study reports, for the first time, the development of overrun-processed porous HA hydrogels for corneal endothelial cell (CEC) sheet transplantation and tissue engineering applications. The hydrogel carriers were characterized to examine their structures and functions. Evaluations of carbodiimide cross-linked air-dried and freeze-dried HA samples were conducted simultaneously for comparison. The results indicated that during the fabrication of freeze-dried HA discs, a technique of introducing gas bubbles in the aqueous biopolymer solutions can be used to enlarge pore structure and prevent dense surface skin formation. Among all the groups studied, the overrun-processed porous HA carriers show the greatest biological stability, the highest freezable water content and glucose permeability, and the minimized adverse effects on ionic pump function of rabbit CECs. After transfer and attachment of bioengineered CEC sheets to the overrun-processed HA hydrogel carriers, the therapeutic efficacy of cell/biopolymer constructs was tested using a rabbit model with corneal endothelial dysfunction. Clinical observations including slit-lamp biomicroscopy, specular microscopy, and corneal thickness measurements showed that the construct implants can regenerate corneal endothelium and restore corneal transparency at 4 weeks postoperatively. Our findings suggest that cell sheet transplantation using overrun-processed porous HA hydrogels offers a new way to reconstruct the posterior corneal surface and improve endothelial tissue function. PMID:26296087

Keratinocyte Motility Is Affected by UVA Radiation-A Comparison between Normal and Dysplastic Cells.

PubMed

Niculiţe, Cristina M; Nechifor, Marina T; Urs, Andreea O; Olariu, Laura; Ceafalan, Laura C; Leabu, Mircea

2018-06-07

UVA radiation induces multiple and complex changes in the skin, affecting epidermal cell behavior. This study reports the effects of UVA exposure on normal (HaCaT) and dysplastic (DOK) keratinocytes. The adherence, spreading and proliferation were investigated by time-lapse measurement of cell layer impedance on different matrix proteins. Prior to UVA exposure, the time required for adherence and spreading did not differ significantly for HaCaT and DOK cells, while spreading areas were larger for HaCaT cells. Under UVA exposure, HaCaT and DOK cells behavior differed in terms of movement and proliferation. The cells' ability to cover the denuded surface and individual cell trajectories were recorded by time-lapse videomicroscopy, during wound healing experiments. Dysplastic keratinocytes showed more sensitivity to UVA, exhibiting transient deficiencies in directionality of movement and a delay in re-coating the denuded area. The actin cytoskeleton displayed a cortical organization immediately after irradiation, in both cell lines, similar to mock-irradiated cells. Post-irradiation, DOK cells displayed a better organization of stress fibers, persistent filopodia, and new, stronger focal contacts. In conclusion, after UVA exposure HaCaT and DOK cells showed a different behavior in terms of adherence, spreading, motility, proliferation, and actin cytoskeleton dynamics, with the dyplastic keratinocytes being more sensitive.

Micropatterned Silica Films with Nanohydroxyapatite for Y-TZP Implants.

PubMed

Miranda, R B P; Grenho, L; Carvalho, A; Fernandes, M H; Monteiro, F J; Cesar, P F

2018-03-01

This investigation aimed at developing micropatterned silica thin films (MSTFs) containing nanohydroxyapatite (nano-HA) microaggregates that were not completely covered by silica so that they could directly interact with the surrounding cells. The objectives were 1) to evaluate the effect of the presence of 2 films (MSTF with or without nano-HA addition) on the characteristic strength (σ 0 ) and Weibull modulus ( m) of a yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) and 2) to evaluate the effect of these 2 films, as applied onto the Y-TZP surface, on the morphology, orientation, and proliferation of MG63 cells. Sol-gel process and soft lithography were used to apply the MSTF onto the Y-TZP specimens. Three experimental groups were produced: Y-TZP, Y-TZP + MSTF, and Y-TZP + MSTF + sprayed nano-HA. All surfaces were characterized by scanning electron microscopy and energy-dispersive X-ray spectroscopy and tested for 4-point flexural strength ( n = 30) in water at 37 °C. Weibull analysis was used to determine m and σ 0 (maximum likelihood method). In vitro biological behavior was performed with human osteoblast-like cells (MG63). Y-TZP was successfully coated with MSFT and MSFT + nano-HA. Scanning electron microscopy micrographs indicated that the microaggregates of nano-HA were not entirely covered by the silica. There was no statistically significant difference among the experimental groups for σ 0 and m. In the groups containing the films, the cells were elongated and aligned along the lines. The MSFT + nano-HA group showed significantly higher cell metabolic activity than that obtained for the Y-TZP group at day 7. This investigation was successful in producing an MSTF containing nano-HA microaggregates that remained exposed to the environment. The developed films did not jeopardize the structural reliability of a commercial Y-TZP, as confirmed by the Weibull statistics. The MG63 cells seeded over the films became elongated and aligned along the films' micropatterned lines. Y-TZP specimens coated with MSTF and nano-HA showed a higher cell metabolic activity and proliferation after 7 d of culture when compared with uncoated Y-TZP.

Fortifying the Bone-Implant Interface Part 1: An In Vitro Evaluation of 3D-Printed and TPS Porous Surfaces

PubMed Central

Lindsey, Derek P.; Bahney, Chelsea S.; Woods, Shane A.; Wolfe, Mark L.; Yerby, Scott A.

2017-01-01

Background An aging society and concomitant rise in the incidence of impaired bone health have led to the need for advanced osteoconductive spinal implant surfaces that promote greater biological fixation (e.g. for interbody fusion cages, sacroiliac joint fusion implants, and artificial disc replacements). Additive manufacturing, i.e. 3D-printing, may improve bone integration by generating biomimetic spinal implant surfaces that mimic bone morphology. Such surfaces may foster an enhanced cellular response compared to traditional implant surfacing processes. Methods This study investigated the response of human osteoblasts to additive manufactured (AM) trabecular-like titanium implant surfaces compared to traditionally machined base material with titanium plasma spray (TPS) coated surfaces, with and without a nanocrystalline hydroxyapatite (HA) coating. For TPS-coated discs, wrought Ti6Al4V ELI was machined and TPS-coating was applied. For AM discs, Ti6Al4V ELI powder was 3D-printed to form a solid base and trabecular-like porous surface. The HA-coating was applied via a precipitation dip-spin method. Surface porosity, pore size, thickness, and hydrophilicity were characterized. Initial cell attachment, proliferation, alkaline phosphatase (ALP) activity, and calcium production of hFOB cells (n=5 per group) were measured. Results Cells on AM discs exhibited expedited proliferative activity. While there were no differences in mean ALP expression and calcium production between TPS and AM discs, calcium production on the AM discs trended 48% higher than on TPS discs (p=0.07). Overall, HA-coating did not further enhance results compared to uncoated TPS and AM discs. Conclusions Results demonstrate that additive manufacturing allows for controlled trabecular-like surfaces that promote earlier cell proliferation and trends toward higher calcium production than TPS coating. Results further showed that nanocrystalline HA may not provide an advantage on porous titanium surfaces. Clinical Relevance Additive manufactured porous titanium surfaces may induce a more osteogenic environment compared to traditional TPS, and thus present as an attractive alternative to TPS-coating for orthopedic spinal implants. PMID:28765799

Fabrication, ultra-structure characterization and in vitro studies of RF magnetron sputter deposited nano-hydroxyapatite thin films for biomedical applications

NASA Astrophysics Data System (ADS)

Surmeneva, Maria A.; Surmenev, Roman A.; Nikonova, Yulia A.; Selezneva, Irina I.; Ivanova, Anna A.; Putlyaev, Valery I.; Prymak, Oleg; Epple, Matthias

2014-10-01

A series of nanostructured low-crystalline hydroxyapatite (HA) coatings averaging 170, 250, and 440 nm in thickness were deposited onto previously etched titanium substrates through radio-frequency (RF) magnetron sputtering. The HA coatings were analyzed using infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning and transmission electron microscopy (SEM and TEM). Cross sections of the thin specimens were prepared by FIB to study the microstructure of the coatings by TEM. The deposition process formed nano-scale grains, generating an amorphous layer at the substrate/coating interface and inducing the growth of a columnar grain structure perpendicular to the substrate surface. A microstructural analysis of the film confirmed that the grain size and crystallinity increased when increasing the deposition time. The nanostructured HA coatings were not cytotoxic, as proven by in vitro assays using primary dental pulp stem cells and mouse fibroblast NCTC clone L929 cells. Low-crystallinity HA coatings with different thicknesses stimulated cells to attach, proliferate and form mineralized nodules on the surface better than uncoated titanium substrates.

Synergistic interaction of platelet derived growth factor (PDGF) with the surface of PLLA/Col/HA and PLLA/HA scaffolds produces rapid osteogenic differentiation.

PubMed

Raghavendran, Hanumantha Rao Balaji; Mohan, Saktiswaren; Genasan, Krishnamurithy; Murali, Malliga Raman; Naveen, Sangeetha Vasudevaraj; Talebian, Sepehr; McKean, Robert; Kamarul, Tunku

2016-03-01

Scaffolds with structural features similar to the extracellular matrix stimulate rapid osteogenic differentiation in favorable microenvironment and with growth factor supplementation. In this study, the osteogenic potential of electrospun poly-l-lactide/hydroxyapatite/collagen (PLLA/Col/HA, PLLA/HA and PLLA/Col) scaffolds were tested in vitro with the supplementation of platelet derived growth factor-BB (PDGF-BB). Cell attachment and topography, mineralization, extracellular matrix protein localization, and gene expression of the human mesenchymal stromal cells were compared between the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA. The levels of osteocalcin, calcium, and mineralization were significantly greater in the PLLA/Col/HA and PLLA/HA compared with PLLA/Col. High expression of fibronectin, intracellular adhesion molecule, cadherin, and collagen 1 (Col1) suggests that PLLA/Col/HA and PLLA/HA scaffolds had superior osteoinductivity than PLLA/Col. Additionally, osteopontin, osteocalcin, osterix, Runt-related transcription factor 2 (Runx2), and bone morphogenic protein (BMP2) expression were higher in PLLA/Col/HA and PLLA/HA compared with PLLA/Col. In comparison with PLLA/Col, the PLLA/Col/HA and PLLA/HA scaffolds presented a significant upregulation of the genes Runx2, Col 1, Integrin, osteonectin (ON), bone gamma-carboxyglutamic acid-containing protein (BGALP), osteopontin (OPN), and BMP2. The upregulation of these genes was further increased with PDGF-BB supplementation. These results show that PDGF-BB acts synergistically with PLLA/Col/HA and PLLA/HA to enhance the osteogenic differentiation potential. Therefore, this combination can be used for the rapid expansion of bone marrow stromal cells into bone-forming cells for tissue engineering. Copyright © 2015 Elsevier B.V. All rights reserved.

Ultrasound-assisted fabrication of a biocompatible magnetic hydroxyapatite.

PubMed

Zhou, Gang; Song, Wei; Hou, Yongzhao; Li, Qing; Deng, Xuliang; Fan, Yubo

2014-10-01

This work describes the fabrication and characterization of a biocompatible magnetic hydroxyapatite (HA) using an ultrasound-assisted co-precipitation method. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and transmission electron microscopy (TEM) were used to characterize the structure and chemical composition of the produced samples. The M-H loops of synthesized materials were traced using a vibrating sample magnetometer (VSM) and the biocompatibility was evaluated by cell culture and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Furthermore, in vivo histopathological examinations were used to evaluate the potential toxicological effects of Fe₃O₄-HA composites on kidney of SD rats injected intraperitoneally with Fe₃O₄-HA particles. The results showed that magnetic iron oxide particles first replace OH ions of HA, which are parallel to the c axis, and then enter the HA crystal lattice which produces changes in the crystal surface of HA. Chemical bond interaction was observed between PO₄³⁻ groups of HA and iron ions of Fe₃O₄. The saturation magnetization (MS ) of Fe₃O₄-HA composites was 46.36 emu/g obtained from VSM data. Cell culture and MTT assays indicated that HA could affect the growth and proliferation of HEK-293 cells. This Fe₃O₄-HA composite produced no negative effects on cell morphology, viability, and proliferation and exhibited remarkable biocompatibility. Moreover, no inflammatory cell infiltration was observed in kidney histopathology slices. Therefore, this study succeeds to develop a Fe₃O₄-HA composite as a prospective biomagnetic material for future applications. © 2013 Wiley Periodicals, Inc.

Pursuing Intracellular Pathogens with Hyaluronan. From a 'Pro-Infection' Polymer to a Biomaterial for 'Trojan Horse' Systems.

PubMed

Montanari, Elita; Di Meo, Chiara; Oates, Angela; Coviello, Tommasina; Matricardi, Pietro

2018-04-18

Hyaluronan (HA) is among the most important bioactive polymers in mammals, playing a key role in a number of biological functions. In the last decades, it has been increasingly studied as a biomaterial for drug delivery systems, thanks to its physico-chemical features and ability to target and enter certain cells. The most important receptor of HA is ‘Cluster of Differentiation 44’ (CD44), a cell surface glycoprotein over-expressed by a number of cancers and heavily involved in HA endocytosis. Moreover, CD44 is highly expressed by keratinocytes, activated macrophages and fibroblasts, all of which can act as ‘reservoirs’ for intracellular pathogens. Interestingly, both CD44 and HA appear to play a key role for the invasion and persistence of such microorganisms within the cells. As such, HA is increasingly recognised as a potential target for nano-carriers development, to pursuit and target intracellular pathogens, acting as a ‘Trojan Horse’. This review describes the biological relationship between HA, CD44 and the entry and survival of a number of pathogens within the cells and the subsequent development of HA-based nano-carriers for enhancing the intracellular activity of antimicrobials.

Biomaterial Property Effects on Platelets and Macrophages: An in Vitro Study.

PubMed

Fernandes, Kelly R; Zhang, Yang; Magri, Angela M P; Renno, Ana C M; van den Beucken, Jeroen J J P

2017-12-11

The purpose of this study was to evaluate the effects of surface properties of bone implants coated with hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) on platelets and macrophages upon implant installation and compare them to grit-blasted Ti and Thermanox used as a control. Surface properties were characterized using scanning electron microscopy, profilometry, crystallography, Fourier transform infrared spectroscopy, and coating stability. For platelets, platelet adherence and morphology were assessed. For macrophages, morphology, proliferation, and polarization were evaluated. Surface characterization showed similar roughness of ∼2.5 μm for grit-blasted Ti discs, both with and without coating. Coating stability assessment showed substantial dissolution of HA and β-TCP coatings. Platelet adherence was significantly higher for grit-blasted Ti, Ti-HA, and Ti-β-TCP coatings compared to that of cell culture control Thermanox. Macrophage cultures revealed a decreased proliferation on both HA and β-TCP coated discs compared to both Thermanox and grit-blasted Ti. In contrast, secretion of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine TGF-β were marginal for grit-blasted Ti and Thermanox, while a coating-dependent increased secretion of pro- and anti-inflammatory cytokines was observed for HA and β-TCP coatings. The results demonstrated a significantly upregulated pro-inflammatory and anti-inflammatory cytokine secretion and marker gene expression of macrophages on HA and β-TCP coatings. Furthermore, HA induced an earlier M1 macrophage polarization but more M2 phenotype potency than β-TCP. In conclusion, our data showed that material surface affects the behaviors of first cell types attached to implants. Due to the demonstrated crucial roles of platelets and macrophages in bone healing and implant integration, this information will greatly aid the design of metallic implants for a higher rate of success in patients.

Electroactive Mg2+-Hydroxyapatite Nanostructured Networks against Drug-Resistant Bone Infection Strains.

PubMed

Andrés, Nancy C; Sieben, Juan M; Baldini, Mónica; Rodríguez, Carlos H; Famiglietti, Ángela; Messina, Paula V

2018-06-13

Surface colonization competition between bacteria and host cells is one of the critical factors involved in tissue/implant integration. Current biomaterials are evaluated for their ability both of withstanding favorable responses of host tissue cells and of resisting bacterial contamination. In this work, the antibacterial ability of biocompatible Mg 2+ -substituted nanostructured hydroxyapatite (HA) was investigated. The densities of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli strains were significantly decreased after culture in the presence of Mg-substituted HA materials in direct correlation with Mg 2+ -Ca 2+ switch in the HA lattice. It was noticed that this decrease was accompanied by a minimal alteration of bacterial environments; therefore, the Mg 2+ -HA antibacterial effect was associated with the material surface topography and it electroactive behavior. It was observed that 2.23 wt % Mg 2+ -HA samples exhibited the best antibacterial performance; it decreased 2-fold the initial population of E. coli, P. aeruginosa, and S. aureus at the intermediate concentration (50 mg mL -1 of broth). Our results reinforce the potential of Mg-HA nanostructured materials to be used in antibacterial coatings for implantable devices and/or medicinal materials to prevent bone infection and to promote wound healing.

Surface topography of hydroxyapatite promotes osteogenic differentiation of human bone marrow mesenchymal stem cells.

PubMed

Yang, Wanlei; Han, Weiqi; He, Wei; Li, Jianlei; Wang, Jirong; Feng, Haotian; Qian, Yu

2016-03-01

Effective and safe induction of osteogenic differentiation is one of the key elements of bone tissue engineering. Surface topography of scaffold materials was recently found to promote osteogenic differentiation. Utilization of this topography may be a safer approach than traditional induction by growth factors or chemicals. The aim of this study is to investigate the enhancement of osteogenic differentiation by surface topography and its mechanism of action. Hydroxyapatite (HA) discs with average roughness (Ra) of surface topography ranging from 0.2 to 1.65 μm and mean distance between peaks (RSm) ranging from 89.7 to 18.6 μm were prepared, and human bone-marrow mesenchymal stem cells (hBMSCs) were cultured on these discs. Optimal osteogenic differentiation was observed on discs with surface topography characterized by Ra ranging from 0.77 to 1.09 μm and RSm ranging from 53.9 to 39.3 μm. On this surface configuration of HA, hBMSCs showed oriented attachment, F-actin arrangement, and a peak in the expression of Yes-associated protein (YAP) and PDZ binding motif (TAZ) (YAP/TAZ). These results indicated that the surface topography of HA promoted osteogenic differentiation of hBMSCs, possibly by increasing cell attachment and promoting the YAP/TAZ signaling pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

Synthesis and investigation of physico-chemical, antibacterial, biomymetic properties of silver and zinc containing hydroxyapatite

NASA Astrophysics Data System (ADS)

Zhuk, Ilya; Rasskazova, Lyudmila; Korotchenko, Natalia; Kozik, Vladimir; Kurzina, Irina

2017-11-01

In the work we carried out microwave synthesis of modified hydroxyapatites (HA) with different content of ions. A solid solution based on HA remains a single-phase sample when the calcium ions are substituted by silver and zinc ions up to 5 % by weight (0.5 mole fraction). The microstructure parameters, morphology and the particle powders size were studied by X-ray diffraction analysis, IR spectroscopy, and scanning electron microscopy (SEM). It is shown that the modification of HA by silver (AgHA) and zinc (ZnHA) ions increases the size of its particles, the degree of crystallinity, and the pore sizes of the samples while reducing their specific surface and uniformity of their forms. Elemental analysis and distribution of elements over the surface of HA, AgHA, and ZnHA powders were performed by X-ray spectral microanalysis (RSMA). The ratio of Ca/P is within the range of 1.66-1.77 and corresponds to the ratio of Ca/P in stoichiometric HA and the HA entering bone tissue. The ability of AgHA- and ZnHA-substrates to form on their surface a calcium-phosphate layer from the simulated body fluid (SBF) at 37 °C is determined. This ability decreases in the order: in ZnHA it is less than in AgHA, but greater than in HA. The antibacterial activity of the samples was analyzed. The AgHA sample has both bactericidal and persistent bacteriostatic properties in the case of direct contact with Escherichia coli cells.

Anti-sessile bacterial and cytocompatibility properties of CHX-loaded nanohydroxyapatite.

PubMed

Barros, J; Grenho, L; Fernandes, M H; Manuel, C M; Melo, L F; Nunes, O C; Monteiro, F J; Ferraz, M P

2015-06-01

Nanohydroxyapatite possesses exceptional biocompatibility and bioactivity regarding bone cells and tissues, justifying its use as a coating material or as a bone substitute. Unfortunately, this feature may also encourage bacterial adhesion and biofilm formation. Surface functionalization with antimicrobials is a promising strategy to reduce the likelihood of bacterial infestation and colonization on medical devices. Chlorhexidine digluconate is a common and effective antimicrobial agent used for a wide range of medical applications. The purpose of this work was the development of a nanoHA biomaterial loaded with CHX to prevent surface bacterial accumulation and, simultaneously, with good cytocompatibility, for application in the medical field. CHX (5-1500 mg/L) was loaded onto nanoHA discs and the materials were evaluated for CHX adsorption and release profile, physic-chemical features, antibacterial activity against Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis, and cytocompatibility toward L929 fibroblasts. Results showed that the adsorption of CHX on nanoHA surface occurred by electrostatic interactions between the cationic group of CHX and the phosphate group of nanoHA. The release of CHX from CHX-loaded nanoHA showed a fast initial rate followed by a slower kinetics release, due to constraints caused by dilution and diffusion-limiting processes. NanoHA.50 to nanoHA.1500 showed strong anti-sessile activity, inhibiting bacterial adhesion and the biofilm formation. CHX-nanoHA caused a dose- and time-dependent inhibitory effect on the proliferation of fibroblasts for nanoHA.100 to nanoHA.1500. Cellular behavior on nanoHA.5 and nanoHA.50 was similar to control. Therefore, CHX-loaded nanoHA surfaces appear as a promising alternative to prevention of devices-related infections. Copyright © 2015 Elsevier B.V. All rights reserved.

Conformational divergence in the HA-33/HA-17 trimer of serotype C and D botulinum toxin complex.

PubMed

Sagane, Yoshimasa; Hayashi, Shintaro; Akiyama, Tomonori; Matsumoto, Takashi; Hasegawa, Kimiko; Yamano, Akihito; Suzuki, Tomonori; Niwa, Koichi; Watanabe, Toshihiro; Yajima, Shunsuke

2016-08-05

Clostridium botulinum produces a large toxin complex (L-TC) comprising botulinum neurotoxin associated with auxiliary nontoxic proteins. A complex of 33- and 17-kDa hemagglutinins (an HA-33/HA-17 trimer) enhances L-TC transport across the intestinal epithelial cell layer via binding HA-33 to a sugar on the cell surface. At least two subtypes of serotype C/D HA-33 exhibit differing preferences for the sugars sialic acid and galactose. Here, we compared the three-dimensional structures of the galactose-binding HA-33 and HA-33/HA-17 trimers produced by the C-Yoichi strain. Comparisons of serotype C/D HA-33 sequences reveal a variable region with relatively low sequence similarity across the C. botulinum strains; the variability of this region may influence the manner of sugar-recognition by HA-33. Crystal structures of sialic acid- and galactose-binding HA-33 are broadly similar in appearance. However, small-angle X-ray scattering revealed distinct solution structures for HA-33/HA-17 trimers. A structural change in the C-terminal variable region of HA-33 might cause a dramatic shift in the conformation and sugar-recognition mode of HA-33/HA-17 trimer. Copyright © 2016 Elsevier Inc. All rights reserved.

Large-scale FMO-MP3 calculations on the surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA)

NASA Astrophysics Data System (ADS)

Mochizuki, Yuji; Yamashita, Katsumi; Fukuzawa, Kaori; Takematsu, Kazutomo; Watanabe, Hirofumi; Taguchi, Naoki; Okiyama, Yoshio; Tsuboi, Misako; Nakano, Tatsuya; Tanaka, Shigenori

2010-06-01

Two proteins on the influenza virus surface have been well known. One is hemagglutinin (HA) associated with the infection to cells. The fragment molecular orbital (FMO) calculations were performed on a complex consisting of HA trimer and two Fab-fragments at the third-order Møller-Plesset perturbation (MP3) level. The numbers of residues and 6-31G basis functions were 2351 and 201276, and thus a massively parallel-vector computer was utilized to accelerate the processing. This FMO-MP3 job was completed in 5.8 h with 1024 processors. Another protein is neuraminidase (NA) involved in the escape from infected cells. The FMO-MP3 calculation was also applied to analyze the interactions between oseltamivir and surrounding residues in pharmacophore.

Mechanical properties and cytocompatibility of carbon fibre reinforced nano-hydroxyapatite/polyamide66 ternary biocomposite.

PubMed

Zhang, Xuesong; Zhang, Yonggang; Zhang, Xuelian; Wang, Yan; Wang, Jiaqi; Lu, Ming; Li, Hong

2015-02-01

Fibre-reinforced composites with good strength and ductility as bone repair biomaterials have been attracting increasing attention in biomedical applications. In the present study, a novel ternary composite was prepared using carbon fibre (CF) to reinforce a nano-hydroxyapatite/polyamide66 composite (HA/PA). The interface and mechanical strength of the ternary composite (CF/HA/PA) were characterised. In addition, to assess the cytocompatibility, the composite was co-cultured with MG-63 cells, and the cell morphology, MTT, and ALP were tested. The results indicated that CFs were uniformly distributed in the HA/PA matrix with random orientation and that the CFs bonded well to the HA/PA matrix. The reinforced ternary composite exhibited a compressive strength of 116-212 MPa, a bending strength of 89-138 MPa, a tensile strength of 109-181 MPa, with the breaking elongation ratio of 6.2-9.1%, and a tensile modulus of 2.9-5.8 GPa, with the values varying with increasing CF content from 5 to 20 (mass fraction). The MG-63 cells of normal phenotype were well extended and spread onto the ternary composite surface. In addition, its proliferation and differentiation on the composite surface were significantly increased with time, indicating that the incorporation of CFs into HA/PA had little negative effects on MG-63 cells. The incorporation of CFs into a HA/PA66 composite improved the strength and ductility and introduced no negative effects on the cytocompatibility. Hence, the CF/HA/PA ternary composite has potential to be used as a bone repair materials and in fixation devices. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of Hyaluronic Acid Incorporation Method on the Stability and Biological Properties of Polyurethane-Hyaluronic Acid Biomaterials

PubMed Central

Ruiz, Amaliris; Rathnam, Kashmila R.; Masters, Kristyn S.

2014-01-01

The high failure rate of small diameter vascular grafts continues to drive the development of new materials and modification strategies that address this clinical problem, with biomolecule incorporation typically achieved via surface-based modification of various biomaterials. In this work, we examined whether the method of biomolecule incorporation (i.e., bulk vs. surface modification) into a polyurethane (PU) polymer impacted biomaterial performance in the context of vascular applications. Specifically, hyaluronic acid (HA) was incorporated into a poly(ether urethane) via bulk copolymerization or covalent surface tethering, and the resulting PU-HA materials characterized with respect to both physical and biological properties. Modification of PU with HA by either surface or bulk methods yielded materials that, when tested under static conditions, possessed no significant differences in their ability to resist protein adsorption, platelet adhesion, and bacterial adhesion, while supporting endothelial cell culture. However, only bulk-modified PU-HA materials were able to fully retain these characteristics following material exposure to flow, demonstrating a superior ability to retain the incorporated HA and minimize enzymatic degradation, protein adsorption, platelet adhesion, and bacterial adhesion. Thus, despite bulk methods rarely being implemented in the context of biomolecule attachment, these results demonstrate improved performance of PU-HA upon bulk, rather than surface, incorporation of HA. Although explored only in the context of PU-HA, the findings revealed by these experiments have broader implications for the design and evaluation of vascular graft modification strategies. PMID:24276670

TAT and HA2 Facilitate Cellular Uptake of Gold Nanoparticles but Do Not Lead to Cytosolic Localisation

PubMed Central

Free, Paul; Lévy, Raphaël

2015-01-01

The methods currently available to deliver functional labels and drugs to the cell cytosol are inefficient and this constitutes a major obstacle to cell biology (delivery of sensors and imaging probes) and therapy (drug access to the cell internal machinery). As cell membranes are impermeable to most molecular cargos, viral peptides have been used to bolster their internalisation through endocytosis and help their release to the cytosol by bursting the endosomal vesicles. However, conflicting results have been reported on the extent of the cytosolic delivery achieved. To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery. We evaluated the number of gold nanoparticles present in cells after internalisation using photothermal microscopy and their subcellular localisation by electron microscopy. While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles. PMID:25836335

Interaction between hyaluronan and CD44 in the development of dimethylnitrosamine-induced liver cirrhosis.

PubMed

Satoh, T; Ichida, T; Matsuda, Y; Sugiyama, M; Yonekura, K; Ishikawa, T; Asakura, H

2000-04-01

A significant increase in serum hyaluronan (HA) levels has been reported in patients with liver cirrhosis. This mechanism is not yet clear, and receptors for HA have not been characterized. In this study, we examined the expression of both HA and its receptors, CD44 and intercellular adhesion molecule-1 (ICAM-1), in dimethylnitrosamine-induced liver cirrhosis. Using biotinylated HA binding protein, HA was detected in the area of periportal fibrosis and around the sinusoidal wall where hepatic fibrosis was developing. Electron microscopy revealed that HA was localized on Ito cells and sinusoidal endothelial cells (SEC). Conversely, CD44, which was only expressed weakly in normal liver, was present in large amounts in cirrhotic liver. The distribution pattern of CD44 was similar to that of HA, however, CD44 was mainly localized on the infiltrating lymphocytes and Kupffer cells. Moreover, CD44 was detected on part of factor VIII-positive SEC. Intercellular adhesion molecule-1, another receptor for HA, was detected on the surface of hepatocytes and around the sinusoidal wall in cirrhotic liver, but its distribution was not accompanied by expression of HA. With respect to CD44 isoforms, the standard form m-RNA predominated in both normal and cirrhotic liver. Variant pMeta-1 mRNA was detected at low levels. An interaction between HA and CD44 may play a role in the recruitment of numerous infiltrating cells and HA accumulation in hepatic sinusoids. Together with phenotypic changes in the SEC, these results may lead to a disturbance in the elimination of HA during the progression of liver cirrhosis.

Inhibition of hyaluronic acid formation sensitizes chronic myelogenous leukemia to treatment with doxorubicin.

PubMed

Uchakina, Olga N; Ban, Hao; Hostetler, Bryan J; McKallip, Robert J

2016-11-01

In the current study we examined the ability of 4-methylumbelliferone (4-MU), which can inhibit hyaluronic acid synthesis, to sensitize K562 chronic myelogenous leukemia (CML) cells to doxorubicin therapy. Exposure of K562 cells to doxorubicin led to increased hyaluronic acid synthase (HAS) gene expression and increased levels of cell surface hyaluronic acid. Furthermore, exposure of K562 cells to exogenous HA caused resistance to doxorubicin-induced cell death. The combination of low dose 4-MU and doxorubicin led to increased apoptosis when compared to higher doses of any agent alone. Additionally, treatment with 4-MU led to a significant reduction in doxorubicin-induced increase in HA cell surface expression. Mechanistically, 4-MU treatment led to an increase in p38 activation and PARP cleavage. The role of p38 in 4-MU/doxorubicin-treated K562 cells was confirmed when p38 inhibitors led to protection from 4-MU/doxorubicin-induced apoptosis. Together, results from this study suggest that treatment with 4-MU increases the sensitivity of CML to chemotherapeutics by decreasing their HA-mediated resistance to apoptosis. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Limitation of biocompatibility of hydrated nanocrystalline hydroxyapatite

NASA Astrophysics Data System (ADS)

Minaychev, V. V.; Teleshev, A. T.; Gorshenev, V. N.; Yakovleva, M. A.; Fomichev, V. A.; Pankratov, A. S.; Menshikh, K. A.; Fadeev, R. S.; Fadeeva, I. S.; Senotov, A. S.; Kobyakova, M. I.; Yurasova, Yu B.; Akatov, V. S.

2018-04-01

Nanostructured hydroxyapatite (HA) in the form of hydrated paste is considered to be a promising material for a minor-invasive surgical curing of bone tissue injure. However questions about adhesion of cells on this material and its biocompatibility still remain. In this study biocompatibility of paste-formed nanosized HA (nano-HA) by in vitro methods is investigated. Nano-HA (particles sized about 20 nm) was synthesized under conditions of mechano-acoustic activation of an aqueous reaction mixture of ammonium hydrophosphate and calcium nitrate. It was ascertained that nanocrystalline paste was not cytotoxic although limitation of adhesion, spreading and growth of the cells on its surface was revealed. The results obtained point on the need of modification of hydrated nano-HA in the aims of increasing its biocompatibility and osteoplastic potential.

Chlorhexidine-loaded hydroxyapatite microspheres as an antimicrobial delivery system and its effect on in vivo osteo-conductive properties.

PubMed

Soriano-Souza, Carlos Alberto; Rossi, Andre L; Mavropoulos, Elena; Hausen, Moema A; Tanaka, Marcelo N; Calasans-Maia, Mônica D; Granjeiro, Jose M; Rocha-Leão, Maria Helena M; Rossi, Alexandre M

2015-04-01

Hydroxyapatite (HA) has been investigated as a delivery system for antimicrobial and antibacterial agents to simultaneously stimulate bone regeneration and prevent infection. Despite evidence supporting the bactericidal efficiency of these HA carriers, few studies have focused on the effect of this association on bone regeneration. In this work, we evaluated the physico-chemical properties of hydroxyapatite microspheres loaded with chlorhexidine (CHX) at two different concentrations, 0.9 and 9.1 μgCHX/cm2 HA, and characterized their effects on in vitro osteoblast viability and bone regeneration. Ultraviolet-visible spectroscopy, scanning and transmission electron microscopy associated with energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to characterize the association of CHX and HA nanoparticles. The high CHX loading dose induced formation of organic CHX plate-like aggregates on the HA surface, whereas a Langmuir film was formed at the low CHX surface concentration. Quantitative evaluation of murine osteoblast viability parameters, including adhesion, mitochondrial activity and membrane integrity of cells exposed to HA/CHX extracts, revealed a cytotoxic effect for both loading concentrations. Histomorphological analysis upon implantation into the dorsal connective tissues and calvaria of rats for 7 and 42 days showed that the high CHX concentration induced the infiltration of inflammatory cells, resulting in retarded bone growth. Despite a strong decrease in in vitro cell viability, the low CHX loading dose did not impair the biocompatibility and osteoconductivity of HA during bone repair. These results indicate that high antimicrobial doses may activate a strong local inflammatory response and disrupt the long-term osteoconductive properties of CHX-HA delivery systems.

Role of bovine serum albumin and humic acid in the interaction between SiO2 nanoparticles and model cell membranes.

PubMed

Wei, Xiaoran; Qu, Xiaolei; Ding, Lei; Hu, Jingtian; Jiang, Wei

2016-12-01

Silica nanoparticles (SiO 2 NPs) can cause health hazard after their release into the environment. Adsorption of natural organic matter and biomolecules on SiO 2 NPs alters their surface properties and cytotoxicity. In this study, SiO 2 NPs were treated by bovine serum albumin (BSA) and humic acid (HA) to study their effects on the integrity and fluidity of model cell membranes. Giant and small unilamellar vesicles (GUVs and SUVs) were prepared as model cell membranes in order to avoid the interference of cellular activities. The microscopic observation revealed that the BSA/HA treated (BSA-/HA-) SiO 2 NPs took more time to disrupt membrane than untreated-SiO 2 NPs, because BSA/HA adsorption covered the surface SiOH/SiO - groups and weakened the interaction between NPs and phospholipids. The deposition of SiO 2 NPs on membrane was monitored by a quartz crystal microbalance with dissipation (QCM-D). Untreated- and HA-SiO 2 NPs quickly disrupted the SUV layer on QCM-D sensor; BSA-SiO 2 NPs attached on the membranes but only caused slow vesicle disruption. Untreated-, BSA- and HA-SiO 2 NPs all caused the gelation of the positively-charged membrane, which was evaluated by the generalized polarity values. HA-SiO 2 NPs caused most serious gelation, and BSA-SiO 2 NPs caused the least. Our results demonstrate that the protein adsorption on SiO 2 NPs decreases the NP-induced membrane damage. Copyright © 2016 Elsevier Ltd. All rights reserved.

Robotic dispensing of composite scaffolds and in vitro responses of bone marrow stromal cells.

PubMed

Hong, Seok-Jung; Jeong, Ishik; Noh, Kyung-Tae; Yu, Hye-Sun; Lee, Gil-Su; Kim, Hae-Won

2009-09-01

The development of bioactive scaffolds with a designed pore configuration is of particular importance in bone tissue engineering. In this study, bone scaffolds with a controlled pore structure and a bioactive composition were produced using a robotic dispensing technique. A poly(epsilon-caprolactone) (PCL) and hydroxyapatite (HA) composite solution (PCL/HA = 1) was constructed into a 3-dimensional (3D) porous scaffold by fiber deposition and layer-by-layer assembly using a computer-aided robocasting machine. The in vitro tissue cell compatibility was examined using rat bone marrow stromal cells (rBMSCs). The adhesion and growth of cells onto the robotic dispensed scaffolds were observed to be limited by applying the conventional cell seeding technique. However, the initially adhered cells were viable on the scaffold surface. The alkaline phosphatase activity of the cells was significantly higher on the HA-PCL than on the PCL and control culture dish, suggesting that the robotic dispensed HA-PCL scaffold should stimulate the osteogenic differentiation of rBMSCs. Moreover, the expression of a series of bone-associated genes, including alkaline phosphatase and collagen type I, was highly up-regulated on the HA-PCL scaffold as compared to that on the pure PCL scaffold. Overall, the robotic dispensed HA-PCL is considered to find potential use as a bioactive 3D scaffold for bone tissue engineering.

Cellular basis for neonatally induced T-suppressor activity. Primary B cell maturation is blocked by suppressor-helper interactions restricted by loci on chromosome 12

PubMed Central

1985-01-01

The cellular mechanism and genetic restriction of neonatally induced HA- specific suppressor T (Ts) cells have been examined. The in vivo effect of these Ts cells on antibody production, primary B cell proliferation, B cell surface marker changes, and helper T (Th) cell priming during primary responses to HA have been determined. The results indicate that, although antigen-induced B cell proliferative responses and surface marker changes occur in the presence of Ts cells, differentiation to Ig secretion, and long-lived memory B cell production are prevented. Further, antigen-specific Th cell priming is completely ablated by Ts cells, suggesting that Ts act by preventing the delivery of Th signals required for both the later stages of primary B cell maturation, and the formation of memory B cell populations. Finally, in vivo cell mixing experiments using congenic mice indicate that this Ts-Th interaction is restricted by loci on mouse chromosome 12. PMID:2580040

Development of hyaluronic acid-Fe2O3 hybrid magnetic nanoparticles for targeted delivery of peptides.

PubMed

Kumar, Arun; Sahoo, Bishwabhusan; Montpetit, Alison; Behera, Sumita; Lockey, Richard F; Mohapatra, Shyam S

2007-06-01

Novel hybrid nanoparticles comprised of hyaluronic acid (HA) and iron oxide were synthesized and characterized for the first time with the average diameter of less than 160 nm. The iron oxide (Fe2O3) particles are hybridized between HA layers by electrostatic interactions between the positive surface charge of the Fe2O3 nanoparticles and the negative charge of the carboxylate groups of HA, forming a corral-like structure. The particles were also characterized by FTIR and NMR to verify the hybridization. The particles were tested for their ability to deliver peptides to the cells using HEK293 and A549 cells. Results show that these particles delivered peptides at about 100% level. These HA-iron oxide nanoparticles are expected to be useful in developing effective tissue and cell targeting systems.

Tumor targeting profiling of hyaluronan-coated lipid based-nanoparticles

NASA Astrophysics Data System (ADS)

Mizrahy, Shoshy; Goldsmith, Meir; Leviatan-Ben-Arye, Shani; Kisin-Finfer, Einat; Redy, Orit; Srinivasan, Srimeenakshi; Shabat, Doron; Godin, Biana; Peer, Dan

2014-03-01

Hyaluronan (HA), a naturally occurring high Mw (HMw) glycosaminoglycan, has been shown to play crucial roles in cell growth, embryonic development, healing processes, inflammation, and tumor development and progression. Low Mw (LMw, <10 kDa) HA has been reported to provoke inflammatory responses, such as induction of cytokines, chemokines, reactive nitrogen species and growth factors. Herein, we prepared and characterized two types of HA coated (LMw and HMw) lipid-based targeted and stabilized nanoparticles (tsNPs) and tested their binding to tumor cells expressing the HA receptor (CD44), systemic immunotoxicity, and biodistribution in tumor bearing mice. In vitro, the Mw of the surface anchored HA had a significant influence on the affinity towards CD44 on B16F10 murine melanoma cells. LMw HA-tsNPs exhibited weak binding, while binding of tsNPs coated with HMw HA was characterized by high binding. Both types of tsNPs had no measured effect on cytokine induction in vivo following intravenous administration to healthy C57BL/6 mice suggesting no immune activation. HMw HA-tsNPs showed enhanced circulation time and tumor targeting specificity, mainly by accumulating in the tumor and its vicinity compared with LMw HA-tsNPs. Finally, we show that methotrexate (MTX), a drug commonly used in cancer chemotherapy, entrapped in HMw HA-tsNPs slowly diffused from the particles with a half-life of 13.75 days, and improved the therapeutic outcome in a murine B16F10 melanoma model compared with NPs suggesting an active cellular targeting beyond the Enhanced Permeability and Retention (EPR) effect. Taken together, these findings have major implications for the use of high molecular weight HA in nanomedicine as a selective and safe active cellular targeting moiety.Hyaluronan (HA), a naturally occurring high Mw (HMw) glycosaminoglycan, has been shown to play crucial roles in cell growth, embryonic development, healing processes, inflammation, and tumor development and progression. Low Mw (LMw, <10 kDa) HA has been reported to provoke inflammatory responses, such as induction of cytokines, chemokines, reactive nitrogen species and growth factors. Herein, we prepared and characterized two types of HA coated (LMw and HMw) lipid-based targeted and stabilized nanoparticles (tsNPs) and tested their binding to tumor cells expressing the HA receptor (CD44), systemic immunotoxicity, and biodistribution in tumor bearing mice. In vitro, the Mw of the surface anchored HA had a significant influence on the affinity towards CD44 on B16F10 murine melanoma cells. LMw HA-tsNPs exhibited weak binding, while binding of tsNPs coated with HMw HA was characterized by high binding. Both types of tsNPs had no measured effect on cytokine induction in vivo following intravenous administration to healthy C57BL/6 mice suggesting no immune activation. HMw HA-tsNPs showed enhanced circulation time and tumor targeting specificity, mainly by accumulating in the tumor and its vicinity compared with LMw HA-tsNPs. Finally, we show that methotrexate (MTX), a drug commonly used in cancer chemotherapy, entrapped in HMw HA-tsNPs slowly diffused from the particles with a half-life of 13.75 days, and improved the therapeutic outcome in a murine B16F10 melanoma model compared with NPs suggesting an active cellular targeting beyond the Enhanced Permeability and Retention (EPR) effect. Taken together, these findings have major implications for the use of high molecular weight HA in nanomedicine as a selective and safe active cellular targeting moiety. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr06102g

Antibacterial activity and biofilm inhibition by surface modified titanium alloy medical implants following application of silver, titanium dioxide and hydroxyapatite nanocoatings.

PubMed

Besinis, A; Hadi, S D; Le, H R; Tredwin, C; Handy, R D

2017-04-01

One of the most common causes of implant failure is peri-implantitis, which is caused by bacterial biofilm formation on the surfaces of dental implants. Modification of the surface nanotopography has been suggested to affect bacterial adherence to implants. Silver nanoparticles are also known for their antibacterial properties. In this study, titanium alloy implants were surface modified following silver plating, anodisation and sintering techniques to create a combination of silver, titanium dioxide and hydroxyapatite (HA) nanocoatings. Their antibacterial performance was quantitatively assessed by measuring the growth of Streptococcus sanguinis, proportion of live/dead cells and lactate production by the microbes over 24 h. Application of a dual layered silver-HA nanocoating to the surface of implants successfully inhibited bacterial growth in the surrounding media (100% mortality), whereas the formation of bacterial biofilm on the implant surfaces was reduced by 97.5%. Uncoated controls and titanium dioxide nanocoatings showed no antibacterial effect. Both silver and HA nanocoatings were found to be very stable in biological fluids with material loss, as a result of dissolution, to be less than 0.07% for the silver nanocoatings after 24 h in a modified Krebs-Ringer bicarbonate buffer. No dissolution was detected for the HA nanocoatings. Thus, application of a dual layered silver-HA nanocoating to titanium alloy implants creates a surface with antibiofilm properties without compromising the HA biocompatibility required for successful osseointegration and accelerated bone healing.

CD44 mediated hyaluronan adhesion of Toxoplasma gondii-infected leukocytes.

PubMed

Hayashi, Takeshi; Unno, Akihiro; Baba, Minami; Ohno, Tamio; Kitoh, Katsuya; Takashima, Yasuhiro

2014-04-01

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects humans and animals. Ingested parasites cross the intestinal epithelium, invade leukocytes and are then disseminated to peripheral organs. However, the mechanism of extravasation of the infected leukocytes remains poorly understood. In this study, we demonstrate that T. gondii-invaded human and mouse leukocytes express higher level of CD44, a ligand of hyaluronan (HA), and its expression on myeloid and non-myeloid leukocytes causes T. gondii-invaded human and mouse leukocyte to adhere to HA more effectively than non-invaded leukocytes. The specific adherence of parasite-invaded leukocytes was inhibited by anti CD44 antibody. Leukocytes of CD44 knockout mice did not show parasite-invaded leukocyte specific adhesion. Our results indicate that parasite-invaded leukocytes, regardless of whether myeloid or not, gain higher ability to adhere to HA than non-invaded leukocytes, via upregulation of CD44 expression and/or selective invasion to CD44 highly expressing cells. The difference in ability to adhere to HA between parasite-invaded cells and non-invaded neighboring cells might facilitate effective delivery of parasite-invaded leukocytes to the HA-producing endothelial cell surface and/or HA-rich extra cellular matrix. © 2013.

Nanocomposite Porous Microcarriers Based on Strontium-Substituted HA- g-Poly(γ-benzyl-l-glutamate) for Bone Tissue Engineering.

PubMed

Yan, Shifeng; Xia, Pengfei; Xu, Shenghua; Zhang, Kunxi; Li, Guifei; Cui, Lei; Yin, Jingbo

2018-05-04

Porous microcarriers have aroused increasing attention recently, which can create a protected environment for sufficient cell seeding density, facilitate oxygen and nutrient transfer, and well support the cell attachment and growth. In this study, porous microcarriers fabricated from the strontium-substituted hydroxyapatite- graft-poly(γ-benzyl-l-glutamate) (Sr10-HA- g-PBLG) hybrid nanocomposite were developed. The surface grating of PBLG, the micromorphology and element distribution, mechanical strength, in vitro degradation, and Sr 2+ ion release of the obtained Sr10-HA- g-PBLG porous microcarriers were investigated, respectively. The grafting ratio and the molecular weight of the grafted PBLG of Sr10-HA- g-PBLG could be effectively controlled by varying the initial ratio of BLG-NCA to Sr10-HA-NH 2 . The microcarriers exhibited a highly porous and interconnected microstructure with the porosity of about 90% and overall density of 1.03-1.06 g/cm 3 . Also, the degradation rate of Sr10-HA-PBLG microcarriers could be effectively controlled and long-term Sr 2+ release was obtained. The Sr10-HA-PBLG microcarriers allowed cells adhesion, infiltration, and proliferation and promoted the osteogenic differentiation of rabbit adipose-derived stem cells (ADSCs). Successful healing of femoral bone defect was proved by injection of the ADSCs-seeded Sr10-HA-PBLG microcarriers in a rabbit model.

Glycosylation at Asn91 of H1N1 haemagglutinin affects binding to glycan receptors

PubMed Central

Jayaraman, Akila; Koh, Xiaoying; Li, Jing; Raman, Rahul; Viswanathan, Karthik; Shriver, Zachary; Sasisekharan, Ram

2012-01-01

The glycoprotein HA (haemagglutinin) on the surface of influenza A virus plays a central role in recognition and binding to specific host cell-surface glycan receptors and in fusion of viral membrane to the host nuclear membrane during viral replication. Given the abundance of HA on the viral surface, this protein is also the primary target for host innate and adaptive immune responses. Although addition of glycosylation sites on HA are a part of viral evolution to evade the host immune responses, there are specific glycosylation sites that are conserved during most of the evolution of the virus. In the present study, it was demonstrated that one such conserved glycosylation site at Asn91 in H1N1 HA critically governs the glycan receptor-binding specificity and hence would potentially impinge on the host adaptation of the virus. PMID:22642577

Glycosylation at Asn91 of H1N1 haemagglutinin affects binding to glycan receptors.

PubMed

Jayaraman, Akila; Koh, Xiaoying; Li, Jing; Raman, Rahul; Viswanathan, Karthik; Shriver, Zachary; Sasisekharan, Ram

2012-06-15

The glycoprotein HA (haemagglutinin) on the surface of influenza A virus plays a central role in recognition and binding to specific host cell-surface glycan receptors and in fusion of viral membrane to the host nuclear membrane during viral replication. Given the abundance of HA on the viral surface, this protein is also the primary target for host innate and adaptive immune responses. Although addition of glycosylation sites on HA are a part of viral evolution to evade the host immune responses, there are specific glycosylation sites that are conserved during most of the evolution of the virus. In the present study, it was demonstrated that one such conserved glycosylation site at Asn(91) in H1N1 HA critically governs the glycan receptor-binding specificity and hence would potentially impinge on the host adaptation of the virus.

Neural stem cell proliferation and differentiation in the conductive PEDOT-HA/Cs/Gel scaffold for neural tissue engineering.

PubMed

Wang, Shuping; Guan, Shui; Xu, Jianqiang; Li, Wenfang; Ge, Dan; Sun, Changkai; Liu, Tianqing; Ma, Xuehu

2017-09-26

Engineering scaffolds with excellent electro-activity is increasingly important in tissue engineering and regenerative medicine. Herein, conductive poly(3,4-ethylenedioxythiophene) doped with hyaluronic acid (PEDOT-HA) nanoparticles were firstly synthesized via chemical oxidant polymerization. A three-dimensional (3D) PEDOT-HA/Cs/Gel scaffold was then developed by introducing PEDOT-HA nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. HA, as a bridge, not only was used as a dopant, but also combined PEDOT into the Cs/Gel via chemical crosslinking. The PEDOT-HA/Cs/Gel scaffold was used as a conductive substrate for neural stem cell (NSC) culture in vitro. The results demonstrated that the PEDOT-HA/Cs/Gel scaffold had excellent biocompatibility for NSC proliferation and differentiation. 3D confocal fluorescence images showed cells attached on the channel surface of Cs/Gel and PEDOT-HA/Cs/Gel scaffolds with a normal neuronal morphology. Compared to the Cs/Gel scaffold, the PEDOT-HA/Cs/Gel scaffold not only promoted NSC proliferation with up-regulated expression of Ki67, but also enhanced NSC differentiation into neurons and astrocytes with up-regulated expression of β tubulin-III and GFAP, respectively. It is expected that this electro-active and bio-active PEDOT-HA/Cs/Gel scaffold will be used as a conductive platform to regulate NSC behavior for neural tissue engineering.

Development of biodegradable PLGA nanoparticles surface engineered with hyaluronic acid for targeted delivery of paclitaxel to triple negative breast cancer cells.

PubMed

Cerqueira, Brenda Brenner S; Lasham, Annette; Shelling, Andrew N; Al-Kassas, Raida

2017-07-01

This study aimed at development of poly (lactic-co-glycolic acid) (PLGA) nanoparticles embedded with paclitaxel and coated with hyaluronic acid (HA-PTX-PLGA) to actively target the drug to a triple negative breast cancer cells. Nanoparticles were successfully fabricated using a modified oil-in-water emulsion method. The effect of various formulations parameters on the physicochemical properties of the nanoparticles was investigated. SEM imaging confirmed the spherical shape and nano-scale size of the nanoparticles. A sustained drug release profile was obtained and enhanced PTX cytotoxicity was observed when MDA-MB-231 cells were incubated with the HA-PTX-PLGA formulation compared to cells incubated with the non-HA coated nanoparticles. Moreover, HA-PLGA nanoparticles exhibited improved cellular uptake, based on a possible receptor mediated endocytosis due to interaction of HA with CD44 receptors when compared to non-coated PLGA nanoparticles. The non-haemolytic potential of the nanoparticles indicated the suitability of the developed formulation for intravenous administration. Copyright © 2017 Elsevier B.V. All rights reserved.

Hyaluronan preserves the proliferation and differentiation potentials of long-term cultured murine adipose-derived stromal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, P.-Y.; Huang, Lynn L.H.; Hsieh, H.-J.

2007-08-17

For long-term culture, murine adipose-derived stromal cells (mADSCs) at latter passages demonstrated a marked decline in proliferative activity, exhibited senescent morphology and reduced differentiation potentials, particularly osteogenesis. To extend the lifespan of mADSCs, two culture conditions containing hyaluronan (HA) was compared in our study, one as a culture medium supplement (SHA), and the other where HA was pre-coated on culture surface (CHA). mADSCs cultivated with SHA exhibited a prolonged lifespan, reduced cellular senescence, and enhanced osteogenic potential compared to regular culture condition (control). Upon CHA treatment, mADSCs tended to form cell aggregates with gradual growth profiles, while their differentiation activitiesmore » remained similar to SHA groups. After transferring mADSCs from CHA to control surface, they were shown to have an extended lifespan and an increase of osteogenic potential. Our results suggested that HA can be useful for preserving the proliferation and differentiation potentials of long-term cultured mADSCs.« less

A novel jet-based nano-hydroxyapatite patterning technique for osteoblast guidance

PubMed Central

Li, Xiang; Koller, Garrit; Huang, Jie; Di Silvio, Lucy; Renton, Tara; Esat, Minoo; Bonfield, William; Edirisinghe, Mohan

2010-01-01

Surface topography is well known to play a crucial role in influencing cellular responses to an implant material and is therefore important in bone tissue regeneration. A novel jet-based patterning technique, template-assisted electrohydrodynamic atomization spraying, was recently devised to control precisely the surface structure as well as its dimensions. In the present study, a detailed investigation of this patterning process was carried out. A range of nano-hydroxyapatite (nHA) line-shaped patterns <20 µm in width were successfully deposited on a commercially pure Ti surface by controlling the flow of an nHA suspension in an electric field. In vitro studies showed that the nHA patterns generated are capable of regulating the human osteoblast cell attachment and orientation. PMID:19493897

Physico-chemical characteristics and protein adsorption potential of hydroxyapatite particles: influence on in vitro biocompatibility of ceramics after sintering.

PubMed

Rouahi, M; Champion, E; Gallet, O; Jada, A; Anselme, K

2006-01-15

Through the example of two HA ceramics prepared from two HA powders (HAD and HAL), we explored the relation between the physico-chemical qualities of the initial HA powder and the final HA ceramic and their influence on the protein adsorption and cell response to the final HA ceramics. The powders were characterized by XRD, FT-IR, zeta potential, and specific surface area (SSA). Their protein adsorption potential was tested after immersion in culture medium +15% of fetal calf serum. These results were correlated with the protein adsorption potential of the two ceramics (cHAD and cHAL) prepared from the HAD and HAL powders respectively and to the cell attachment after 4, 24 and 72 h on the ceramics. From our results, it appears that a relation can be established between the physico-chemical characteristics of the initial HA powders and the final biological response to the sintered ceramics prepared from these powders. An inverse relation exists between the SSA and the protein adsorption capacity of HA powders and the protein adsorption and cell attachment on HA ceramics. This inverse relation is related to phenomenon occurring during the sintering phase and the formation of inter-granular micro-porosity.

Thrombomodulin exerts cytoprotective effect on low-dose UVB-irradiated HaCaT cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwata, Masahiro; Laboratory of Vascular Medicine, Department of Cardiovascular and Respiratory Disorders Advanced Therapeutics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520; Kawahara, Ko-ichi

Thrombomodulin (TM) is an endothelial cell surface anticoagulant glycoprotein that performs antimetastatic, angiogenic, adhesive, and anti-inflammatory functions in various tissues. It is also expressed in epidermal keratinocytes. We found that a physiological dose (10 mJ/cm{sup 2}) of mid-wavelength ultraviolet irradiation (UVB) significantly induced TM expression via the p38mitogen-activated protein kinase (MAPK)/cyclic AMP response element (CRE) signaling pathway in the epidermal keratinocyte cell line HaCaT; this shows that TM regulates the survival of HaCaT cells. SB203580, a p38MAPK inhibitor, significantly decreased TM expression and the viability of cells exposed to UVB. Furthermore, overexpression of TM markedly increased cell viability, and itmore » was abrogated by TM small interfering RNA (siRNA), suggesting that TM may play an important role in exerting cytoprotective effect on epidermal keratinocytes against low-dose UVB.« less

Electrospun fibrous scaffolds combined with nanoscale hydroxyapatite induce osteogenic differentiation of human periodontal ligament cells

PubMed Central

Wu, Xiaonan; Miao, Leiying; Yao, Yingfang; Wu, Wenlei; Liu, Yu; Chen, Xiaofeng; Sun, Weibin

2014-01-01

Periodontal repair is a complex process in which regeneration of alveolar bone is a vital component. The aim of this study was to develop a biodegradable scaffold with good biocompatibility and osteoinductive ability. Two types of composite fibrous scaffolds were produced by electrospinning, ie, type I collagen/poly(ε-caprolactone) (COL/PCL) and type I collagen/poly(ε-caprolactone)/nanoscale hydroxyapatite (COL/PCL/nHA) with an average fiber diameter of about 377 nm. After a simulated body fluid (SBF) immersion test, the COL/PCL/nHA-SBF scaffold developed a rough surface because of the calcium phosphate deposited on the fibers, suggesting that the presence of nHA promoted the mineralization potential of the scaffold. Energy dispersive X-ray spectroscopy clearly showed the calcium and phosphorus content in the COL/PCL/nHA and COL/PCL/nHA-SBF scaffolds, confirming the findings of nHA and calcium phosphate precipitation on scanning electron micrographs. Water contact analysis revealed that nHA could improve the hydrophilic nature of the COL/PCL/nHA-SBF scaffold. The morphology of periodontal ligament cells cultured on COL/PCL-SBF and COL/PCL/nHA-SBF was evaluated by scanning electron microscopy. The results showed that cells adhered to either type of scaffold and were slightly spindle-shaped in the beginning, then extended gradually with stretched filopodia, indicating an ability to fill the fiber pores. A Cell Counting Kit-8 assay showed that both scaffolds supported cell proliferation. However, real-time quantitative polymerase chain reaction analysis showed that expression of the bone-related markers, alkaline phosphatase and osteocalcin, was upregulated only on the COL/PCL/nHA-SBF scaffold, indicating that this scaffold had the ability to induce osteogenic differentiation of periodontal ligament cells. In this study, COL/PCL/nHA-SBF produced by electrospinning followed by biomimetic mineralization had combined electrospun fibers with nHA in it. This scaffold has good biocompatibility and osteoinductive ability as a result of the characteristics of nHA, so could be innovatively applied to periodontal tissue engineering as a potential scaffold. PMID:25206304

Specific targeting to B cells by lipid-based nanoparticles conjugated with a novel CD22-ScFv.

PubMed

Loomis, Kristin; Smith, Brandon; Feng, Yang; Garg, Himanshu; Yavlovich, Amichai; Campbell-Massa, Ryan; Dimitrov, Dimiter S; Blumenthal, Robert; Xiao, Xiaodong; Puri, Anu

2010-04-01

The CD22 antigen is a viable target for therapeutic intervention for B-cell lymphomas. Several therapeutic anti-CD22 antibodies as well as an anti-CD22-based immunotoxin (HA22) are currently under investigation in clinical settings. Coupling of anti-CD22 reagents with a nano-drug delivery vehicle is projected to significantly improve treatment efficacies. Therefore, we generated a mutant of the targeting segment of HA22 (a CD22 scFv) to increase its soluble expression (mut-HA22), and conjugated it to the surface of sonicated liposomes to generate immunoliposomes (mut-HA22-liposomes). We examined liposome binding and uptake by CD22(+) B-lymphocytes (BJAB) by using calcein and/or rhodamine PE-labeled liposomes. We also tested the effect of targeting on cellular toxicity with doxorubicin-loaded liposomes. We report that: (i) Binding of mut-HA22-liposomes to BJAB cells was significantly greater than liposomes not conjugated with mut-HA22 (control liposomes), and mut-HA22-liposomes bind to and are taken in by BJAB cells in a dose and temperature-dependent manner, respectively; (ii) This binding occurred via the interaction with the cellular CD22 as pre-incubation of the cells with mut-HA22 blocked subsequent liposome binding; (iii) Intracellular localization of mut-HA22-liposomes at 37 degrees C but not at 4 degrees C indicated that our targeted liposomes were taken up through an energy dependent process via receptor-mediated endocytosis; and (iv) Mut-HA22-liposomes loaded with doxorubicin exhibited at least 2-3 fold more accumulation of doxorubicin in BJAB cells as compared to control liposomes. Moreover, these liposomes showed at least a 2-4 fold enhanced killing of BJAB or Raji cells (CD22(+)), but not SUP-T1 cells (CD22(-)). Taken together these data suggest that these 2nd-generation liposomes may serve as promising carriers for targeted drug delivery to treat patients suffering from B-cell lymphoma. Published by Elsevier Inc.

Molecular mechanisms underlying protection against H9N2 influenza virus challenge in mice by recombinant Lactobacillus plantarum with surface displayed HA2-LTB.

PubMed

Jiang, Yanlong; Yang, Guilian; Wang, Qi; Wang, Zhannan; Yang, Wentao; Gu, Wei; Shi, Chunwei; Wang, Jianzhong; Huang, Haibin; Wang, Chunfeng

2017-10-10

It has been considered that the Avian influenza virus (AIV) causes severe threats to poultry industry. In this study, we constructed a series of recombinant Lactobacillus plantarum (L. plantarum) with surface displayed hemagglutinin subunit 2 (HA2) alone or together with heat-labile toxin B subunit (LTB) from enterotoxigenic Escherichia coli. Balb/c mice were used as model to evaluate the protective effects of recombinant L. plantarum strains against H9N2 subtype challenge. The results showed that the presence of LTB significantly increased the percentages of CD3 + CD4 + IL-4 + , CD3 + CD4 + IFN-γ + and CD3 + CD4 + IL-17 + T cells, as well as CD3 + CD8 + IFN-γ + T cells in spleen and MLNs determined by Fluorescence-Activated Cell Sorting assay. Similar increased production of serum IFN-γ was also confirmed by enzyme linked immunosorbent assay (ELISA). The L. plantarum with surface displayed HA2-LTB also dramatically increased the percentages of B220 + IgA + B cells in peyer patch, in consistent with elevated production of mucosal SIgA antibody determined by ELISA. Finally, the orally administrated HA2-LTB expressing strain efficiently protected mice against H9N2 subtype AIV challenge shown by increased survival percentages, body weight gains and decreased lung lesions in histopathologic analysis. In conclusion, this study provides more detail mechanisms underlying the adjuvant effects of LTB on heterologous antigen produced in recombinant lactic acid bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

The effects of nanostructured hydroxyapatite coating on the biodegradation and cytocompatibility of magnesium implants.

PubMed

Iskandar, Maria Emil; Aslani, Arash; Liu, Huinan

2013-08-01

Magnesium (Mg) alloys, a novel class of degradable, metallic biomaterials, have attracted growing interest as a promising alternative for medical implant and device applications due to their advantageous mechanical and biological properties. Although its biodegradability is an attractive property, rapid degradation of Mg in the physiological environments imposes a major obstacle that limits the translation of Mg-based implants to clinical applications. Therefore, the objective of this study was to develop a nanostructured hydroxyapatite (nHA) coating on polished Mg substrates to mediate the rapid degradation of Mg while improving its integration with bone tissue for orthopedic applications. The nHA coatings were deposited on polished Mg using the patented transonic particle acceleration (Spire Biomedical) process. Surface morphology, elemental compositions, and crystal structures were characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction (XRD) analysis, respectively. The degradation of nHA-coated and non-coated Mg samples was investigated by incubating the samples in phosphate buffered saline and revised simulated body fluid, under standard cell culture conditions. Rat bone marrow stromal cells (BMSCs) were harvested and cultured with nHA-coated and non-coated Mg samples to determine cytocompatibility. The degradation results suggested that the nHA coatings decreased Mg degradation. Improved BMSC adhesion was observed on the surfaces of the nHA-coated and non-coated Mg samples, in comparison with the cells on the culture plate surrounding the Mg samples. In conclusion, nHA coatings showed promise for improving the biodegradation and cytocompatibility properties of Mg-based orthopedic implants and should be further studied. Copyright © 2013 Wiley Periodicals, Inc.

Biological response of Sr-containing coating with various surface treatments on titanium substrate for medical applications

NASA Astrophysics Data System (ADS)

Yang, Shih-Ping; Lee, Tzer-Min; Lui, Truan-Sheng

2015-08-01

An implant requires a suitable surface to trigger osteointegration. The surface characteristics and chemical composition are important factors in this process. Plasma spraying and micro-arc oxidation can be used to fabricate rough and porous structures for medical applications. Strontium (Sr) has been shown to prevent osteoporosis in vitro and in vivo. However, few scientists have evaluated the biological response of Sr-containing coatings on different surface treatments. In this study, a sand-blasted (SB) surface (as the control), plasma-sprayed hydroxyapatite (HA) and Sr-substituted HA coatings (HAPS and SrHAPS, respectively), calcium phosphate and Sr-containing calcium phosphate micro-arc oxidation surface (CPM and SrCPM, respectively) were analyzed in terms of human osteoblastic cell (MG63) response. Sr was confirmed to be incorporated into the surface. SrHAPS and SrCPM specimens had higher cell responses than those of the HAPS and CPM groups, respectively. The cells cultured on SrCPM and SrHAPS specimens exhibited high proliferation and differentiation. However, CPM and SrCPM specimens stimulated more ECM-like structures than other specimens. The results show that Sr-containing coatings have good characteristics that enhance cell response. The SrCPM coating is a suitable implant surface treatment for clinical applications.

Laser fabrication of Ag-HA nanocomposites on Ti6Al4V implant for enhancing bioactivity and antibacterial capability.

PubMed

Liu, Xiangmei; Man, H C

2017-01-01

For titanium alloy implants, both surface bioactivity and antibacterial infection are the two critical factors in determining the success of clinical implantation of these metallic implants. In the present work, a novel nanocomposite layer of nano-silver-containing hydroxyapatite (Ag-HA) was prepared on the surface of biomedical Ti6Al4V by laser processing. Analysis using SEM, EDS and XRD shows the formation of an Ag-HA layer of about 200μm fusion bonded to the substrate. Mineralization tests in simulated body fluid (SBF) showed that laser fabricated Ag-HA nanocomposite layer favors the deposition of apatite on the surface of the implants. Antibacterial tests confirmed that all Ag-HA nanocomposite layers can kill bacteria while a higher Ag content would lower the cytocompatibility of these coatings. Cell viability decreases when the Ag content reaches 5% in these coatings, due to the larger amount of Ag leached out, as confirmed by ion release evaluation. Our results reveal that laser fabricated Ag-HA nanocomposite coatings containing 2% Ag show both excellent cytocompatibility and antibacterial capability. Copyright © 2016 Elsevier B.V. All rights reserved.

Differences in antigen presentation to MHC class I-and class II- restricted influenza virus-specific cytolytic T lymphocyte clones

PubMed Central

1986-01-01

We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA- specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II- restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets. PMID:3485173

Porous alumina-hydroxyapatite composites through protein foaming-consolidation method.

PubMed

Sopyan, I; Fadli, A; Mel, M

2012-04-01

This report presents physical characterization and cell culture test of porous alumina-hydroxyapatite (HA) composites fabricated through protein foaming-consolidation technique. Alumina and HA powders were mixed with yolk and starch at an adjusted ratio to make slurry. The resulting slip was poured into cylindrical shaped molds and followed by foaming and consolidation via 180 °C drying for 1 h. The obtained green bodies were burned at 600 °C for 1 h, followed by sintering at temperatures of 1200-1550 °C for 2 h. Porous alumina-HA bodies with 26-77 vol.% shrinkage, 46%-52% porosity and 0.1-6.4 MPa compressive strength were obtained. The compressive strength of bodies increased with the increasing sintering temperatures. The addition of commercial HA in the body was found to increase the compressive strength, whereas the case is reverse for sol-gel derived HA. Biocompatibility study of porous alumina-HA was performed in a stirred tank bioreactor using culture of Vero cells. A good compatibility of the cells to the porous microcarriers was observed as the cells attached and grew at the surface of microcarriers at 8-120 cultured hours. The cell growth on porous alumina microcarrier was 0.015 h(-1) and increased to 0.019 h(-1) for 0.3 w/w HA-to-alumina mass ratio and decreased again to 0.017 h(-1) for 1.0 w/w ratio. Copyright © 2011 Elsevier Ltd. All rights reserved.

Improved biocompatibility of poly (styrene-b-(ethylene-co-butylene)-b-styrene) elastomer by a surface graft polymerization of hyaluronic acid.

PubMed

Li, Xiaomeng; Luan, Shifang; Shi, Hengchong; Yang, Huawei; Song, Lingjie; Jin, Jing; Yin, Jinghua; Stagnaro, Paola

2013-02-01

Hyaluronic acid (HA) is an important component of extracellular matrix (ECM) in many tissues, providing a hemocompatible and supportive environment for cell growth. In this study, glycidyl methacrylate-hyaluronic acid (GMHA) was first synthesized and verified by proton nuclear magnetic resonance ((1)H NMR) spectroscopy. GMHA was then grafted to the surface of biomedical elastomer poly (styrene-b-(ethylene-co-butylene)-b-styrene) (SEBS) via an UV-initiated polymerization, monitored by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). The further improvement of biocompatibility of the GMHA-modified SEBS films was assessed by platelet adhesion experiments and in vitro response of murine osteoblastic cell line MC-3T3-E1 with the virgin SEBS surface as the reference. It showed that the surface modification with HA strongly resisted platelet adhesion whereas improved cell-substrate interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Fluorescence polarization-based assay using N-glycan-conjugated quantum dots for screening in hemagglutinin blockers for influenza A viruses.

PubMed

Okamatsu, Masatoshi; Feng, Fei; Ohyanagi, Tatsuya; Nagahori, Noriko; Someya, Kazuhiko; Sakoda, Yoshihiro; Miura, Nobuaki; Nishimura, Shin-Ichiro; Kida, Hiroshi

2013-02-01

Attachment of influenza virus to susceptible cells is mediated by viral protein hemagglutinin (HA), which recognizes cell surface glycoconjugates that terminate in α-sialosides. To develop anti-influenza drugs based on inhibition of HA-mediated infection, novel fluorescent nanoparticles displaying multiple biantennary N-glycan chains with α-sialosides (A2-PC-QDs) that have high affinity for the HA were designed and constructed. The A2-PC-QDs enabled an easy and efficient fluorescence polarization (FP) assay for detection of interaction with the HA and competitive inhibition even by small molecule compounds against A2-PC-QDs-HA binding. The quantum dot (QD)-based FP assay established in the present study is a useful tool for high-throughput screening and to accelerate the development of novel and more effective blockers of the viral attachment of influenza virus. Copyright © 2012 Elsevier B.V. All rights reserved.

Improving osteointegration and osteogenesis of three-dimensional porous Ti6Al4V scaffolds by polydopamine-assisted biomimetic hydroxyapatite coating.

PubMed

Li, Yong; Yang, Wei; Li, Xiaokang; Zhang, Xing; Wang, Cairu; Meng, Xiangfei; Pei, Yifeng; Fan, Xiangli; Lan, Pingheng; Wang, Chunhui; Li, Xiaojie; Guo, Zheng

2015-03-18

Titanium alloys with various porous structures can be fabricated by advanced additive manufacturing techniques, which are attractive for use as scaffolds for bone defect repair. However, modification of the scaffold surfaces, particularly inner surfaces, is critical to improve the osteointegration of these scaffolds. In this study, a biomimetic approach was employed to construct polydopamine-assisted hydroxyapatite coating (HA/pDA) onto porous Ti6Al4V scaffolds fabricated by the electron beam melting method. The surface modification was characterized with the field emission scanning electron microscopy, energy dispersive spectroscopy, water contact angle measurement, and confocal laser scanning microscopy. Attachment and proliferation of MC3T3-E1 cells on the scaffold surface were significantly enhanced by the HA/pDA coating compared to the unmodified surfaces. Additionally, MC3T3-E1 cells grown on the HA/pDA-coated Ti6Al4V scaffolds displayed significantly higher expression of runt-related transcription factor-2, alkaline phosphatase, osteocalcin, osteopontin, and collagen type-1 compared with bare Ti6Al4V scaffolds after culture for 14 days. Moreover, microcomputed tomography analysis and Van-Gieson staining of histological sections showed that HA/pDA coating on surfaces of porous Ti6Al4V scaffolds enhanced osteointegration and significantly promoted bone regeneration after implantation in rabbit femoral condylar defects for 4 and 12 weeks. Therefore, this study provides an alternative to biofunctionalized porous Ti6Al4V scaffolds with improved osteointegration and osteogenesis functions for orthopedic applications.

Biocompatibility of hydroxyapatite coatings deposited by pulse electrodeposition technique on the Nitinol superelastic alloy.

PubMed

Marashi-Najafi, F; Khalil-Allafi, J; Etminanfar, M R

2017-07-01

The present study deals with pulse electrochemical deposition of HA on NiTi alloy and in vitro evaluation of coatings. At first step, a thermo-chemical surface modification process was applied to control the Ni release of the alloy. The electrochemical deposition of CaP coatings was examined at both dilute and concentrated solutions. The morphology and the composition of coatings were studied using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Plate like and needle like morphologies were formed for dilute and concentrated solution respectively and HA phase was formed by increasing the pulse current density for both electrolyte. The thickness of the samples was measured using cross sectioning technique. Fibroblast cell culture test on the coated samples revealed that the HA coating obtained by dilute solution shows the best biocompatibility. Also, MTT assay showed the highest cell density and cell proliferation after 5days for the HA coating of dilute solution. The contact angle of samples was measured and the coated samples showed a hydrophilic surface. Soaking the sample in SBF revealed that the crystallization rate of calcium-phosphate compounds is higher on the plate like HA coating as compared to the needle like morphology. The P release of the HA coated samples was measured in a physiological saline solution and the results show that the ions releasing in the plate like coating are less than the needle like coating. It seems that the stability of the plate like coating in biological environments is responsible for the better biocompatibility of the coating. Copyright © 2017 Elsevier B.V. All rights reserved.

Transplantation of human placenta-derived mesenchymal stem cells in a silk fibroin/hydroxyapatite scaffold improves bone repair in rabbits.

PubMed

Jin, Jun; Wang, Jun; Huang, Jian; Huang, Fang; Fu, Jianhong; Yang, Xinjing; Miao, Zongning

2014-11-01

The main requirements for successful tissue engineering of the bone are non-immunogenic cells with osteogenic potential and a porous biodegradable scaffold. The purpose of this study is to evaluate the potential of a silk fibroin/hydroxyapatite (SF/HA) porous material as a delivery vehicle for human placenta-derived mesenchymal stem cells (PMSCs) in a rabbit radius defect model. In this study, we randomly assigned 16 healthy adult New Zealand rabbits into two groups, subjected to transplantation with either SF/HA and PMSCs (experimental group) or SF/HA alone (control group). To evaluate fracture healing, we assessed the extent of graft absorption, the quantity of newly formed bone, and re-canalization of the cavitas medullaris using radiographic and histological tools. We performed flow cytometric analysis to characterize PMSCs, and found that while they express CD90, CD105 and CD73, they stain negative for HLA-DR and the hematopoietic cell surface markers CD34 and CD45. When PMSCs were exposed to osteogenic induction medium, they secreted calcium crystals that were identified by von Kossa staining. Furthermore, when seeded on the surface of SF/HA scaffold, they actively secreted extracellular matrix components. Here, we show, through radiographic and histological analyses, that fracture healing in the experimental group is significantly improved over the control group. This strongly suggests that transplantation of human PMSCs grown in an SF/HA scaffold into injured radius segmental bone in rabbits, can markedly enhance tissue repair. Our finding provides evidence supporting the utility of human placenta as a potential source of stem cells for bone tissue engineering. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

CD44-tropic polymeric nanocarrier for breast cancer targeted rapamycin chemotherapy.

PubMed

Zhao, Yunqi; Zhang, Ti; Duan, Shaofeng; Davies, Neal M; Forrest, M Laird

2014-08-01

In contrast with the conventional targeting of nanoparticles to cancer cells with antibody or peptide conjugates, a hyaluronic acid (HA) matrix nanoparticle with intrinsic-CD44-tropism was developed to deliver rapamycin for localized CD44-positive breast cancer treatment. Rapamycin was chemically conjugated to the particle surface via a novel sustained-release linker, 3-amino-4-methoxy-benzoic acid. The release of the drug from the HA nanoparticle was improved by 42-fold compared to HA-temsirolimus in buffered saline. In CD44-positive MDA-MB-468 cells, using HA as drug delivery carrier, the cell viability was significantly decreased compared to free rapamycin and CD44-blocked controls. Rat pharmacokinetics showed that the area under the curve of HA nanoparticle formulation was 2.96-fold greater than that of the free drug, and the concomitant total body clearance was 8.82-fold slower. Moreover, in immunocompetent BALB/c mice bearing CD44-positive 4T1.2neu breast cancer, the rapamycin-loaded HA particles significantly improved animal survival, suppressed tumor growth and reduced the prevalence of lung metastasis. This study demonstrates increased efficiency of rapamycin delivery and consequential treatment effects in a breast cancer model by hyaluronic acid - L-rapamycin conjugates with intrinsic tropism for CD44-positive cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Microstructural characteristics and biocompatibility of a Type-B carbonated hydroxyapatite coating deposited on NiTi shape memory alloy.

PubMed

Chu, Chenglin; Hu, T; Yin, L H; Pu, Y P; Dong, Y S; Lin, P H; Chung, C Y; Yeung, K W K; Chu, P K

2009-01-01

Microstructural characteristics and biocompatibility of a Type-B carbonated hydroxyapatite (HA) coating prepared on NiTi SMA by biomimetic deposition were characterized using XRD, SEM, XPS, FTIR and in vitro studies including hemolysis test, MTT cytotoxicity test and fibroblasts cytocompatibility test. It is found CO(3)(2-) groups were present as substitution of PO(4)(3-) anions in HA crystal lattice due to Type-B carbonate. The growth of Type-B carbonated HA coating in SBF containing HCO(3)(-) ions is stable during all periods of biomimetic deposition. The carbonated HA coating has better blood compatibility than the chemically-polished NiTi SMA. There was a good cell adhesion to this HA coating surface and cell proliferation in the vicinity of the coating was better than that for the chemically-polished NiTi SMA. Thus biomimetic deposition of this carbonated HA coating is a promising way to improve the biocompatibility of NiTi SMA for implant applications.

Osteoconductive hydroxyapatite coated PEEK for spinal fusion surgery

NASA Astrophysics Data System (ADS)

Hahn, Byung-Dong; Park, Dong-Soo; Choi, Jong-Jin; Ryu, Jungho; Yoon, Woon-Ha; Choi, Joon-Hwan; Kim, Jong-Woo; Ahn, Cheol-Woo; Kim, Hyoun-Ee; Yoon, Byung-Ho; Jung, In-Kwon

2013-10-01

Polyetheretherketone (PEEK) has attracted much interest as biomaterial for interbody fusion cages due to its similar stiffness to bone and good radio-transparency for post-op visualization. Hydroxyapatite (HA) coating stimulates bone growth to the medical implant. The objective of this work is to make an implant consisting of biocompatible PEEK with an osteoconductive HA surface for spinal or orthopedic applications. Highly dense and well-adhered HA coating was developed on medical-grade PEEK using aerosol deposition (AD) without thermal degradation of the PEEK. The HA coating had a dense microstructure with no cracks or pores, and showed good adhesion to PEEK at adhesion strengths above 14.3 MPa. The crystallinity of the HA coating was remarkably enhanced by hydrothermal annealing as post-deposition heat-treatment. In addition, in vitro and in vivo biocompatibility of PEEK, in terms of cell adhesion morphology, cell proliferation, differentiation, and bone-to-implant contact ratio, were remarkably enhanced by the HA coating through AD.

The synthesis, characterization and in vivo study of mineral substituted hydroxyapatite for prospective bone tissue rejuvenation applications.

PubMed

Govindaraj, Dharman; Rajan, Mariappan; Munusamy, Murugan A; Alarfaj, Abdullah A; Sadasivuni, Kishor Kumar; Kumar, S Suresh

2017-11-01

Minerals substituted apatite (M-HA) nanoparticles were prepared by the precipitation of minerals and phosphate reactants in choline chloride-Thiourea (ChCl-TU) deep eutectic solvent (DESs) as a facile and green way approach. After preparation of nanoparticles (F-M-HA (F=Fresh solvent)), the DESs was recovered productively and reprocess for the preparation of R-M-HA nanoparticles (R=Recycle solvent).The functional groups, phase, surface texture and the elemental composition of the M-HA nanoparticles were evaluated by advance characterization methods. The physicochemical results of the current work authoritative the successful uses of the novel (ChCl-TU) DESs as eco-friendly recuperate and give the medium for the preparation of M-HA nanoparticles. Moreover, the as-synthesized both M-HA nanoparticles exhibit excellent biocompatibility, consisting of cell co-cultivation and cell adhesion, in vivo according to surgical implantation of Wistar rats. Copyright © 2017 Elsevier Inc. All rights reserved.

C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein

PubMed Central

Popa, Andreea; Pager, Cara Teresia; Dutch, Rebecca Ellis

2011-01-01

The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the viral-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of HA tag addition varied with other fusion proteins, as parainfluenza virus 5 F-HA showed decreased surface expression and no stimulation in fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in modulation of the membrane fusion reaction promoted by these viral glycoproteins. PMID:21175223

Effect of Induced Periimplantitis on Dental Implants With and Without Ultrathin Hydroxyapatite Coating.

PubMed

Madi, Marwa; Zakaria, Osama; Ichinose, Shizuko; Kasugai, Shohei

2016-02-01

The aim of this study was to compare the effect of ligature-induced periimplantitis on dental implants with and without hydroxyapatite (HA) coat. Thirty-two dental implants (3.3 mm wide, 13 mm long) with 4 surface treatments (8 implant/group) (M: machined, SA: sandblasted acid etched, S: sputter HA coat and P: plasma-sprayed HA coat) were inserted into canine mandibles. After 12 weeks, oral hygiene procedures were stopped and silk ligatures were placed around the implant abutments to allow plaque accumulation for the following 16 weeks. Implants with the surrounding tissues were retrieved and prepared for histological examination. Bone-to-implant contact (BIC) and implant surfaces were examined using scanning electron microscopy and energy dispersive x-ray spectroscopy. Histological observation revealed marginal bone loss and large inflammatory cell infiltrates in the periimplant soft tissue. Sputter HA implants showed the largest BIC (98.1%) and machined implant showed the smallest values (70.4%). After 28 weeks, thin sputter HA coat was almost completely dissolved, whereas plasma-sprayed HA coat showed complete thickness preservation. Thin sputter HA-coated implants showed more bone implant contact and less marginal bone loss than thick HA-coated implants under periimplantitis condition.

Mag-seeding of rat bone marrow stromal cells into porous hydroxyapatite scaffolds for bone tissue engineering.

PubMed

Shimizu, Kazunori; Ito, Akira; Honda, Hiroyuki

2007-09-01

Bone tissue engineering has been investigated as an alternative strategy for autograft transplantation. In the process of tissue engineering, cell seeding into three-dimensional (3-D) scaffolds is the first step for constructing 3-D tissues. We have proposed a methodology of cell seeding into 3-D porous scaffolds using magnetic force and magnetite nanoparticles, which we term Mag-seeding. In this study, we applied this Mag-seeding technique to bone tissue engineering using bone marrow stromal cells (BMSCs) and 3-D hydroxyapatite (HA) scaffolds. BMSCs were magnetically labeled with our original magnetite cationic liposomes (MCLs) having a positive surface charge to improve adsorption to cell surface. Magnetically labeled BMSCs were seeded onto a scaffold, and a 1-T magnet was placed under the scaffold. By using Mag-seeding, the cells were successfully seeded into the internal space of scaffolds with a high cell density. The cell seeding efficiency into HA scaffolds by Mag-seeding was approximately threefold larger than that by static-seeding (conventional method, without a magnet). After a 14-d cultivation period using the osteogenic induction medium by Mag-seeding, the level of two representative osteogenic markers (alkaline phosphatase and osteocalcin) were significantly higher than those by static-seeding. These results indicated that Mag-seeding of BMSCs into HA scaffolds is an effective approach to bone tissue engineering.

Mechanisms involved in enhancement of the expression and function of aggrecanases by hyaluronan oligosaccharides

PubMed Central

Ariyoshi, Wataru; Takahashi, Nobunori; Hida, Daisuke; Knudson, Cheryl B.; Knudson, Warren

2011-01-01

Objective Small hyaluronan (HA) oligosaccharides serve as competitive receptor antagonists to displace HA from the cell surface and induce cell signaling events. In articular chondrocytes this cell signaling is mediated by the HA receptor CD44 and induces stimulation of genes involved in matrix degradation such as matrix metalloproteinases as well as matrix repair genes including collagen type II, aggrecan and HA synthase-2. The objective of this study was to determine changes in the expression and function of aggrecanases after disruption of chondrocyte CD44-HA interactions. Methods Bovine articular chondrocytes or bovine cartilage tissue were pre-treated with a variety of inhibitors of major signaling pathways prior to the addition of HA oligosaccharides. Changes in aggrecanase were monitored by real time reverse transcriptase-polymerase chain reaction and western blot analysis of ADAMTS4, ADAMTS5 and aggrecan proteolytic fragments. To test the interactions between ADAMTS4 and MT4-MMP, protein lysates purified from stimulated chondrocytes were subjected to co-immunoprecipitation. Results Disruption of chondrocyte CD44-HA interactions with HA oligosaccharides induced the transcription of ADAMTS4 and ADAMTS5 in time- and dose-dependent manner. The association of GPI-anchored MT4-MMP with ADAMTS4 was also induced in articular chondrocytes by HA oligosaccharides. Inhibition of the NF-κB pathway blocked HA oligosaccharides-mediated stimulation of aggrecanases. Conclusions Disruptive changes in chondrocyte-matrix interactions by HA oligosaccharides induce matrix degradation and elevate aggrecanases via the activation of the NF-κB signaling pathway. PMID:21905012

The ultrastructural surface morphology of oral cancer cells and keratinocytes after exposure to chitosan

NASA Astrophysics Data System (ADS)

Fatimah; Sarsito, A. S.; Wimardhani, Y. S.

2017-08-01

Low-molecular-weight chitosan (LMWC) has the same selective cytotoxic effects on oral cancer cells as cisplatin. The cell deaths caused by the anticancer characteristics of chitosan show that apoptosis is not the death pathway of the primary cells involved. The interactions between LMWC and the cells need to be explored. The objective of this study was to compare the ultrastructural morphology of oral Squamous Cell Carcinoma (SCC Ca)-922 and noncancer keratinocyte HaCaT cell lines after exposure to LMWC and cisplatin. The cells were treated with LMWC and cisplatin, and their ultrastructural morphology was analyzed using scanning electron micrographs. Features of early apoptosis, seen as the loss of microvilli, were detected in the LMWC-exposed Ca9-22 cells, and there was a material surrounding the cells. In contrast, the LMWC-exposed HaCaT cells showed no changes related to apoptosis. The results were the opposite when cisplatin was used. This study confirms that there are differences in the ultrastructural surface morphology of LMWC-exposed and cisplatin-exposed oral cancer cells and keratinocytes that could be correlated with their biological activity.

Allogeneic Mesenchymal Stem Cells in Combination with Hyaluronic Acid for the Treatment of Osteoarthritis in Rabbits.

PubMed

Chiang, En-Rung; Ma, Hsiao-Li; Wang, Jung-Pan; Liu, Chien-Lin; Chen, Tain-Hsiung; Hung, Shih-Chieh

2016-01-01

Mesenchymal stem cell (MSC)-based therapies may aid in the repair of articular cartilage defects. The purpose of this study was to investigate the effects of intraarticular injection of allogeneic MSCs in an in vivo anterior cruciate ligament transection (ACLT) model of osteoarthritis in rabbits. Allogeneic bone marrow-derived MSCs were isolated and cultured under hypoxia (1% O2). After 8 weeks following ACLT, MSCs suspended in hyaluronic acid (HA) were injected into the knees, and the contralateral knees were injected with HA alone. Additional controls consisted of a sham operation group as well as an untreated osteoarthritis group. The tissues were analyzed by macroscopic examination as well as histologic and immunohistochemical methods at 6 and 12 weeks post-transplantation. At 6 and 12 weeks, the joint surface showed less cartilage loss and surface abrasion after MSC injection as compared to the tissues receiving HA injection alone. Significantly better histological scores and cartilage content were observed with the MSC transplantation. Furthermore, engraftment of allogenic MSCs were evident in surface cartilage. Thus, injection of the allogeneic MSCs reduced the progression of osteoarthritis in vivo.

Effects of anodic oxidation and hydrothermal treatment on surface characteristics and biocompatibility of Ti-30Nb-1Fe-1Hf alloy

NASA Astrophysics Data System (ADS)

Ou, Shih-Fu; Chou, Hsin-Hua; Lin, Chao-Sung; Shih, Ching-Jui; Wang, Kuang-Kuo; Pan, Yung-Ning

2012-06-01

Anodic oxidation followed by hydrothermal treatment has been widely applied for surface modification of titanium alloys to precipitate a crystalline hydroxyapatite (HA) layer in order to achieve improved osteoconduction. A majority of the studies in the literature imposed relatively high powers to enhance Ca and P in the anodic oxide film (AOF). However, high powers have been found to cause deterioration of the adhesive strength in one of the author's previous study. In this study, a new electrolyte comprising calcium acetate monohydrate (CA), β-glycerophosphate disodium pentahydrate (β-GP) and HA powder was developed, and the Ti-30Nb-1Fe-1Hf alloy was anodized in this HA-containing electrolyte to a relatively low voltage. Results show that the AOF anodized in the HA-containing electrolyte exhibits a better HA forming ability during hydrothermal treatment, attributing to the presence of HA powder in the electrolyte that effectively enhances both the Ca content and Ca/P ratio in the AOF. On the other hand, the adhesive strength was little affected due to the decrease in size of the craters residing in the AOF. With respect to the biological responses, not much difference in biocompatibility of the treated and untreated Ti-Nb surfaces was obtained. However, the anodized and hydrothermally treated surface promotes the attachment of cells.

Polyurethane/nano-hydroxyapatite composite films as osteogenic platforms.

PubMed

Jackson, Bailey K; Bow, Austin J; Kannarpady, Ganesh; Biris, Alexandru S; Anderson, David E; Dhar, Madhu; Bourdo, Shawn E

2018-08-01

A wide variety of biomaterials are utilized in tissue engineering to promote cell proliferations in vitro or tissue growth in vivo. The combination of cells, extracellular matrices, and biocompatible materials may make it possible to grow functional living tissues ranging from bone to nerve cells. In bone regeneration, polymeric scaffolds can be enhanced by the addition of bioactive materials. To this end, this study designed several ratios of polyurethane (PU) and nano-hydroxyapatite (nHA) composites (PU-nHA ratios: 100/0, 90/10, 80/20, 70/30, 60/40 w/w). The physical and mechanical properties of these composites and their relative cellular compatibility in vitro were determined. The chemical composition and crystallinity of the composites were confirmed using X-ray diffraction, X-ray photoelectron spectroscopy, and thermogravimetric analyses. Atomic force microscopy, nano-indentation, and contact angle measurements were used to evaluate surface properties. The results showed a significant increase in surface roughness and a decrease in contact angle when the nHA concentration increased above 20%, resulting in a significant increase in hydrophilicity. These surface property changes influenced cellular behavior when MC 3T3-E1 cells were seeded on the composites. All composites were cytocompatible. There was a linear increase in cell proliferation on the 80/20 and 70/30 composites only, whereas subjective evaluation demonstrated noticeable clusters or nodules of cells (considered hallmarks of osteogenic differentiation) in the absence of any osteogenic inducers only on the 90/10 and 80/20 composites. Cellular data suggests that the 80/20 composite was an optimal environment for cell adhesion, proliferation, and, potentially, osteogenic differentiation in vitro.

The influence of sintering temperature on the proliferation of fibroblastic cells in contact with HA-bioceramics.

PubMed

Frayssinet, P; Rouquet, N; Fages, J; Durand, M; Vidalain, P O; Bonel, G

1997-06-05

HA-ceramics used in human surgery as osteoconductive surfaces show a great variety of characteristics. Certain characteristics such as grain size, porosity, and surface area, are controlled by the sintering temperature of the slurry. We grew L-929 fibroblast cells on HA-ceramic disks that had been sintered at different temperatures ranging from 850 degrees-1350 degrees C. The cell line growth rate was lower on ceramic disks than on the culture-grade polystyrene used as a negative control. Cell growth correlated with the ceramic sintering temperature although no significant difference in the cell adhesion to the different ceramics was shown. Growth rate on ceramics sintered at low temperatures (850 degrees and 950 degrees C) was negative whereas it was positive on disks sintered at higher temperatures. When the cells were separated from the disks by a polycarbonate membrane, the growth rate was negative on those membranes in contact with low-temperature sintered disks and positive on the high-temperature sintered disks. The calcium and phosphorus concentration in the culture medium in contact with ceramics sintered below 1050 degrees C decreased during the culture period. Ceramics sintered between 1100 degrees and 1250 degrees C brought about an increase in Ca and P concentrations while ceramics sintered at higher temperatures did not induce any changes. SEM examination of the 850 degrees and 1200 degrees C sintered ceramics showed that the 850 degrees C sintered ceramics consisted of small grains with pores between them and the 1200 degrees C sintered ceramics were made of larger grains without any visible pores, thereby decreasing the surface of material in contact with the culture medium. This difference in surface area was confirmed by the fact that the amount of albumin absorbed onto the ceramic was dependent on the sintering temperature. In conclusion, the modification of the culture medium brought about by high-surfaced ceramics could influence the growth of cells with which such ceramics come in contact.

Transparent, biocompatible nanostructured surfaces for cancer cell capture and culture

PubMed Central

Cheng, Boran; He, Zhaobo; Zhao, Libo; Fang, Yuan; Chen, Yuanyuan; He, Rongxiang; Chen, Fangfang; Song, Haibin; Deng, Yuliang; Zhao, Xingzhong; Xiong, Bin

2014-01-01

Circulating tumor cells (CTCs) in the blood which have detached from both the primary tumor and any metastases may be considered as a “liquid biopsy” and are expected to replace tumor biopsies in the monitoring of treatment response and determining patient prognosis. Here, we introduce a facile and efficient CTC detection material made of hydroxyapatite/chitosan (HA/CTS), which is beneficial because of its transparency and excellent biological compatibility. Atomic force microscopy images show that the roughness of the HA/CTS nanofilm (HA/CTSNF) substrates can be controlled by changing the HA:CTS ratio. Enhanced local topographic interactions between nano-components on cancer cell membranes, and the antibody coated nanostructured substrate lead to improved CTC capture and separation. This remarkable nanostructured substrate has the potential for CTC culture in situ and merits further analysis. CTCs captured from artificial blood samples were observed in culture on HA/CTSNF substrates over a period of 14 days by using conventional staining methods (hematoxylin eosin and Wright’s stain). We conclude that these substrates are multifunctional materials capable of isolating and culturing CTCs for subsequent studies. PMID:24904216

Enhancement of stem cell differentiation to osteogenic lineage on hydroxyapatite-coated hybrid PLGA/gelatin nanofiber scaffolds.

PubMed

Sanaei-Rad, Parisa; Jafarzadeh Kashi, Tahereh-Sadat; Seyedjafari, Ehsan; Soleimani, Masoud

2016-11-01

A combination of polymeric materials and bioceramics has recently received a great deal of attention for bone tissue engineering applications. In the present study, hybrid nanofibrous scaffolds were fabricated from PLGA and gelatin via electrospinning and then were coated with hydroxyapatite (HA). They were then characterized and used in stem cell culture studies for the evaluation of their biological behavior and osteogenic differentiation in vitro. This study showed that all PLGA, hybrid PLGA/gelatin and HA-PLGA/gelatin scaffolds were composed of ultrafine fibers with smooth morphology and interconnected pores. The MTT assay confirmed that the scaffolds can support the attachment and proliferation of stem cells. During osteogenic differentiation, bone-related gene expression, ALP activity and biomineralization on HA-PLGA/gelatin scaffolds were higher than those observed on other scaffolds and TCPS. PLGA/gelatin electrospun scaffolds also showed higher values of these markers than TCPS. Taking together, it was shown that nanofibrous structure enhanced osteogenic differentiation of adipose-tissue derived stem cells. Furthermore, surface-coated HA stimulated the effect of nanofibers on the commitment of stem cells toward osteolineage. In conclusion, HA-PLGA/gelatin electrospun scaffolds were demonstrated to have significant potential for bone tissue engineering applications. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Comparison of new bone formation, implant integration, and biocompatibility between RGD-hydroxyapatite and pure hydroxyapatite coating for cementless joint prostheses--an experimental study in rabbits.

PubMed

Bitschnau, Achim; Alt, Volker; Böhner, Felicitas; Heerich, Katharina Elisabeth; Margesin, Erika; Hartmann, Sonja; Sewing, Andreas; Meyer, Christof; Wenisch, Sabine; Schnettler, Reinhard

2009-01-01

This is the first work to report on additional Arginin-Glycin-Aspartat (RGD) coating on precoated hydroxyapatite (HA) surfaces regarding new bone formation, implant bone contact, and biocompatibility compared to pure HA coating and uncoated stainless K-wires. There were 39 rabbits in total with 6 animals for the RGD-HA and HA group for the 4 week time period and 9 animals for each of the 3 implant groups for the 12 week observation. A 2.0 K-wire either with RGD-HA or with pure HA coating or uncoated was placed into the intramedullary canal of the tibia. After 4 and 12 weeks, the tibiae were harvested and three different areas of the tibia were assessed for quantitative and qualitative histology for new bone formation, direct implant bone contact, and formation of multinucleated giant cells. Both RGD-HA and pure HA coating showed statistically higher new bone formation and implant bone contact after 12 weeks than the uncoated K-wire. There were no significant differences between the RGD-HA and the pure HA coating in new bone formation and direct implant bone contact after 4 and 12 weeks. The number of multinucleated giant did not differ significantly between the RGD-HA and HA group after both time points. Overall, no significant effects of an additional RGD coating on HA surfaces were detected in this model after 12 weeks. (c) 2008 Wiley Periodicals, Inc.

Physico-Mechanical Properties of HA/TCP Pellets and Their Three-Dimensional Biological Evaluation In Vitro.

PubMed

Kamalaldin, Nurulain 'Atikah; Jaafar, Mariatti; Zubairi, Saiful Irwan; Yahaya, Badrul Hisham

2018-01-04

The use of bioceramics, especially the combination of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), as a three-dimensional scaffold in bone engineering is essential because together these elements constitute 60% of the bone content. Different ratios of HA and β-TCP were previously tested for their ability to produce suitable bioceramic scaffolds, which must be able to withstand high mechanical load. In this study, two ratios of HA/TCP (20:80 and 70:30) were used to create pellets, which then were evaluated in vitro to identify any adverse effects of using the material in bone grafting. Diametral tensile strength (DTS) and density testing was conducted to assess the mechanical strength and porosity of the pellets. The pellets then were tested for their toxicity to normal human fibroblast cells. In the toxicity assay, cells were incubated with the pellets for 3 days. At the end of the experiment, cell morphological changes were assessed, and the absorbance was read using PrestoBlue Cell Viability Reagent™. An inversely proportional relationship between DTS and porosity percentage was detected. Fibroblasts showed normal cell morphology in both treatments, which suggests that the HA/TCP pellets were not toxic. In the osteoblast cell attachment assay, cells were able to attach to the surface of both ratios, but cells were also able to penetrate inside the scaffold of the 70:30 pellets. This finding suggests that the 70:30 ratio had better osteoconduction properties than the 20:80 ratio.

Internalized compartments encapsulated nanogels for targeted drug delivery

NASA Astrophysics Data System (ADS)

Yu, Jicheng; Zhang, Yuqi; Sun, Wujin; Wang, Chao; Ranson, Davis; Ye, Yanqi; Weng, Yuyan; Gu, Zhen

2016-04-01

Drug delivery systems inspired by natural particulates hold great promise for targeted cancer therapy. An endosome formed by internalization of plasma membrane has a massive amount of membrane proteins and receptors on the surface, which is able to specifically target the homotypic cells. Herein, we describe a simple method to fabricate an internalized compartments encapsulated nanogel with endosome membrane components (EM-NG) from source cancer cells. Following intracellular uptake of methacrylated hyaluronic acid (m-HA) adsorbed SiO2/Fe3O4 nanoparticles encapsulating a crosslinker and a photoinitiator, EM-NG was readily prepared through in situ crosslinking initiated under UV irradiation after internalization. The resulting nanogels loaded with doxorubicin (DOX) displayed enhanced internalization efficiency to the source cells through a specific homotypic affinity in vitro. However, when treated with the non-source cells, the EM-NGs exhibited insignificant difference in therapeutic efficiency compared to a bare HA nanogel with DOX. This study illustrates the potential of utilizing an internalized compartments encapsulated formulation for targeted cancer therapy, and offers guidelines for developing a natural particulate-inspired drug delivery system.Drug delivery systems inspired by natural particulates hold great promise for targeted cancer therapy. An endosome formed by internalization of plasma membrane has a massive amount of membrane proteins and receptors on the surface, which is able to specifically target the homotypic cells. Herein, we describe a simple method to fabricate an internalized compartments encapsulated nanogel with endosome membrane components (EM-NG) from source cancer cells. Following intracellular uptake of methacrylated hyaluronic acid (m-HA) adsorbed SiO2/Fe3O4 nanoparticles encapsulating a crosslinker and a photoinitiator, EM-NG was readily prepared through in situ crosslinking initiated under UV irradiation after internalization. The resulting nanogels loaded with doxorubicin (DOX) displayed enhanced internalization efficiency to the source cells through a specific homotypic affinity in vitro. However, when treated with the non-source cells, the EM-NGs exhibited insignificant difference in therapeutic efficiency compared to a bare HA nanogel with DOX. This study illustrates the potential of utilizing an internalized compartments encapsulated formulation for targeted cancer therapy, and offers guidelines for developing a natural particulate-inspired drug delivery system. Electronic supplementary information (ESI) available: Synthesis of m-HA; synthesis of rhodamine-HA derivative; supplementary data on relative fluorescence intensity of DOX-EN-NGs on HeLa cells. See DOI: 10.1039/c5nr08895j

Hyaluronic acid oligosaccharide modified redox-responsive mesoporous silica nanoparticles for targeted drug delivery.

PubMed

Zhao, Qinfu; Geng, Hongjian; Wang, Ying; Gao, Yikun; Huang, Jiahao; Wang, Yan; Zhang, Jinghai; Wang, Siling

2014-11-26

A redox-responsive delivery system based on colloidal mesoporous silica (CMS) has been developed, in which 6-mercaptopurine (6-MP) was conjugated to vehicles by cleavable disulfide bonds. The oligosaccharide of hyaluronic acid (oHA) was modified on the surface of CMS by disulfide bonds as a targeting ligand and was able to increase the stability and biocompatibility of CMS under physiological conditions. In vitro release studies indicated that the cumulative release of 6-MP was less than 3% in the absence of glutathione (GSH), and reached nearly 80% within 2 h in the presence of 3 mM GSH. Confocal microscopy and fluorescence-activated cell sorter (FACS) methods were used to evaluate the cellular uptake performance of fluorescein isothiocyanate (FITC) labeled CMS, with and without oHA modification. The CMS-SS-oHA exhibited a higher cellular uptake performance via CD44 receptor-mediated endocytosis in HCT-116 (CD44 receptor-positive) cells than in NIH-3T3 (CD44 receptor-negative) cells. 6-MP loaded CMS-SS-oHA exhibited greater cytotoxicity against HCT-116 cells than NIH-3T3 cells due to the enhanced cell uptake behavior of CMS-SS-oHA. This study provides a novel strategy to covalently link bioactive drug and targeting ligand to the interiors and exteriors of mesoporous silica to construct a stimulus-responsive targeted drug delivery system.

Targeting CD44 with nanoparticles in head and neck squamous cell carcinoma: A novel therapeutic strategy against cancer stem cells

NASA Astrophysics Data System (ADS)

Thapa, Ranjeeta

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide and is associated with significant morbidity and mortality. Advances in multi-modality treatments have only minimally improved survival rates in the past several years. Recent attention has been focused on the hypothesis that cancer stem cells (CSCs) may be responsible for the failure of current treatments. In HNSCC, a CSC population is contained within the cell fraction that expresses high levels of CD44. CD44 is a cell surface glycoprotein and was the first CSC marker to be described in solid malignancies. in this study, hyaluronan conjugated, dextran-coated super paramagnetic iron-oxide nanoparticles (HA-DESPIONs) were used to target the CD44 population in CD44-overexpressed HNSCC cell lines for treatment by establishing the interaction of HA-DESPIONs with radiation and hyperthermia therapy. The first part of this dissertation studied the cytotoxic, radiosensitizing, and hyperthermic properties of the HA-DESPIONs using cell proliferation and clonogenic survival assays. Cells were grown, plated, treated with HA-DESPIONs, irradiated/exposed to local hyperthermia, and then analyzed for apoptosis. HA-DESPIONs proved to be relatively non-toxic and nonradiosensitizing. However, temperature-dependent cell survival reduction upon incubation with HA-DESPIONs was observed with evidence of apoptotic cell death. These results supported further development of an alternating magnetic field (AMF) approach to activate the HADESPIONs attached to CSCs. In the second part of the dissertation, an AMF generator was constructed and its heat generating effect was tested via kinetic and dose-dependent bulk heating experiments by exposing magnetic nanoparticles to AMF. For elimination of the CD44 population, cells were treated with HA-DESPIONs/DESPIONs, exposed to AMF, and processed for flow cytometrybased apoptosis analysis. Magnetic nanoparticles caused concentration-dependent bulk heating in response to AMF resulting in a significant temperature rise. Following exposure to AMF, DESPIONs were unable to induce targeted hyperthermia and hence had no effect on CD44 cell death in HNSCC cells. However, there was significant cell death in the CD44 population treated with HA-DESPIONs and exposed to AMF. This effect was observed only when the AMF was turned on. These results demonstrated that HA-DESPIONs caused targeted cell-death in CD44overexpressing cells. This may be a promising strategy to specifically target CSCs for the treatment of HNSCC.

Preparation and Characterization of Hyaluronic Acid-Polycaprolactone Copolymer Micelles for the Drug Delivery of Radioactive Iodine-131 Labeled Lipiodol.

PubMed

Chen, Shih-Cheng; Yang, Ming-Hui; Chung, Tze-Wen; Jhuang, Ting-Syuan; Yang, Jean-Dean; Chen, Ko-Chin; Chen, Wan-Jou; Huang, Ying-Fong; Jong, Shiang-Bin; Tsai, Wan-Chi; Lin, Po-Chiao; Tyan, Yu-Chang

2017-01-01

Micelles, with the structure of amphiphilic molecules including a hydrophilic head and a hydrophobic tail, are recently developed as nanocarriers for the delivery of drugs with poor solubility. In addition, micelles have shown many advantages, such as enhanced permeation and retention (EPR) effects, prolonged circulation times, and increased endocytosis through surface modification. In this study, we measured the critical micelle concentrations, diameters, stability, and cytotoxicity and the cell uptake of micelles against hepatic cells with two kinds of hydrophilic materials: PEG-PCL and HA-g-PCL. We used 131 I as a radioactive tracer to evaluate the stability, drug delivery, and cell uptake activity of the micelles. The results showed that HA-g-PCL micelles exhibited higher drug encapsulation efficiency and stability in aqueous solutions. In addition, the 131 I-lipiodol loaded HA-g-PCL micelles had better affinity and higher cytotoxicity compared to HepG2 cells.

Effects of surface active agents on DNAPL migration and distribution in saturated porous media.

PubMed

Cheng, Zhou; Gao, Bin; Xu, Hongxia; Sun, Yuanyuan; Shi, Xiaoqing; Wu, Jichun

2016-11-15

Dissolved surface active agents such as surfactant and natural organic matter can affect the distribution and fate of dense nonaqueous liquids (DNAPLs) in soil and groundwater systems. This work investigated how two common groundwater surface active agents, humic acid (HA) and Tween 80, affected tetrachloroethylene (PCE) migration and source zone architecture in saturated porous media under environmentally relevant conditions. Batch experiments were first conducted to measure the contact angles and interfacial tensions (IFT) between PCE and quartz surface in water containing different amount of surface active agents. Results showed that the contact angle increased and IFT decreased with concentration of surface active agent increasing, and Tween 80 was much more effective than HA. Five 2-D flow cell experiments were then conducted. Correspondingly, Tween 80 showed strong effects on the migration and distribution of PCE in the porous media due to its ability to change the medium wettability from water-wet into intermediate/NAPL-wet. The downward migration velocities of the PCE in three Tween 80 cells were slower than those in the other two cells. In addition, the final saturation of the PCE in the cells containing surface active agents was higher than that in the water-only cell. Results from this work indicate that the presence of surface active agents in groundwater may strongly affect the fate and distribution of DNAPL through altering porous medium wettability. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of nano-hydroxyapatite coating on the osteoinductivity of porous biphasic calcium phosphate ceramics.

PubMed

Hu, Jianzhong; Zhou, Yongchun; Huang, Lihua; Liu, Jun; Lu, Hongbin

2014-04-01

Porous biphasic calcium phosphate (BCP) ceramics exhibit good biocompatibility and bone conduction but are not inherently osteoinductive. To overcome this disadvantage, we coated conventional porous BCP ceramics with nano-hydroxyapatite (nHA). nHA was chosen as a coating material due to its high osteoinductive potential. We used a hydrothermal deposition method to coat conventional porous BCP ceramics with nHA and assessed the effects of the coating on the physical and mechanical properties of the underlying BCP. Next, its effects on mesenchymal stem cell (MSC) attachment, proliferation, viability, and osteogenic differentiation were investigated. nHA formed a deposited layer on the BCP surface, and synthesized nHA had a rod-like shape with lengths ranging from ~50-200 nm and diameters from ~15-30 mm. The nHA coating did not significantly affect the density, porosity, flexural strength, or compressive strength of the underlying BCP (P > 0.1). Scanning electron microscopy showed MSC attachment to the scaffolds, with a healthy morphology and anchorage to nHA crystals via cytoplasmic processes. The densities of MSCs attached on BCP and nHA-coated BCP scaffolds were 62 ± 26 cells/mm2 and 63 ± 27 cells/mm2 (P > 0.1), respectively, after 1 day and 415 ± 62 cells/mm2 and 541 ± 35 cells/mm2 (P < 0.05) respectively, after 14 days. According to an MTT assay, MSC viability was higher on nHA-coated BCP scaffolds than on BCP scaffolds (P < 0.05). In addition, MSCs on nHA-coated BCP scaffolds produced more alkaline phosphatase, collagen type I, and osteocalcin than MSCs on BCP scaffolds (P < 0.05). Our results demonstrate that BCP scaffolds coated with nHA were more conducive for MSC adhesion, proliferation, and osteogenic differentiation than conventional, uncoated BCP scaffolds, indicating that nHA coating can enhance the osteoinductive potential of BCP ceramics, making this material more suitable for applications in bone tissue engineering.

Effect of nano-hydroxyapatite coating on the osteoinductivity of porous biphasic calcium phosphate ceramics

PubMed Central

2014-01-01

Background Porous biphasic calcium phosphate (BCP) ceramics exhibit good biocompatibility and bone conduction but are not inherently osteoinductive. To overcome this disadvantage, we coated conventional porous BCP ceramics with nano-hydroxyapatite (nHA). nHA was chosen as a coating material due to its high osteoinductive potential. Methods We used a hydrothermal deposition method to coat conventional porous BCP ceramics with nHA and assessed the effects of the coating on the physical and mechanical properties of the underlying BCP. Next, its effects on mesenchymal stem cell (MSC) attachment, proliferation, viability, and osteogenic differentiation were investigated. Results nHA formed a deposited layer on the BCP surface, and synthesized nHA had a rod-like shape with lengths ranging from ~50–200 nm and diameters from ~15–30 mm. The nHA coating did not significantly affect the density, porosity, flexural strength, or compressive strength of the underlying BCP (P > 0.1). Scanning electron microscopy showed MSC attachment to the scaffolds, with a healthy morphology and anchorage to nHA crystals via cytoplasmic processes. The densities of MSCs attached on BCP and nHA-coated BCP scaffolds were 62 ± 26 cells/mm2 and 63 ± 27 cells/mm2 (P > 0.1), respectively, after 1 day and 415 ± 62 cells/mm2 and 541 ± 35 cells/mm2 (P < 0.05) respectively, after 14 days. According to an MTT assay, MSC viability was higher on nHA-coated BCP scaffolds than on BCP scaffolds (P < 0.05). In addition, MSCs on nHA-coated BCP scaffolds produced more alkaline phosphatase, collagen type I, and osteocalcin than MSCs on BCP scaffolds (P < 0.05). Conclusions Our results demonstrate that BCP scaffolds coated with nHA were more conducive for MSC adhesion, proliferation, and osteogenic differentiation than conventional, uncoated BCP scaffolds, indicating that nHA coating can enhance the osteoinductive potential of BCP ceramics, making this material more suitable for applications in bone tissue engineering. PMID:24690170

Development of osteoblast colonies on new bioactive coatings

NASA Astrophysics Data System (ADS)

Legoux, J. G.; Chellat, F.; Lima, R. S.; Marple, B. R.; Bureau, M. N.; Shen, H.; Candeliere, G. A.

2006-12-01

The aging baby boomer population coupled with an increase in life expectancy is leading to a rising number of active elderly persons in occidental countries. As a result, the orthopedic implant industry is facing numerous challenges such as the need to extend implant life, reduce the incidence of revision surgery, and improve implant performance. This paper reports results of an investigation on the bioperformance of newly developed coating-substrate systems. Hydroxyapatite (HA) and nano-titania (nano-TiO2) coatings were produced on Ti-6Al-4V and fiber reinforced polymer composite substrates. In vitro studies were conducted to determine the capacity of bioactive coatings developed to sustain osteoblast cells (fetal rat calvaria) adherence, growth, and differentiation. As revealed by scanning electron microscopy (SEM) observations and alkaline phosphatase activity, cell adhesion and proliferation demonstrated that HA coatings over a polymer composite are at least as good as HA coatings made over Ti-6Al-4V substrate in terms of osteoblast cell activity. Nano-TiO2 coatings produced by high-velocity oxyfuel (HVOF) spraying led to different results. For short-term cell culture (4.5 and 24 h), the osteoblasts appeared more flattened when grown on nano-TiO2 than on HA. The surface cell coverage after seven days of incubation was also more complete on nano-TiO2 than HA. Preliminary results indicate that osteoblast activity after 15 days of incubation on nano-TiO2 is equivalent to or greater than that observed on HA.

The influence of hydroxyapatite particles on in vitro degradation behavior of poly epsilon-caprolactone-based composite scaffolds.

PubMed

Guarino, Vincenzo; Taddei, Paola; Di Foggia, Michele; Fagnano, Concezio; Ciapetti, Gabriela; Ambrosio, Luigi

2009-11-01

The design of composite scaffolds with slow degradation kinetics imposes the assessment of the time-course of degradation to predict the long-term in vitro behavior. In this work, the effect of hydroxyapatite (HA) particles on the hydrolytic degradation of poly epsilon-caprolactone composite scaffold was investigated. The study of accelerated degradation mechanisms in alkaline medium enabled analysing comparable degradation profiles at different times. The accurate qualitative and quantitative study of morphology by scanning electron microscopy supported by image analysis demonstrated only a negligible effect on the structural porosity, to be ascribed to the addition of micrometric HA as a filler. Moreover, by comparing the Raman spectra with thermal analysis(thermogravimetry and differential scanning calorimetry) the role of HA on the composite degradation mechanism was defined, by separately quantifying the contribution of HA particles in the bulk and on the surface, on the bone formation as a function of modifications induced in the pore morphology, as well as physical and chemical properties of the polymer matrix. Indeed, HA particles alter the poly epsilon-caprolactone crystallinity inducing a "shielding" effect of the polymer matrix. Meanwhile, the slight reduction of pore size as a function of the increasing HA content and the improvement of the effective hydrophilicity of the scaffolds also influence the degradation by faster mechanisms. Finally, it has been proven that the presence of HA enhances the scaffold bioactivity and human osteoblast cell response, remarking the active role of bioactive signals on the promotion of the surface mineralization and, as a consequence, on the cell-material interaction.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nur, Adrian; Rahmawati, Alifah; Ilmi, Noor Izzati

Synthesis of nanosized of hydroxyapatite (HA) by electrochemical pulsed direct current (PDC) method has been studied. The aim of this work is to study the influence of various PDC parameters (pH initial, electrode distance, duty cycle, frequency, and amplitude) on particle surface area of HA powders. The electrochemical synthesis was prepared in solution Ca{sup 2+}/EDTA{sup 4−}/PO{sub 4}{sup 3+} at concentration 0.25/0.25/0.15 M for 24 h. The electrochemical cell was consisted of two carbon rectangular electrodes connected to a function generator to produce PDC. There were two treatments for particles after electrosynthesized, namely without aging and aged for 2 days atmore » 40 °C. For both cases, the particles were filtered and washed by demineralized water to eliminate the impurities and unreacted reactants. Then, the particles were dried at 100 °C for 2 days. The dried particles were characterized by X-ray diffraction, surface area analyzer, scanning electron microscopy (SEM), Fourier transform infrared spectra and thermogravimetric and differential thermal analysis. HA particles can be produced when the initial pH > 6. The aging process has significant effect on the produced HA particles. SEM images of HA particles showed that the powders consisted of agglomerates composed of fine crystallites and have morphology plate-like and sphere. The surface area of HA particles is in the range of 25 – 91 m{sup 2}/g. The largest particle surface area of HA was produced at 4 cm electrode distance, 80% cycle duty, frequency 0.1 Hz, amplitude 9 V and with aging process.« less

Generating Chondromimetic Mesenchymal Stem Cell Spheroids by Regulating Media Composition and Surface Coating.

PubMed

Sridharan, BanuPriya; Laflin, Amy D; Detamore, Michael S

2018-04-01

Spheroids of mesenchymal stem cells (MSCs) in cartilage tissue engineering have been shown to enhance regenerative potential owing to their 3D structure. In this study, we explored the possibility of priming spheroids under different media to replace the use of inductive surface coatings for chondrogenic differentiation. Rat bone marrow-derived MSCs were organized into cell spheroids by the hanging drop technique and subsequently cultured on hyaluronic acid (HA) coated or non-coated well plates under different cell media conditions. Endpoint analysis included cell viability, DNA and Glycosaminoglycan (GAG) and collagen content, gene expression and immunohistochemistry. For chondrogenic applications, MSC spheroids derived on non-coated surfaces outperformed the spheroids derived from HA-coated surfaces in matrix synthesis and collagen II gene expression. Spheroids on non-coated surfaces gave rise to the highest collagen and GAG when primed with medium containing insulin-like growth factor (IGF) for 1 week during spheroid formation. Spheroids that were grown in chondroinductive raw material-inclusive media such as aggrecan or chondroitin sulfate exhibited the highest Collagen II gene expression in the non-coated surface at 1 week. Media priming by growth factors and raw materials might be a more predictive influencer of chondrogenesis compared to inductive-surfaces. Such tailored bioactivity of the stem cell spheroids in the stage of the spheroid formation may give rise to a platform technology that may eventually produce spheroids capable of chondrogenesis achieved by mere media manipulation, skipping the need for additional culture on a modified surface, that paves the way for cost-effective technologies.

Enhanced alveolar growth and remodeling in Guinea pigs raised at high altitude.

PubMed

Hsia, Connie C W; Carbayo, Juan J Polo; Yan, Xiao; Bellotto, Dennis J

2005-05-12

To examine the effects of chronic high altitude (HA) exposure on lung structure during somatic maturation, we raised male weanling guinea pigs at HA (3800m) for 1, 3, or 6 months, while their respective male littermates were simultaneously raised at low altitude (LA, 1200m). Under anaesthesia, airway pressure was measured at different lung volumes. The right lung was fixed at a constant airway pressure for morphometric analysis under light and electron microscopy. In animals raised at HA for 1 month, lung volume, alveolar surface area and alveolar-capillary blood volume (V(c)) were elevated above LA control values. Following 3-6 months of HA exposure, increases in lung volume and alveolar surface area persisted while the initial increase in V(c) normalized. Additional adaptation occurred, including a higher epithelial cell volume, septal tissue volume and capillary surface area, a lower alveolar duct volume and lower harmonic mean diffusion barrier resulting in higher membrane and lung diffusing capacities. These data demonstrate enhanced alveolar septal growth and progressive acinar remodeling during chronic HA exposure with long-term augmentation of alveolar dimensions as well as functional compensation in lung compliance and diffusive gas transport.

Tumor targeting profiling of hyaluronan-coated lipid based-nanoparticles.

PubMed

Mizrahy, Shoshy; Goldsmith, Meir; Leviatan-Ben-Arye, Shani; Kisin-Finfer, Einat; Redy, Orit; Srinivasan, Srimeenakshi; Shabat, Doron; Godin, Biana; Peer, Dan

2014-04-07

Hyaluronan (HA), a naturally occurring high Mw (HMw) glycosaminoglycan, has been shown to play crucial roles in cell growth, embryonic development, healing processes, inflammation, and tumor development and progression. Low Mw (LMw, <10 kDa) HA has been reported to provoke inflammatory responses, such as induction of cytokines, chemokines, reactive nitrogen species and growth factors. Herein, we prepared and characterized two types of HA coated (LMw and HMw) lipid-based targeted and stabilized nanoparticles (tsNPs) and tested their binding to tumor cells expressing the HA receptor (CD44), systemic immunotoxicity, and biodistribution in tumor bearing mice. In vitro, the Mw of the surface anchored HA had a significant influence on the affinity towards CD44 on B16F10 murine melanoma cells. LMw HA-tsNPs exhibited weak binding, while binding of tsNPs coated with HMw HA was characterized by high binding. Both types of tsNPs had no measured effect on cytokine induction in vivo following intravenous administration to healthy C57BL/6 mice suggesting no immune activation. HMw HA-tsNPs showed enhanced circulation time and tumor targeting specificity, mainly by accumulating in the tumor and its vicinity compared with LMw HA-tsNPs. Finally, we show that methotrexate (MTX), a drug commonly used in cancer chemotherapy, entrapped in HMw HA-tsNPs slowly diffused from the particles with a half-life of 13.75 days, and improved the therapeutic outcome in a murine B16F10 melanoma model compared with NPs suggesting an active cellular targeting beyond the Enhanced Permeability and Retention (EPR) effect. Taken together, these findings have major implications for the use of high molecular weight HA in nanomedicine as a selective and safe active cellular targeting moiety.

Porous biomorphic silicon carbide ceramics coated with hydroxyapatite as prospective materials for bone implants.

PubMed

Gryshkov, Oleksandr; Klyui, Nickolai I; Temchenko, Volodymyr P; Kyselov, Vitalii S; Chatterjee, Anamika; Belyaev, Alexander E; Lauterboeck, Lothar; Iarmolenko, Dmytro; Glasmacher, Birgit

2016-11-01

Porous and cytocompatible silicon carbide (SiC) ceramics derived from wood precursors and coated with bioactive hydroxyapatite (HA) and HA-zirconium dioxide (HA/ZrO2) composite are materials with promising application in engineering of bone implants due to their excellent mechanical and structural properties. Biomorphic SiC ceramics have been synthesized from wood (Hornbeam, Sapele, Tilia and Pear) using a forced impregnation method. The SiC ceramics have been coated with bioactive HA and HA/ZrO2 using effective gas detonation deposition approach (GDD). The surface morphology and cytotoxicity of SiC ceramics as well as phase composition and crystallinity of deposited coatings were analyzed. It has been shown that the porosity and pore size of SiC ceramics depend on initial wood source. The XRD and FTIR studies revealed the preservation of crystal structure and phase composition of in the HA coating, while addition of ZrO2 to the initial HA powder resulted in significant decomposition of the final HA/ZrO2 coating and formation of other calcium phosphate phases. In turn, NIH 3T3 cells cultured in medium exposed to coated and uncoated SiC ceramics showed high re-cultivation efficiency as well as metabolic activity. The recultivation efficiency of cells was the highest for HA-coated ceramics, whereas HA/ZrO2 coating improved the recultivation efficiency of cells as compared to uncoated SiC ceramics. The GDD method allowed generating homogeneous HA coatings with no change in calcium to phosphorus ratio. In summary, porous and cytocompatible bio-SiC ceramics with bioactive coatings show a great promise in construction of light, robust, inexpensive and patient-specific bone implants for clinical application. Copyright © 2016 Elsevier B.V. All rights reserved.

Targeted Delivery of Hyaluronan-Immobilized Magnetic Ceramic Nanocrystals.

PubMed

Wu, Hsi-Chin; Wang, Tzu-Wei; Hsieh, Shun-Yu; Sun, Jui-Sheng; Kang, Pei-Leun

2016-01-01

Effective cancer therapy relies on delivering the therapeutic agent precisely to the target site to improve the treatment outcome and to minimize side effects. Although surgery, chemotherapy, and radiotherapy are the standard methods commonly used in clinics, hyperthermia has been developed as a new and promising strategy for cancer therapy. In this study, magnetic bioceramic hydroxyapatite (mHAP) nanocrystals have been developed as heat mediator for intracellular hyperthermia. Hyaluronic acid (HA) modified mHAP nanocrystals are synthesized by a wet chemical precipitation process to achieve active targeting. The results demonstrate that the HA targeting moiety conjugated by a poly(ethylene glycol) (PEG) spacer arm is successfully immobilized on the surface of mHAP. The HA-modified mHAP possesses relatively good biocompatibility, an adequate biodegradation rate and superparamagnetic properties. The HA-modified mHAP could be localized and internalized into HA receptor-overexpressed malignant cells (e.g., MDA-MB-231 cell) and used as the heat generating agent for intracellular hyperthermia. The results from this study indicate that biocompatible HA-modified mHAP shows promise as a novel heat mediator and a specific targeting nanoagent for intracellular hyperthermia cancer therapy.

In vitro characterisation of a sol-gel derived in situ silica-coated silicate and carbonate co-doped hydroxyapatite nanopowder for bone grafting.

PubMed

Latifi, Seyed Mohsen; Fathi, Mohammadhossein; Sharifnabi, Ali; Varshosaz, Jaleh

2017-06-01

Design and synthesis of materials with better properties and performance are essential requirements in the field of biomaterials science that would directly improve patient quality of life. For this purpose, in situ silica-coated silicate and carbonate co-doped hydroxyapatite (Sc/S.C.HA) nanopowder was synthesized via the sol-gel method. Characterisation of the prepared nanopowder was carried out by XRD, FTIR, TEM, SEM, EDX, ICP, zeta potential, acid dissolution test, and cell culture test. The substitution of the silicate and carbonate ions into hydroxyapatite structure was confirmed by FTIR analysis. XRD analysis showed that silica is an amorphous phase, which played a role in covering the surface of the S.C.HA nanoparticles as confirmed by acid dissolution test. Low thickness and low integrity of the amorphous silica surface layer facilitated ions release from S.C.HA nanoparticles into physiological saline solution. Zeta potential of the prepared nanopowder suspended in physiological saline solution was -27.3±0.2mV at pH7.4. This negatively charged surface, due to the presence of amorphous silica layer upon the S.C.HA nanoparticles, not only had an accelerating effect on in vitro biomineralization of apatite, but also had a positive effect on cell attachment. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro testing of calcium phosphate (HA, TCP, and biphasic HA-TCP) whiskers.

PubMed

Jalota, Sahil; Bhaduri, Sarit B; Tas, A Cuneyt

2006-09-01

Calcium phosphate [single-phase hydroxyapatite (HA, Ca(10)(PO(4))(6)(OH)(2)), single-phase tricalcium phosphate (beta-TCP, Ca(3)(PO(4))(2)), and biphasic HA-TCP] whiskers were formed by using a novel microwave-assisted molten salt mediated process. Aqueous solutions containing NaNO(3), HNO(3), Ca(NO(3))(2) x 4H(2)O, and KH(2)PO(4) (with or without urea) were used as starting reagents. These solutions were irradiated in a household microwave oven for 5 min. As-recovered precursors were then simply stirred in water at room temperature for 1 h to obtain the whiskers of the desired calcium phosphate (CaP) bioceramics. These whiskers were evaluated, respectively, in vitro by (1) soaking those in synthetic body fluid (SBF) solutions at 37 degrees C for one week, and (2) performing cell attachment and total protein assay tests on the neat whiskers by using a mouse osteoblast cell line (7F2). beta-TCP, HA, and HA-TCP biphasic whiskers were all found to possess apatite-inducing ability when soaked in SBF. SBF-soaked whiskers were found to have BET surface areas ranging from 45 to 112 m(2)/g. Although the osteoblast viability and protein concentrations were found to be the highest on the neat HA whiskers, cells were attached and proliferated on all the whiskers.

Preparation and Characterization of Lanthanum-Incorporated Hydroxyapatite Coatings on Titanium Substrates

PubMed Central

Lou, Weiwei; Dong, Yiwen; Zhang, Hualin; Jin, Yifan; Hu, Xiaohui; Ma, Jianfeng; Liu, Jinsong; Wu, Gang

2015-01-01

Titanium (Ti) has been widely used in clinical applications for its excellent biocompatibility and mechanical properties. However, the bioinertness of the surface of Ti has motivated researchers to improve the physicochemical and biological properties of the implants through various surface modifications, such as coatings. For this purpose, we prepared a novel bioactive material, a lanthanum-incorporated hydroxyapatite (La-HA) coating, using a dip-coating technique with a La-HA sol along with post-heat treatment. The XRD, FTIR and EDX results presented in this paper confirmed that lanthanum was successfully incorporated into the structure of HA. The La-HA coating was composed of rod-like particles which densely compacted together without microcracks. The results of the interfacial shear strength test indicated that the incorporation of lanthanum increased the bonding strength of the HA coating. The mass loss ratios under acidic conditions (pH = 5.5) suggested that the La-HA coatings have better acid resistance. The cytocompatibility of the La-HA coating was also revealed by the relative activity of alkaline phosphatase, cellular morphology and cell proliferation assay in vitro. The present study suggested that La-HA coated on Ti has promising potential for applications in the development of a new type of bioactive coating for metal implants. PMID:26404255

Functionalized scaffolds to enhance tissue regeneration

PubMed Central

Guo, Baolin; Lei, Bo; Li, Peng; Ma, Peter X.

2015-01-01

Tissue engineering scaffolds play a vital role in regenerative medicine. It not only provides a temporary 3-dimensional support during tissue repair, but also regulates the cell behavior, such as cell adhesion, proliferation and differentiation. In this review, we summarize the development and trends of functional scaffolding biomaterials including electrically conducting hydrogels and nanocomposites of hydroxyapatite (HA) and bioactive glasses (BGs) with various biodegradable polymers. Furthermore, the progress on the fabrication of biomimetic nanofibrous scaffolds from conducting polymers and composites of HA and BG via electrospinning, deposition and thermally induced phase separation is discussed. Moreover, bioactive molecules and surface properties of scaffolds are very important during tissue repair. Bioactive molecule-releasing scaffolds and antimicrobial surface coatings for biomedical implants and scaffolds are also reviewed. PMID:25844177

Multifunctional Role of 35 Kilodalton Hyaluronan in Promoting Defense of the Intestinal Epithelium.

PubMed

Kessler, Sean P; Obery, Dana R; Nickerson, Kourtney P; Petrey, Aaron C; McDonald, Christine; de la Motte, Carol A

2018-04-01

Intestinal epithelium plays a critical role in host defense against orally acquired pathogens. Dysregulation of this protective barrier is a primary driver of inflammatory bowel diseases (Crohn's and ulcerative colitis) and also infant gastrointestinal infections. Previously, our lab reported that hyaluronan (HA) isolated from human milk induces the expression of the antimicrobial peptide β-defensin in vivo and protects against Salmonella Typhimurium infection of epithelial cells in vitro. In addition, we demonstrated that commercially available 35 kDa size HA induces the expression of β-defensin, upregulates the expression of tight junction protein zonula occludens-1 (ZO-1), and attenuates murine Citrobacter rodentium infection in vivo. In this current study, we report that HA35 remains largely intact and biologically active during transit through the digestive tract where it directly induces β-defensin expression upon epithelial cell contact. We also demonstrate HA35 abrogation of murine Salmonella Typhimurium infection as well as downregulation of leaky tight junction protein claudin-2 expression. Taken together, we propose a dual role for HA in host innate immune defense at the epithelial cell surface, acting to induce antimicrobial peptide production and also block pathogen-induced leaky gut. HA35 is therefore a promising therapeutic in the defense against bacterially induced colitis in compromised adults and vulnerable newborns.

Fabrication of human hair keratin/jellyfish collagen/eggshell-derived hydroxyapatite osteoinductive biocomposite scaffolds for bone tissue engineering: From waste to regenerative medicine products.

PubMed

Arslan, Yavuz Emre; Sezgin Arslan, Tugba; Derkus, Burak; Emregul, Emel; Emregul, Kaan C

2017-06-01

In the present study, we aimed at fabricating an osteoinductive biocomposite scaffold using keratin obtained from human hair, jellyfish collagen and eggshell-derived nano-sized spherical hydroxyapatite (nHA) for bone tissue engineering applications. Keratin, collagen and nHA were characterized with the modified Lowry method, free-sulfhydryl groups and hydroxyproline content analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR) and thermal gravimetric analysis (TGA) which confirmed the success of the extraction and/or isolation processes. Human adipose mesenchymal stem cells (hAMSCs) were isolated and the cell surface markers were characterized via flow cytometry analysis in addition to multilineage differentiation capacity. The undifferentiated hAMSCs were highly positive for CD29, CD44, CD73, CD90 and CD105, but were not seen to express hematopoietic cell surface markers such as CD14, CD34 and CD45. The cells were successfully directed towards osteogenic, chondrogenic and adipogenic lineages in vitro. The microarchitecture of the scaffolds and cell attachment were evaluated using scanning electron microscopy (SEM). The cell viability on the scaffolds was assessed by the MTT assay which revealed no evidence of cytotoxicity. The osteogenic differentiation of hAMSCs on the scaffolds was determined histologically using alizarin red S, osteopontin and osteonectin stainings. Early osteogenic differentiation markers of hAMSCs were significantly expressed on the collagen-keratin-nHA scaffolds. In conclusion, it is believed that collagen-keratin-nHA osteoinductive biocomposite scaffolds have the potential of being used in bone tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

Influence of humic acid imposed changes of ferrihydrite aggregation on microbial Fe(III) reduction

NASA Astrophysics Data System (ADS)

Amstaetter, Katja; Borch, Thomas; Kappler, Andreas

2012-05-01

Microbial reduction of Fe(III) minerals at neutral pH is faced by the problem of electron transfer from the cells to the solid-phase electron acceptor and is thought to require either direct cell-mineral contact, the presence of Fe(III)-chelators or the presence of electron shuttles, e.g. dissolved or solid-phase humic substances (HS). In this study we investigated to which extent the ratio of Pahokee Peat Humic Acids (HA) to ferrihydrite in the presence and absence of phosphate influences rates of Fe(III) reduction by Shewanella oneidensis MR-1 and the identity of the minerals formed. We found that phosphate generally decreased reduction rates by sorption to the ferrihydrite and surface site blocking. In the presence of low ferrihydrite concentrations (5 mM), the addition of HA helped to overcome this inhibiting effect by functioning as electron shuttle between cells and the ferrihydrite. In contrast, at high ferrihydrite concentrations (30 mM), the addition of HA did not lead to an increase but rather to a decrease in reduction rates. Confocal laser scanning microscopy images and ferrihydrite sedimentation behaviour suggest that the extent of ferrihydrite surface coating by HA influences the aggregation of the ferrihydrite particles and thereby their accessibility for Fe(III)-reducing bacteria. We further conclude that in presence of dissolved HA, iron reduction is stimulated through electron shuttling while in the presence of only sorbed HA, no stimulation by electron shuttling takes place. In presence of phosphate the stimulation effect did not occur until a minimum concentration of 10 mg/l of dissolved HA was reached followed by increasing Fe(III) reduction rates up to dissolved HA concentrations of approximately 240 mg/l above which the electron shuttling effect ceased. Not only Fe(III) reduction rates but also the mineral products changed in the presence of HA. Sequential extraction, XRD and 57Fe-Mössbauer spectroscopy showed that crystallinity and grain size of the magnetite produced by Fe(III) reduction in the presence of HA is lower than the magnetite produced in the absence of HA. In summary, this study shows that both the concentration of HA and Fe(III) minerals strongly influence microbial Fe(III) reduction rates and the mineralogy of the reduction products. Thus, deviations in iron (hydr)oxide reactivity with changes in aggregation state, such as HA induced ferrihydrite aggregation, need to be considered within natural environments.

Formation of bone-like mineralized matrix by periodontal ligament cells in vivo: a morphological study in rats.

PubMed

Hiraga, Toru; Ninomiya, Tadashi; Hosoya, Akihiro; Takahashi, Masafumi; Nakamura, Hiroaki

2009-01-01

Periodontal ligament (PDL) is a unique connective tissue that not only connects cementum and alveolar bone to support teeth, but also plays an important role in reconstructing periodontal tissues. Previous studies have suggested that PDL cells have osteogenic potential; however, they lack precise histological examinations. Here, we studied bone-like matrix formation by PDL cells in rats using morphological techniques. Rat and human PDL cells exhibited substantial alkaline phosphatase activity and induced mineralization in vitro. RT-PCR analyses showed that PDL cells expressed the osteoblast markers, Runx2, osterix, and osteocalcin. These results suggest that PDL cells share similar phenotypes with osteoblasts. To examine the bone-like matrix formation in vivo, PDL cells isolated from green fluorescent protein (GFP)-transgenic rats were inoculated with hydroxyapatite (HA) disks into wild-type rats. Five weeks after the implantation, the pores in HA disks were occupied by GFP-positive cells. Mineralized matrix formation was also found on the surface of HA pores. At 12 weeks, some of the pores were filled with bone-like mineralized matrices (BLMM), which were positive for the bone matrix proteins, osteopontin, bone sialoprotein, and osteocalcin. Immunohistochemical examination revealed that most of the osteoblast- and osteocyte-like cells on or in the BLMM were GFP-positive, suggesting that the BLMM were directly formed by the inoculated PDL cells. On the pore surfaces, Sharpey's fiber-like structures embedded in cementum-like mineralized layers were also observed. These results collectively suggest that PDL cells have the ability to form periodontal tissues and could be a useful source for regenerative therapies of periodontal diseases.

Keratinocytes negatively regulate the N-cadherin levels of melanoma cells via contact-mediated calcium regulation.

PubMed

Chung, Heesung; Jung, Hyejung; Jho, Eek-Hoon; Multhaupt, Hinke A B; Couchman, John R; Oh, Eok-Soo

2018-06-14

In human skin, melanocytes and their neighboring keratinocytes have a close functional interrelationship. Keratinocytes, which represent the prevalent cell type of human skin, regulate melanocytes through various mechanisms. Here, we use a keratinocyte and melanoma co-culture system to show for the first time that keratinocytes regulate the cell surface expression of N-cadherin through cell-cell contact. Compared to mono-cultured human melanoma A375 cells, which expressed high levels of N-cadherin, those co-cultured with the HaCaT human keratinocyte cell line showed reduced levels of N-cadherin. This reduction was most evident in areas of A375 cells that underwent cell-cell contact with the HaCaT cells, whereas HaCaT cell-derived extracellular matrix and conditioned medium both failed to reduce N-cadherin levels. The intracellular level of calcium in co-cultured A375 cells was lower than that in mono-cultured A375 cells, and treatment with a cell-permeant calcium chelator (BAPTA) reduced the N-cadherin level of mono-cultured A375 cells. Furthermore, co-culture with HaCaT cells reduced the expression levels of transient receptor potential cation channel (TRPC) 1, -3 and -6 in A375 cells, and siRNA-mediated multi-depletion of TRPC1, -3 and -6 reduced the N-cadherin level in these cells. Taken together, these data suggest that keratinocytes negatively regulate the N-cadherin levels of melanoma cells via cell-to-cell contact-mediated calcium regulation. Copyright © 2018. Published by Elsevier Inc.

Surface localization of the nuclear receptor CAR in influenza A virus-infected cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Tadanobu; Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, CREST, JST, and COE Program in the 21st Century, Shizuoka 422-8526; Moriyama, Yusuke

Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism.

Induction Plasma Sprayed Nano Hydroxyapatite Coatings on Titanium for Orthopaedic and Dental Implants

PubMed Central

Roy, Mangal; Bandyopadhyay, Amit; Bose, Susmita

2011-01-01

This paper reports preparation of a highly crystalline nano hydroxyapatite (HA) coating on commercially pure titanium (Cp-Ti) using inductively coupled radio frequency (RF) plasma spray and their in vitro and in vivo biological response. HA coatings were prepared on Ti using normal and supersonic plasma nozzles at different plate powers and working distances. X-ray diffraction (XRD) and Fourier transformed infrared spectroscopic (FTIR) analysis show that the normal plasma nozzle lead to increased phase decomposition, high amorphous calcium phosphate (ACP) phase formation, and severe dehydroxylation of HA. In contrast, coatings prepared using supersonic nozzle retained the crystallinity and phase purity of HA due to relatively short exposure time of HA particles in the plasma. In addition, these coatings exhibited a microstructure that varied from porous and glassy structure at the coating-substrate interface to dense HA at the top surface. The microstructural analysis showed that the coating was made of multigrain HA particles of ~200 nm in size, which consisted of recrystallized HA grains in the size range of 15– 20 nm. Apart from the type of nozzle, working distance was also found to have a strong influence on the HA phase decomposition, while plate power had little influence. Depending on the plasma processing conditions, a coating thickness between 300 and 400 μm was achieved where the adhesive bond strengths were found to be between 4.8 MPa to 24 MPa. The cytotoxicity of HA coatings was examined by culturing human fetal osteoblast cells (hFOB) on coated surfaces. In vivo studies, using the cortical defect model in rat femur, evaluated the histological response of the HA coatings prepared with supersonic nozzle. After 2 weeks of implantation, osteoid formation was evident on the HA coated implant surface, which could indicate early implant- tissue integration in vivo. PMID:21552358

Hyaluronic acid modified pH-sensitive liposomes for targeted intracellular delivery of doxorubicin.

PubMed

Paliwal, Shivani Rai; Paliwal, Rishi; Agrawal, Govind Prasad; Vyas, Suresh Prasad

2016-12-01

Surface-modified pH-sensitive liposomal system may be useful for intracellular delivery of chemotherapeutics. Achieving site-specific targeting with over-expressed hyaluronic acid (HA) receptors along with using pH sensitive liposome carrier for intracellular drug delivery was the aim of this study. Stealth HA-targeted pH-sensitive liposomes (SL-pH-HA) were developed and evaluated to achieve effective intracellular delivery of doxorubicin (DOX) vis-a-vis enhanced antitumor activity. The in vitro release studies demonstrated that the release of DOX from SL-pH-HA was pH-dependent, i.e. faster at mildly acidic pH ∼5, compared to physiological pH ∼7.4. SLpH-HA was evaluated for their cytotoxicity potential on CD44 receptor expressing MCF-7 cells. The half maximal inhibitory concentration (IC50) of SL-pH-HA and SL-HA were about 1.9 and 2.5 μM, respectively, after 48 h of incubation. The quantitative uptake study revealed higher localization of targeted liposomes in the receptor positive cells, which was further confirmed by fluorescent microscopy. The antitumor efficacy of the DOX-loaded HA-targeted pH-sensitive liposomes was also verified in a tumor xenograft mouse model. DOX was efficiently delivered to the tumor site by active targeting via HA and CD44 receptor interaction. The major side-effect of conventional DOX formulation, i.e. cardiotoxicity was also estimated by measuring serum enzyme levels of LDH and CPK and found to be minimized with developed formulation. Overall, HA targeted pH-sensitive liposomes were significantly more potent than the non-targeted liposomes in cells expressing high levels of CD44. Results strongly implies the promise of such liposomal system as an intracellular drug delivery carrier developed for potential anticancer treatment.

Targeted and controlled drug delivery system loading artersunate for effective chemotherapy on CD44 overexpressing cancer cells.

PubMed

Tran, Tuan Hiep; Nguyen, Tuan Duc; Van Nguyen, Han; Nguyen, Hanh Thuy; Kim, Jong Oh; Yong, Chul Soon; Nguyen, Chien Ngoc

2016-05-01

Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with negative surface charge were reversed to positive by cationic surfactant-DDAB before being coated with an anionic polymer, hyaluronic acid, to improve their site-specific intracellular delivery against CD44 receptor overexpressing cancer cells. Incorporating artesunate (ART)-a promising anticancer drug into PLGA/HA nanoparticles, is expected not only to overcome its poor aqueous solubility and stability but also enhance the activities. The obtained particles were characterized by dynamic light scattering, zeta potential measurements, and transmission electron microscopy (TEM). Cancer cell internalization of the NPs was evaluated by flow cytometry and cytotoxicity of the NPs was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay. PLGA/HA nanoparticles showed greater extent of cellular uptake to SCC-7 and MCF-7 cells, indicating their affinity with CD44 receptor-mediated endocytosis. Almost 60 % of ART was released into the outer media after 48 h. In vitro fluorescence sorting demonstrated that PLGA/HA had highly efficient targeting and accumulation into CD44 receptor overexpression cells. The significant reduction in cell viability as well as greater induction of apoptosis suggested a potential in anticancer therapy of ART loaded PLGA/HA.

Bioinspired Design of Polycaprolactone Composite Nanofibers as Artificial Bone Extracellular Matrix for Bone Regeneration Application.

PubMed

Gao, Xiang; Song, Jinlin; Zhang, Yancong; Xu, Xiao; Zhang, Siqi; Ji, Ping; Wei, Shicheng

2016-10-07

The design and development of functional biomimetic systems for programmed stem cell response is a field of topical interest. To mimic bone extracellular matrix, we present an innovative strategy for constructing drug-loaded composite nanofibrous scaffolds in this study, which could integrate multiple cues from calcium phosphate mineral, bioactive molecule, and highly ordered fiber topography for the control of stem cell fate. Briefly, inspired by mussel adhesion mechanism, a polydopamine (pDA)-templated nanohydroxyapatite (tHA) was synthesized and then surface-functionalized with bone morphogenetic protein-7-derived peptides via catechol chemistry. Afterward, the resulting peptide-loaded tHA (tHA/pep) particles were blended with polycaprolactone (PCL) solution to fabricate electrospun hybrid nanofibers with random and aligned orientation. Our research demonstrated that the bioactivity of grafted peptides was retained in composite nanofibers. Compared to controls, PCL-tHA/pep composite nanofibers showed improved cytocompatibility. Moreover, the incorporated tHA/pep particles in nanofibers could further facilitate osteogenic differentiation potential of human mesenchymal stem cells (hMSCs). More importantly, the aligned PCL-tHA/pep composite nanofibers showed more osteogenic activity than did randomly oriented counterparts, even under nonosteoinductive conditions, indicating excellent performance of biomimetic design in cell fate decision. After in vivo implantation, the PCL-tHA/pep composite nanofibers with highly ordered structure could significantly promote the regeneration of lamellar-like bones in a rat calvarial critical-sized defect. Accordingly, the presented strategy in our work could be applied for a wide range of potential applications in not only bone regeneration application but also pharmaceutical science.

Osteoblast and osteoclast responses to A/B type carbonate-substituted hydroxyapatite ceramics for bone regeneration.

PubMed

Germaini, Marie-Michèle; Detsch, Rainer; Grünewald, Alina; Magnaudeix, Amandine; Lalloue, Fabrice; Boccaccini, Aldo R; Champion, Eric

2017-06-06

The influence of carbonate substitution (4.4 wt%, mixed A/B type) in hydroxyapatite ceramics for bone remodeling scaffolds was investigated by separately analyzing the response of pre-osteoblasts and osteoclast-like cells. Carbonated hydroxyapatite (CHA) (Ca 9.5 (PO 4 ) 5.5 (CO 3 ) 0.5 (OH)(CO 3 ) 0.25 -CHA), mimicking the chemical composition of natural bone mineral, and pure hydroxyapatite (HA) (Ca 10 (PO 4 ) 6 (OH) 2 -HA) porous ceramics were processed to obtain a similar microstructure and surface physico-chemical properties (grain size, porosity ratio and pore size, surface roughness and zeta potential). The biological behavior was studied using MC3T3-E1 pre-osteoblastic and RAW 264.7 monocyte/macrophage cell lines. Chemical dissolution in the culture media and resorption lacunae produced by osteoclasts occur with both HA and CHA ceramics, but CHA exhibits much higher dissolution and greater bioresorption ability. CHA ceramics promoted a significantly higher level of pre-osteoblast proliferation. Osteoblastic differentiation, assessed by qRT-PCR of RUNX2 and COLIA2, and pre-osteoclastic proliferation and differentiation were not significantly different on CHA or HA ceramics but cell viability and metabolism were significantly greater on CHA ceramics. Thus, the activity of both osteoclast-like and osteoblastic cells was influenced by the carbonate substitution in the apatite structure. Furthermore, CHA showed a particularly interesting balance between biodegradation, by osteoclasts and chemical dissolution, and osteogenesis through osteoblasts' activity, to stimulate bone regeneration. It is hypothesized that this amount of 4.4 wt% carbonate substitution leads to an adapted concentration of calcium in the fluid surrounding the ceramic to stimulate the activity of cells. These results highlight the superior biological behavior of microporous 4.4 wt% A/B CHA ceramics that could beneficially replace the commonly used HA of biphasic calcium phosphates for future applications in bone tissue engineering.

Yeast Surface-Displayed H5N1 Avian Influenza Vaccines

PubMed Central

Lei, Han; Jin, Sha; Karlsson, Erik; Schultz-Cherry, Stacey

2016-01-01

Highly pathogenic H5N1 avian influenza viruses pose a pandemic threat to human health. A rapid vaccine production against fast outbreak is desired. We report, herein, a paradigm-shift influenza vaccine technology by presenting H5N1 hemagglutinin (HA) to the surface of yeast. We demonstrated, for the first time, that the HA surface-presented yeast can be used as influenza vaccines to elicit both humoral and cell-mediated immunity in mice. The HI titer of antisera reached up to 128 in vaccinated mice. A high level of H5N1 HA-specific IgG1 and IgG2a antibody production was detected after boost immunization. Furthermore, we demonstrated that the yeast surface-displayed HA preserves its antigenic sites. It preferentially binds to both avian- and human-type receptors. In addition, the vaccine exhibited high cross-reactivity to both homologous and heterologous H5N1 viruses. A high level production of anti-HA antibodies was detected in the mice five months after vaccination. Finally, our animal experimental results indicated that the yeast vaccine offered complete protection of mice from lethal H5N1 virus challenge. No severe side effect of yeast vaccines was noted in animal studies. This new technology allows for rapid and large-scale production of influenza vaccines for prepandemic preparation. PMID:28078309

Surface modification for interaction study with bacteria and preosteoblast cells

NASA Astrophysics Data System (ADS)

Song, Qing

Surface modification plays a pivotal role in bioengineering. Polymer coatings can provide biocompatibility and biofunctionalities to biomaterials through surface modification. In this dissertation, initiated chemical vapor deposition (iCVD) was utilized to coat two-dimensional (2D) and three-dimensional (3D) substrates with differently charged polyelectrolytes in order to generate antimicrobial and osteocompatible biomaterials. ICVD is a modified CVD technique that enables surface modification in an all-dry condition without substrate damage and solvent contamination. The free-radical polymerization allows the vinyl polymers to conformally coat on various micro- and nano-structured substrates and maintains the delicate structure of the functional groups. The vapor deposition of polycations provided antimicrobial activity to planar and porous substrates through destroying the negatively charged bacterial membrane and brought about high contact-killing efficiency (99.99%) against Gram-positive Bacillus subtilis and Gram-negative Escherichia coli. Additionally, the polyampholytes synthesized by iCVD exhibited excellent antifouling performance against the adhesion of Gram-positive Listeria innocua and Gram-negative E. coli in phosphate buffered saline (PBS). Their antifouling activities were attributed to the electrostatic interaction and hydration layers that served as physical and energetic barriers to prevent bacterial adhesion. The contact-killing and antifouling polymers synthesized by iCVD can be applied to surface modification of food processing equipment and medical devices with the aim of reducing foodborne diseases and medical infections. Moreover, the charged polyelectrolyte modified 2D polystyrene surfaces displayed good osteocompatibility and enhanced osteogenesis of preosteoblast cells than the un-modified polystyrene surface. In order to promote osteoinduction of hydroxyapatite (HA) scaffolds, bioinspired polymer-controlled mineralization was conducted on the polyelectrolyte modified HA scaffolds. The mineralized scaffolds stimulated osteogenesis of preosteoblast cells compared with the control HA scaffolds. Therefore, the surface modification through vapor deposition of polyelectrolytes and polymer-controlled mineralization can improve osteoinduction of bone materials. In summary, the iCVD-mediated surface modification is a simple and promising approach to biofunctionalizing various structured substrates and generating antimicrobial and biocompatible biomaterials.

Interactions between meniscal cells and a self assembled biomimetic surface composed of hyaluronic acid, chitosan and meniscal extracellular matrix molecules.

PubMed

Tan, Guak-Kim; Dinnes, Donna L M; Butler, Lauren N; Cooper-White, Justin J

2010-08-01

Menisci are one of the most commonly injured parts of the knee with a limited healing potential. This study focuses on fabrication and characterization of biomimetic surfaces for meniscal tissue engineering. To achieve this, a combination of hyaluronic acid/chitosan (HA/CH) mutilayers with covalently immobilized major extracellular matrix (ECM) components of native meniscus, namely collagen I/II (COL.I/II) and chondroitin-6-sulfate (C6S) was employed. Adsorption of the biomolecules was monitored using a quartz crystal microbalance with dissipation (QCM-D) and fourier transform-surface plasmon resonance (FT-SPR). Immobilization of the biomolecules onto HA/CH mutilayers was achieved by sequential adsorption, with optimum binding at a molar ratio of 1.4:1 (COL.I/II: C6S). After adding COL.I/II, the layers became relatively more rigid and large aggregates were evident. The effects of the modified surfaces on cell proliferation, gene expression and proteoglycan production of rat meniscal cells were examined. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) analysis showed that primary meniscal cells dedifferentiated rapidly after one passage in monolayer culture. This process could be reversed by culturing the cells on C6S surfaces, as indicated by increases in both collagen II and aggrecan gene expression, as well as proteoglycan production. Cells with abundant lipid vacuoles were evident on all the surfaces over an extended culture period. The results demonstrate the feasibility of C6S surfaces to avoid the dedifferentiation that normally occurs during monolayer expansion of meniscal cells. Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.

Bone plate composed of a ternary nano-hydroxyapatite/polyamide 66/glass fiber composite: biomechanical properties and biocompatibility

PubMed Central

Qiao, Bo; Li, Jidong; Zhu, Qingmao; Guo, Shuquan; Qi, Xiaotong; Li, Weichao; Wu, Jun; Liu, Yang; Jiang, Dianming

2014-01-01

An ideal bone plate for internal fixation of bone fractures should have good biomechanical properties and biocompatibility. In this study, we prepared a new nondegradable bone plate composed of a ternary nano-hydroxyapatite/polyamide 66/glass fiber (n-HA/PA66/GF) composite. A breakage area on the n-HA/PA66/GF plate surface was characterized by scanning electron microscopy. Its mechanical properties were investigated using bone-plate constructs and biocompatibility was evaluated in vitro using bone marrow-derived mesenchymal stem cells. The results confirmed that adhesion between the n-HA/PA66 matrix and the glass fibers was strong, with only a few fibers pulled out at the site of breakage. Fractures fixed by the n-HA/PA66/GF plate showed lower stiffness and had satisfactory strength compared with rigid fixation using a titanium plate. Moreover, the results with regard to mesenchymal stem cell morphology, MTT assay, Alizarin Red S staining, enzyme-linked immunosorbent assay, and reverse transcription polymerase chain reaction for alkaline phosphatase and osteocalcin showed that the n-HA/PA66/GF composite was suitable for attachment and proliferation of mesenchymal stem cells, and did not have a negative influence on matrix mineralization or osteogenic differentiation of mesenchymal stem cells. These observations indicate that the n-HA/PA66/GF plate has good biomechanical properties and biocompatibility, and may be considered a new option for internal fixation in orthopedic surgery. PMID:24669191

Bone plate composed of a ternary nano-hydroxyapatite/polyamide 66/glass fiber composite: biomechanical properties and biocompatibility.

PubMed

Qiao, Bo; Li, Jidong; Zhu, Qingmao; Guo, Shuquan; Qi, Xiaotong; Li, Weichao; Wu, Jun; Liu, Yang; Jiang, Dianming

2014-01-01

An ideal bone plate for internal fixation of bone fractures should have good biomechanical properties and biocompatibility. In this study, we prepared a new nondegradable bone plate composed of a ternary nano-hydroxyapatite/polyamide 66/glass fiber (n-HA/PA66/GF) composite. A breakage area on the n-HA/PA66/GF plate surface was characterized by scanning electron microscopy. Its mechanical properties were investigated using bone-plate constructs and biocompatibility was evaluated in vitro using bone marrow-derived mesenchymal stem cells. The results confirmed that adhesion between the n-HA/PA66 matrix and the glass fibers was strong, with only a few fibers pulled out at the site of breakage. Fractures fixed by the n-HA/PA66/GF plate showed lower stiffness and had satisfactory strength compared with rigid fixation using a titanium plate. Moreover, the results with regard to mesenchymal stem cell morphology, MTT assay, Alizarin Red S staining, enzyme-linked immunosorbent assay, and reverse transcription polymerase chain reaction for alkaline phosphatase and osteocalcin showed that the n-HA/PA66/GF composite was suitable for attachment and proliferation of mesenchymal stem cells, and did not have a negative influence on matrix mineralization or osteogenic differentiation of mesenchymal stem cells. These observations indicate that the n-HA/PA66/GF plate has good biomechanical properties and biocompatibility, and may be considered a new option for internal fixation in orthopedic surgery.

The synergistic effect of nano-hydroxyapatite and dexamethasone in the fibrous delivery system of gelatin and poly(l-lactide) on the osteogenesis of mesenchymal stem cells.

PubMed

Amjadian, Sara; Seyedjafari, Ehsan; Zeynali, Bahman; Shabani, Iman

2016-06-30

Recently, electrospun nanofibrous scaffolds are vastly taken into consideration in the bone tissue engineering due to mimicking the natural structure of native tissue. In our study, surface features of nanofibers were modified through simultaneous electrospining of the synthetic and natural polymers using poly l-lactide (PLLA) and gelatin to fabricate the hybrid scaffold (PLLA/gelatin). Then, hydroxyapatite nanoparticles (nHA) were loaded in electrospun PLLA nanofibers (PLLA,nHA/gelatin) and also dexamethasone (DEX) was incorporated in these fibers (PLLA,nHA,DEX/gelatin) in the second experiment. Fabricated nanofibrous composite scaffolds were characterized via SEM, FTIR spectroscopy, contact angle, tensile strength measurements, DEX release profile and MTT assay. After seeding adipose derived mesenchymal stem cells, osteoinductivity and osteoconductivity of fabricated scaffolds were analyzed using common osteogenic markers such as alkaline phosphatase activity, calcium depositions and gene expression. These results confirmed that all properties of nanofibers were improved by modifications. Moreover, osteogenic differentiation of stem cells increased in PLLA,nHA/gelatin group in comparison with PLLA/gelatin. The sustained release of DEX was obtained from PLLA,nHA,DEX/gelatin which subsequently led to more osteogenic differentiation. Taken together, PLLA,nHA,DEX/gelatin showed significant potential to support the stem cell proliferation and ostogenic differentiation, and can be a good candidates for tissue engineering and regenerative medicine applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of model hydroxyapatite bone scaffolds with multiscale porosity for potential load bearing applications

NASA Astrophysics Data System (ADS)

Dellinger, Jennifer Gwynne

2005-11-01

Model hydroxyapatite (HA) bone scaffolds consisting of a latticed pattern of rods were fabricated by a solid freeform fabrication (SFF) technique based on the robotic deposition of colloidal pastes. An optimal HA paste formulation for this method was developed. Local porosity, i.e. microporosity (1--30 mum) and sintering porosity (less than 1 mum), were produced by including polymer microsphere porogens in the HA pastes and by controlling the sintering of the scaffolds. Scaffolds with and without local porosity were evaluated with and without in vitro accelerated degradation. Percent weight loss of the scaffolds and calcium and phosphorus concentrations in solution increased with degradation time. After degradation, compressive strength and modulus decreased significantly for scaffolds with local porosity, but did not change significantly for scaffolds without local porosity. The compressive strength and modulus of scaffolds without local porosity were comparable to human cortical bone and were significantly greater than the scaffolds with local porosity. Micropores in HA disks caused surface pits that increased the surface roughness as compared to non-microporous HA disks. Mouse mesenchymal stem cells extended their cell processes into these microporous pits on HA disks in vitro. ALP expression was prolonged, cell attachment strength increased, and ECM production appeared greater on microporous HA disks compared to non-microporous HA disks and tissue culture treated polystyrene controls. Scaffolds with and without microporosity were implanted in goats bones. Microporous scaffolds with rhBMP-2 increased the percent of the scaffold filled with bone tissue compared to microporous scaffolds without rhBMP-2. Lamellar bone inside scaffolds was aligned near the rods junctions whereas lamellar bone was aligned in a more random configuration away from the rod junctions. Microporous scaffolds stained darkly with toluidine blue beneath areas of contact with new bone. This staining might indicate either extracellular matrix (ECM) in the rods or dye bound to the degrading scaffold. Although the presence of microporous topography alone did not influence bone healing in vivo, micropores were shown to provide tailorability of scaffold mechanical properties, provide a location for the storage and controlled release of a growth factor, and provide a location for bone integration inside the scaffold rods.

Incorporation of cerium oxide into hydroxyapatite coating regulates osteogenic activity of mesenchymal stem cell and macrophage polarization.

PubMed

Li, Kai; Shen, Qingyi; Xie, Youtao; You, Mingyu; Huang, Liping; Zheng, Xuebin

2017-02-01

Biomedical coatings for orthopedic implants should facilitate osseointegration and mitigate implant-induced inflammatory reactions. Cerium oxide (CeO 2 ) ceramics possess anti-oxidative properties and can be used to decrease mediators of inflammation, which makes them attractive for biomedical applications. In our work, two kinds of CeO 2 incorporated hydroxyapatite coatings (HA-10Ce and HA-30Ce) were prepared via plasma spraying technique and the effects of CeO 2 addition on the responses of bone mesenchymal stem cells (BMSCs) and RAW264.7 macrophages were investigated. An increase in CeO 2 content in the HA coatings resulted in better osteogenic behaviors of BMSCs in terms of cell proliferation, alkaline phosphatase (ALP) activity and mineralized nodule formation. RT-PCR and western blot analysis suggested that the incorporation of CeO 2 may promote the osteogenic differentiation of BMSCs through the Smad-dependent BMP signaling pathway, which activated Runx2 expression and subsequently enhanced the expression of ALP and OCN. The expression profiles of macrophages cultured on the CeO 2 modified coating revealed a tendency toward a M2 phenotype, because of an upregulation of M2 surface markers (CD163 and CD206), anti-inflammatory cytokines (TNF-α and IL-6) and osteoblastogenesis-related genes (BMP2 and TGF-β1) as well as a downregulation of M1 surface markers (CCR7 and CD11c), proinflammatory cytokines (IL-10 and IL-1ra) and reactive oxygen species production. The results suggested the regulation of BMSCs behaviors and macrophage-mediated responses at the coating's surface were associated with CeO 2 incorporation. The incorporation of CeO 2 in HA coatings can be a valuable strategy to promote osteogenic responses and reduce inflammatory reactions.

Synergistic Effect of Artificial Tears Containing Epigallocatechin Gallate and Hyaluronic Acid for the Treatment of Rabbits with Dry Eye Syndrome.

PubMed

Tseng, Ching-Li; Hung, Ya-Jung; Chen, Zhi-Yu; Fang, Hsu-Wei; Chen, Ko-Hua

2016-01-01

Dry eye syndrome (DES) is a common eye disease. Artificial tears (AT) are used to treat DES, but they are not effective. In this study, we assessed the anti-inflammatory effect of AT containing epigallocatechin gallate (EGCG) and hyaluronic acid (HA) on DES. Human corneal epithelial cells (HCECs) were used in the WST-8 assay to determine the safe dose of EGCG. Lipopolysaccharide-stimulated HCECs showing inflammation were treated with EGCG/HA. The expression of IL-1ß, IL-6, IL-8, and TNF-α was assessed by real-time PCR and AT physical properties such as the viscosity, osmolarity, and pH were examined. AT containing EGCG and HA were topically administered in a rabbit DES model established by treatment with 0.1% benzalkonium chloride (BAC). Tear secretion was assessed and fluorescein, H&E, and TUNEL staining were performed. Inflammatory cytokine levels in the corneas were also examined. The non-toxic optimal concentration of EGCG used for the treatment of HCECs in vitro was 10 μg/mL. The expression of several inflammatory genes, including IL-1ß, IL-6, IL-8, and TNF-α, was significantly inhibited in inflamed HCECs treated with 10 μg/mL EGCG and 0.1% (w/v) HA (E10/HA) compared to that in inflamed HCECs treated with either EGCG or HA alone. AT containing E10/HA mimic human tears, with similar osmolarity and viscosity and a neutral pH. Fluorescence examination of the ocular surface of mouse eyes showed that HA increased drug retention on the ocular surface. Topical treatment of DES rabbits with AT plus E10/HA increased tear secretion, reduced corneal epithelial damage, and maintained the epithelial layers and stromal structure. Moreover, the corneas of the E10/HA-treated rabbits showed fewer apoptotic cells, lower inflammation, and decreased IL-6, IL-8, and TNF-α levels. In conclusion, we showed that AT plus E10/HA had anti-inflammatory and mucoadhesive properties when used as topical eye drops and were effective for treating DES in rabbits.

Influence of nanotopography on periodontal ligament stem cell functions and cell sheet based periodontal regeneration

PubMed Central

Gao, Hui; Li, Bei; Zhao, Lingzhou; Jin, Yan

2015-01-01

Periodontal regeneration is an important part of regenerative medicine, with great clinical significance; however, the effects of nanotopography on the functions of periodontal ligament (PDL) stem cells (PDLSCs) and on PDLSC sheet based periodontal regeneration have never been explored. Titania nanotubes (NTs) layered on titanium (Ti) provide a good platform to study this. In the current study, the influence of NTs of different tube size on the functions of PDLSCs was observed. Afterward, an ectopic implantation model using a Ti/cell sheets/hydroxyapatite (HA) complex was applied to study the effect of the NTs on cell sheet based periodontal regeneration. The NTs were able to enhance the initial PDLSC adhesion and spread, as well as collagen secretion. With the Ti/cell sheets/HA complex model, it was demonstrated that the PDLSC sheets were capable of regenerating the PDL tissue, when combined with bone marrow mesenchymal stem cell (BMSC) sheets and HA, without the need for extra soluble chemical cues. Simultaneously, the NTs improved the periodontal regeneration result of the ectopically implanted Ti/cell sheets/HA complex, giving rise to functionally aligned collagen fiber bundles. Specifically, much denser collagen fibers, with abundant blood vessels as well as cementum-like tissue on the Ti surface, which well-resembled the structure of natural PDL, were observed in the NT5 and NT10 sample groups. Our study provides the first evidence that the nanotopographical cues obviously influence the functions of PDLSCs and improve the PDLSC sheet based periodontal regeneration size dependently, which provides new insight to the periodontal regeneration. The Ti/cell sheets/HA complex may constitute a good model to predict the effect of biomaterials on periodontal regeneration. PMID:26150714

Influence of nanotopography on periodontal ligament stem cell functions and cell sheet based periodontal regeneration.

PubMed

Gao, Hui; Li, Bei; Zhao, Lingzhou; Jin, Yan

2015-01-01

Periodontal regeneration is an important part of regenerative medicine, with great clinical significance; however, the effects of nanotopography on the functions of periodontal ligament (PDL) stem cells (PDLSCs) and on PDLSC sheet based periodontal regeneration have never been explored. Titania nanotubes (NTs) layered on titanium (Ti) provide a good platform to study this. In the current study, the influence of NTs of different tube size on the functions of PDLSCs was observed. Afterward, an ectopic implantation model using a Ti/cell sheets/hydroxyapatite (HA) complex was applied to study the effect of the NTs on cell sheet based periodontal regeneration. The NTs were able to enhance the initial PDLSC adhesion and spread, as well as collagen secretion. With the Ti/cell sheets/HA complex model, it was demonstrated that the PDLSC sheets were capable of regenerating the PDL tissue, when combined with bone marrow mesenchymal stem cell (BMSC) sheets and HA, without the need for extra soluble chemical cues. Simultaneously, the NTs improved the periodontal regeneration result of the ectopically implanted Ti/cell sheets/HA complex, giving rise to functionally aligned collagen fiber bundles. Specifically, much denser collagen fibers, with abundant blood vessels as well as cementum-like tissue on the Ti surface, which well-resembled the structure of natural PDL, were observed in the NT5 and NT10 sample groups. Our study provides the first evidence that the nanotopographical cues obviously influence the functions of PDLSCs and improve the PDLSC sheet based periodontal regeneration size dependently, which provides new insight to the periodontal regeneration. The Ti/cell sheets/HA complex may constitute a good model to predict the effect of biomaterials on periodontal regeneration.

Immobilized heavy chain-hyaluronic acid polarizes lipopolysaccharide-activated macrophages toward M2 phenotype.

PubMed

He, Hua; Zhang, Suzhen; Tighe, Sean; Son, Ji; Tseng, Scheffer C G

2013-09-06

Despite the known anti-inflammatory effect of amniotic membrane, its action mechanism remains largely unknown. HC-HA complex (HC-HA) purified from human amniotic membrane consists of high molecular weight hyaluronic acid (HA) covalently linked to the heavy chain (HC) 1 of inter-α-trypsin inhibitor. In this study, we show that soluble HC-HA also contained pentraxin 3 and induced the apoptosis of both formyl-Met-Leu-Phe or LPS-activated neutrophils and LPS-activated macrophages while not affecting the resting cells. This enhanced apoptosis was caused by the inhibition of cell adhesion, spreading, and proliferation caused by HC-HA binding of LPS-activated macrophages and preventing adhesion to the plastic surface. Preferentially, soluble HC-HA promoted phagocytosis of apoptotic neutrophils in resting macrophages, whereas immobilized HC-HA promoted phagocytosis in LPS-activated macrophages. Upon concomitant LPS stimulation, immobilized HC-HA but not HA polarized macrophages toward the M2 phenotype by down-regulating IRF5 protein and preventing its nuclear localization and by down-regulating IL-12, TNF-α, and NO synthase 2. Additionally, IL-10, TGF-β1, peroxisome proliferator-activated receptor γ, LIGHT (TNF superfamily 14), and sphingosine kinase-1 were up-regulated, and such M2 polarization was dependent on TLR ligation. Collectively, these data suggest that HC-HA is a unique matrix component different from HA and uses multiple mechanisms to suppress M1 while promoting M2 phenotype. This anti-inflammatory action of HC-HA is highly desirable to promote wound healing in diseases heightened by unsuccessful transition from M1 to M2 phenotypes.

A DNA Aptamer Against Influenza A Virus: An Effective Inhibitor to the Hemagglutinin-Glycan Interactions.

PubMed

Li, Wenkai; Feng, Xinru; Yan, Xing; Liu, Keyi; Deng, Le

2016-06-01

Most therapeutical nucleic acid aptamers tend to inhibit protein-protein interactions and thereby function as antagonists. Attachment of the influenza virus surface glycoprotein hemagglutinin (HA) to sialic acid-containing host cell receptors (glycan) facilitates the initial stage of viral infection. Inhibition of the attachment may result in an antiviral effect on the proliferation of the influenza virus. To develop therapeutically interesting agents, we selected two single-stranded DNA (ssDNA) aptamers specific to the HA protein of H1N1 influenza virus (A/Puerto Rico/8/1934) through a procedure of systematic evolution of ligands by exponential enrichment. As it showed a higher binding affinity for HA protein (Kd = 78 ± 1 nM), aptamer 1 was tested for its ability to interfere with HA-glycan interactions using chicken red blood cell hemagglutination and microneutralization assays, which demonstrated that it significantly suppressed the viral infection in host cells. These results indicate that the isolated ssDNA aptamer may be developed as an antiviral agent against influenza through appropriate therapeutic formulation.

In vitro osteogenic cell proliferation, mineralization, and in vivo osseointegration of injection molded high-density polyethylene-based hybrid composites in rabbit animal model.

PubMed

Tripathi, Garima; Basu, Bikramjit

2014-07-01

The present work reports the biocompatibility property of injection molded HDPE-HA-Al2O3 hybrid composites. In vitro cytocompatibility results reveal that osteogenic cell viability and bone mineralization are favorably supported in a statistically significant manner on HDPE-20% HA-20% Al2O3 composite, in comparison to HDPE-40 wt.% HA or HDPE-40 wt.% Al2O3 The difference in cytocompatibility property is explained in terms of difference in substrate wettability/surface energy and importantly, both the cell proliferation at 7 days or bone mineralization at 21 days on HDPE-20% HA-20% Al2O3 composite are either comparable or better than sintered HA. The progressive healing of cylindrical femoral bone defects in rabbit animal model was assessed by implantation experiments over 1, 4 and 12 weeks. Based on the histological analysis as well as histomorphometrical evaluation, a better efficacy of HDPE-20% HA-20% Al2O3 over high-density polyethylene (HDPE) for bone regeneration and neobone formation at host bone-implant interface was established. Taken together, the present study unequivocally establishes that despite the presence of 20% Al2O3, HDPE-based hybrid composites are as biocompatible as HA in vitro or better than HDPE in vivo. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Electrophoretic deposition of hydroxyapatite-hexagonal boron nitride composite coatings on Ti substrate.

PubMed

Göncü, Yapıncak; Geçgin, Merve; Bakan, Feray; Ay, Nuran

2017-10-01

In this study, commercial pure titanium samples were coated with nano hydroxyapatite-nano hexagonal boron nitride (nano HA-nano hBN) composite by electrophoretic deposition (EPD). The effect of process parameters (applied voltage, deposition time and solid concentration) on the coating morphology, thickness and the adhesion behavior were studied systematically and crack free nano hBN-nano HA composite coating production was achieved for developing bioactive coatings on titanium substrates for orthopedic applications. For the examination of structural and morphological characteristics of the coating surfaces, various complementary analysis methods were performed. For the structural characterization, XRD and Raman Spectroscopy were used while, Scanning Electron Microscopy (SEM) equipped with an energy dispersive spectrometer (EDS) and Transmission Electron Microscopy (TEM) techniques were carried out for revealing the morphological characterization. The results showed that nano HA-nano hBN were successfully deposited on Ti surface with uniform, crack-free coating by EPD. The amounts of hBN in suspension are considered to have no effect on coating thickness. By adding hBN into HA, the morphology of HA did not change and hBN has no significant effect on porous structure. These nanostructured surfaces are expected to be suitable for proliferation of cells and have high potential for bioactive materials. Copyright © 2017 Elsevier B.V. All rights reserved.

Inducing bioactivity of dental ceramic/bioactive glass composites by Nd:YAG laser.

PubMed

Beketova, Anastasia; Poulakis, Nikolaos; Bakopoulou, Athina; Zorba, Triantafillia; Papadopoulou, Lambrini; Christofilos, Dimitrios; Kantiranis, Nikolaos; Zachariadis, George A; Kontonasaki, Eleana; Kourouklis, Gerasimos A; Paraskevopoulos, Konstantinos M; Koidis, Petros

2016-11-01

Aims of this study were to investigate the optimal conditions of laser irradiation of a novel Bioactive Glass/Dental Ceramic-BP67 composite for acceleration of hydroxyapatite-HA formation and to assess cellular responses on the precipitated HA region. BP67 (Bioactive Glass: 33.3%, Dental Ceramic: 66.7%) was fabricated by the sol-gel method. A laser assisted biomimetic-LAB process was applied to BP67 sintered specimens immersed in 1.5-times concentrated simulated body fluid-1.5×-SBF. The effect of various energy densities of pulsed nanosecond Nd-YAG (1064nm) laser and irradiation exposure times (30min, 1 and 3h) were evaluated for HA precipitation. The HA film was characterized by FTIR, XRD, SEM and micro Raman techniques. ICP-AES was used for revealing changes in chemical composition of the 1.5×-SBF during irradiation. Cell viability and morphological characteristics of periodontal ligament fibroblasts-PDLFs, human gingival fibroblasts-HGFs and SAOS-2 osteoblasts on the HA surface were evaluated by MTT assays and SEM. At optimal energy fluence of 1.52J/cm 2 and irradiation time for 3h followed by immersion in 1.5×-SBF at 60°C, a dense HA layer was formed on laser-irradiated BP67 within 7 days. The resulting HA film was tightly bonded to the underlying substrate and had mineral composition similar to cementum. MTT assay showed a consistent reduction of cell proliferation on the HA layer in comparison to conventional control ceramic and BP67 for all 3 cell lines studied. These findings suggest LAB is an effective method for acceleration of HA formation on materials with low bioactivity, while cellular responses need further investigation. Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Response of Human Osteoblast to n-HA/PEEK—Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite

NASA Astrophysics Data System (ADS)

Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

2016-03-01

Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.

Hierarchical Microspheres Constructed from Chitin Nanofibers Penetrated Hydroxyapatite Crystals for Bone Regeneration.

PubMed

Duan, Bo; Shou, Kangquan; Su, Xiaojuan; Niu, Yahui; Zheng, Guan; Huang, Yao; Yu, Aixi; Zhang, Yu; Xia, Hong; Zhang, Lina

2017-07-10

Chitin exists abundantly in crab and shrimp shells as the template of the minerals, which inspired us to mineralize it for fabricating bone grafting materials. In the present work, chitin nanofibrous microspheres were used as the matrix for in situ synthesis of hydroxyapatite (HA) crystals including microflakes, submicron-needles, and submicron-spheres, which were penetrated by long chitin nanofibers, leading to the hierarchical structure. The shape and size of the HA crystals could be controlled by changing the HA synthesis process. The tight interface adhesion between chitin and HA through the noncovanlent bonds occurred in the composite microspheres, and HAs were homogeneously dispersed and bounded to the chitin nanofibers. In our findings, the inherent biocompatibilities of the both chitin and HA contributed the bone cell adhesion and osteoconduction. Moreover, the chitin microsphere with submicron-needle and submicron-sphere HA crystals remarkably promoted in vitro cell adhesion and in vivo bone healing. It was demonstrated that rabbits with 1.5 cm radius defect were almost cured completely within three months in a growth factor- and cell-free state, as a result of the unique surface microstructure and biocompatibilities of the composite microspheres. The microsphere scaffold displayed excellent biofunctions and an appropriate biodegradability. This work opened up a new avenue to construct natural polymer-based organic-inorganic hybrid microspheres for bone regeneration.

Response of Human Osteoblast to n-HA/PEEK--Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite.

PubMed

Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

2016-03-09

Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn't increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca(2+) concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.

Prevalence of methicillin resistant Staphylococcus aureus [MRSA] colonization or carriage among health-care workers.

PubMed

Pathare, Nirmal A; Asogan, Harshini; Tejani, Sara; Al Mahruqi, Gaitha; Al Fakhri, Salma; Zafarulla, Roshna; Pathare, Anil V

2016-01-01

In Oman, the prevalence of health care associated methicillin resistant Staphylococcus aureus [HA-MRSA] is unknown. Therefore, to estimate the prevalence of HA-MRSA, we collected nasal swabs and swabs from cell phones on sterile polyester swabs and immediately inoculated on the mannitol salt agar containing oxacillin from medical students and hospital health care providers. Antibiotic susceptibility testing of the isolates was then performed using the Kirby Bauer's disc diffusion method. Additionally, a brief survey questionnaire was used to acquire demographic data. Amongst the 311 participants enrolled, nasal colonization with HA-MRSA was found in 47 individuals (15.1%, 95% confidence interval [CI]=11.1%, 19.1%). HA-MRSA was also isolated from the cell phone surfaces in 28 participants (9.0%, 95% CI=8.6%, 9.3%). 5 participants (1.6%) showed positive results both from their nasal swabs and from their cell phones. Antibiotic resistance to erythromycin [48%] and clindamycin [29%] was relatively high. 9.3% HA-MRSA isolates were vancomycin resistant [6.6% nasal carriage]. There was no statistically significant correlation between HA-MRSA isolates and the demographic characteristics or the risk factors namely gender, underlying co-morbidities like diabetes, hypertension, skin/soft tissue infections, skin ulcers/wounds, recent exposure to antibiotics, or hospital visits (p>0.05, Chi-square test). Copyright © 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins

PubMed Central

Zhang, Jie; Pekosz, Andrew; Lamb, Robert A.

2000-01-01

Influenza viruses encoding hemagglutinin (HA) and neuraminidase (NA) glycoproteins with deletions in one or both cytoplasmic tails (HAt− or NAt−) have a reduced association with detergent-insoluble glycolipids (DIGs). Mutations which eliminated various combinations of the three palmitoylation sites in HA exhibited reduced amounts of DIG-associated HA in virus-infected cells. The influenza virus matrix (M1) protein was also found to be associated with DIGs, but this association was decreased in cells infected with HAt− or NAt− virus. Regardless of the amount of DIG-associated protein, the HA and NA glycoproteins were targeted primarily to the apical surface of virus-infected, polarized cells. The uncoupling of DIG association and apical transport was augmented by the observation that the influenza A virus M2 protein as well as the influenza C virus HA-esterase-fusion glycoprotein were not associated with DIGs but were apically targeted. The reduced DIG association of HAt− and NAt− is an intrinsic property of the glycoproteins, as similar reductions in DIG association were observed when the proteins were expressed from cDNA. Examination of purified virions indicated reduced amounts of DIG-associated lipids in the envelope of HAt− and NAt− viruses. The data indicate that deletion of both the HA and NA cytoplasmic tails results in reduced DIG association and changes in both virus polypeptide and lipid composition. PMID:10775599

Low-magnitude, high-frequency vibration promotes the adhesion and the osteogenic differentiation of bone marrow-derived mesenchymal stem cells cultured on a hydroxyapatite-coated surface: The direct role of Wnt/β-catenin signaling pathway activation.

PubMed

Chen, Bailing; Lin, Tao; Yang, Xiaoxi; Li, Yiqiang; Xie, Denghui; Zheng, Wenhui; Cui, Haowen; Deng, Weimin; Tan, Xin

2016-11-01

The positive effect of low-magnitude, high‑frequency (LMHF) vibration on implant osseointegration has been demonstrated; however, the underlying cellular and molecular mechanisms remain unknown. The aim of this study was to explore the effect of LMHF vibration on the adhesion and the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) cultured on hydroxyapatite (HA)-coated surfaces in an in vitro model as well as to elucidate the molecular mechanism responsible for the effects of LMHF vibration on osteogenesis. LMHF vibration resulted in the increased expression of fibronectin, which was measured by immunostaining and RT-qPCR. Stimulation of BMSCs by LMHF vibration resulted in the rearrangement of the actin cytoskeleton with more prominent F-actin. Moreover, the expression of β1 integrin, vinculin and paxillin was notably increased following LMHF stimulation. Scanning electron microscope observations revealed that there were higher cell numbers and more extracellular matrix attached to the HA-coated surface in the LMHF group. Alkaline phosphatase activity as well as the expression of osteogenic-specific genes, namely Runx2, osterix, collagen I and osteocalcin, were significantly elevated in the LMHF group. In addition, the protein expression of Wnt10B, β-catenin, Runx2 and osterix was increased following exposure to LMHF vibration. Taken together, the findings of this study indicate that LMHF vibration promotes the adhesion and the osteogenic differentiation of BMSCs on HA-coated surfaces in vitro, and LMHF vibration may directly induce osteogenesis by activating the Wnt/β‑catenin signaling pathway. These data suggest that LMHF vibration enhances the osseointegration of bone to a HA-coated implant, and provide a scientific foundation for improving bone-implant osseointegration through the application of LMHF vibration.

Preparation, characterization and in vitro response of bioactive coatings on polyether ether ketone.

PubMed

Durham, John W; Allen, Matthew J; Rabiei, Afsaneh

2017-04-01

Polyether ether ketone (PEEK) is a highly heat-resistant thermoplastic with excellent strength and elastic modulus similar to human bone, making it an attractive material for orthopedic implants. However, the hydrophobic surface of PEEK implants induces fibrous encapsulation which is unfavorable for stable implant anchorage. In this study, PEEK was coated via ion-beam-assisted deposition (IBAD) using a two-layer design of yttria-stabilized zirconia (YSZ) as a heat-protection layer, and hydroxyapatite (HA) as a top layer to improve osseointegration. Microstructural analysis of the coatings showed a dense, uniform columnar grain structure in the YSZ layer and no delamination from the substrate. The HA layer was found to be amorphous and free of porosities in its as-deposited state. Subsequent heat treatment via microwave energy followed by autoclaving crystallized the HA layer, confirmed by SEM and XRD analysis. An in vitro study using MC3T3 preosteoblast cells showed improved bioactivity in heat-treated sample groups. Cell proliferation, differentiation, and mineralization were analyzed by MTT assay and DNA content, osteocalcin expression, and Alizarin Red S (AR-S) content, respectively. Initial cell growth was increased, and osteogenic maturation and mineralization were accelerated most on coatings that underwent a combined microwave and autoclave heat treatment process as compared to uncoated PEEK and amorphous HA surfaces. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 560-567, 2017. © 2015 Wiley Periodicals, Inc.

Preparation of porous collagen/hyaluronic acid hybrid scaffolds for biomimetic functionalization through biochemical binding affinity.

PubMed

Lee, Su Jin; Kim, So Yeon; Lee, Young Moo

2007-08-01

This study demonstrated the feasibility of introducing an avidin-biotin system to three-dimensional and highly porous scaffolds for the purpose of designing scaffolds that have binding affinity with bioactive molecules for various biomimetic modifications. Porous hybrid scaffolds composed of collagen and hyaluronic acid (HA) were prepared by a novel overrun process. The overrun-processed scaffolds showed a uniform dual-pore structure because of the injection of gas bubbles and ice recrystallization during the fabrication process and had a higher porosity than scaffolds prepared by a conventional freeze-drying method. The mechanical strength and biodegradation kinetics were controlled by the method of preparation and the composition of collagen/HA. Collagen/HA scaffolds did not show any significant adverse effects on cell viability even after 10 days of incubation. The fibroblasts cultured in the overrun-processed scaffolds were widely distributed and had proliferated on the surfaces of the macropores in the scaffolds, whereas the cells that were seeded in the freeze-dried scaffolds had attached mainly on the dense surface of the scaffolds. As the collagen content in the scaffolds increased, the cellular ingrowth into the inner pores of the scaffolds decreased because of the high affinity between the collagen and the cells. Measurements obtained via confocal microscopy revealed that the porous collagen/HA scaffolds could be functionalized with the biotin by incorporating avidin. Therefore, the present biotinylation approach may allow the incorporation of various bioactive molecules (DNA, growth factors, drug, peptide, etc) into the three-dimensional porous scaffolds.

Activation of Influenza A Viruses by Host Proteases from Swine Airway Epithelium

PubMed Central

Peitsch, Catharina; Klenk, Hans-Dieter; Garten, Wolfgang

2014-01-01

Pigs are important natural hosts of influenza A viruses, and due to their susceptibility to swine, avian, and human viruses, they may serve as intermediate hosts supporting adaptation and genetic reassortment. Cleavage of the influenza virus surface glycoprotein hemagglutinin (HA) by host cell proteases is essential for viral infectivity. Most influenza viruses, including human and swine viruses, are activated at a monobasic HA cleavage site, and we previously identified TMPRSS2 and HAT to be relevant proteases present in human airways. We investigated the proteolytic activation of influenza viruses in primary porcine tracheal and bronchial epithelial cells (PTEC and PBEC, respectively). Human H1N1 and H3N2 viruses replicated efficiently in PTECs and PBECs, and viruses containing cleaved HA were released from infected cells. Moreover, the cells supported the proteolytic activation of HA at the stage of entry. We found that swine proteases homologous to TMPRSS2 and HAT, designated swTMPRSS2 and swAT, respectively, were expressed in several parts of the porcine respiratory tract. Both proteases cloned from primary PBECs were shown to activate HA with a monobasic cleavage site upon coexpression and support multicycle replication of influenza viruses. swAT was predominantly localized at the plasma membrane, where it was present as an active protease that mediated activation of incoming virus. In contrast, swTMPRSS2 accumulated in the trans-Golgi network, suggesting that it cleaves HA in this compartment. In conclusion, our data show that HA activation in porcine airways may occur by similar proteases and at similar stages of the viral life cycle as in human airways. PMID:24155384

Differential effect of hydroxyapatite nano-particle versus nano-rod decorated titanium micro-surface on osseointegration.

PubMed

Bai, Long; Liu, Yanlian; Du, Zhibin; Weng, Zeming; Yao, Wei; Zhang, Xiangyu; Huang, Xiaobo; Yao, Xiaohong; Crawford, Ross; Hang, Ruiqiang; Huang, Di; Tang, Bin; Xiao, Yin

2018-06-15

Coating materials applied for intraosseous implants must be optimized to stimulate osseointegration. Osseointegration is a temporal and spatial physiological process that not only requires interactions between osteogenesis and angiogenesis but also necessitates a favorable immune microenvironment. It is now well-documented that hierarchical nano-micro surface structures promote the long-term stability of implants, the interactions between nano-micro structure and the immune response are largely unknown. Here, we report the effects of microporous titanium (Ti) surfaces coated with nano-hydroxyapatite (HA) produced by micro-arc oxidation and steam-hydrothermal treatment (SHT) on multiple cell behavior and osseointegration. By altering the processing time of SHT it was possible to shift HA structures from nano-particles to nano-rods on the microporous Ti surfaces. Ti surfaces coated with HA nano-particles were found to modulate the inflammatory response resulting in an osteoimmune microenvironment more favorable for osteo-/angio-genesis, most likely via the activation of certain key signaling pathways (TGF-β, OPG/RANKL, and VEGF). By contrast, Ti surfaces coated with nano-rod shaped HA particles had a negative impact on osteo-/angio-genesis and osteoimmunomodulation. In vivo results further demonstrated that Ti implant surfaces decorated with HA nano-particles can stimulate new bone formation and osseointegration with enhanced interaction between osteocytes and implant surfaces. This study demonstrated that Ti implants with micro-surfaces coated with nano-particle shaped HA have a positive impact on osseointegration. Osteo-/angio-genesis are of importance during osteointegration of the implants. Recent advances unravel that immune response of macrophages and its manipulated osteoimmunomodulation also exerts a pivotal role to determine the fate of the implant. Surface nano-micro modification has evidenced to be efficient to influence osteogenesis, however, little is known links nano-microstructured surface to immune response, as well the osteoimmunomodulation. This study demonstrates that the nano-particles decorated micro-surface, compared with the nano-rods decorated micro-surface enables osteogenesis and angiogenesis concurrently that has not been investigated previously. This study also unravels that the immune response of macrophages can be manipulated by the nano-micro surface, especially the nano-dimension matters, leading to a differential effect on osteointegration. The additional knowledge obtained from this study may provide foundation and reference for future design of the coating materials for implantable materials. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Context-specific target definition in influenza a virus hemagglutinin-glycan receptor interactions.

PubMed

Shriver, Zachary; Raman, Rahul; Viswanathan, Karthik; Sasisekharan, Ram

2009-08-28

Protein-glycan interactions are important regulators of a variety of biological processes, ranging from immune recognition to anticoagulation. An important area of active research is directed toward understanding the role of host cell surface glycans as recognition sites for pathogen protein receptors. Recognition of cell surface glycans is a widely employed strategy for a variety of pathogens, including bacteria, parasites, and viruses. We present here a representative example of such an interaction: the binding of influenza A hemagglutinin (HA) to specific sialylated glycans on the cell surface of human upper airway epithelial cells, which initiates the infection cycle. We detail a generalizable strategy to understand the nature of protein-glycan interactions both structurally and biochemically, using HA as a model system. This strategy combines a top-down approach using available structural information to define important contacts between glycans and HA, with a bottom-up approach using data-mining and informatics approaches to identify the common motifs that distinguish glycan binders from nonbinders. By probing protein-glycan interactions simultaneously through top-down and bottom-up approaches, we can scientifically validate a series of observations. This in turn provides additional confidence and surmounts known challenges in the study of protein-glycan interactions, such as accounting for multivalency, and thus truly defines concepts such as specificity, affinity, and avidity. With the advent of new technologies for glycomics-including glycan arrays, data-mining solutions, and robust algorithms to model protein-glycan interactions-we anticipate that such combination approaches will become tractable for a wide variety of protein-glycan interactions.

Facile incorporation of hydroxyapatite onto an anodized Ti surface via a mussel inspired polydopamine coating

NASA Astrophysics Data System (ADS)

Zhe, Wang; Dong, Chaofang; Sefei, Yang; Dawei, Zhang; Kui, Xiao; Xiaogang, Li

2016-08-01

Inspired by the porous morphology of anodized Ti and the adhesive versatility of polydopamine (PDA), which can induce apatite mineralization, we fabricated a novel interface by coating a porous anodized TiO2 layer with PDA to rapidly immobilize HA on Ti-based substrates. It was found that the as-prepared PDA/anodized (HD) surface exhibited nanoscale roughness, which possessed an excellent ability to form apatite when immersed in 1.5× simulated body fluid (SBF), as observed by AFM and FE-SEM. The morphology and composition of each layer were further confirmed by XPS, XRD and FTIR. The corrosion resistance of the multilayer was investigated using potentiodynamic polarization curve and electrochemical impedance spectra (EIS) measurements in a 0.9 wt% NaCl solution, the results suggested that the HA/PDA/anodized (HDA) layer increased the corrosion resistance of pure Ti with higher corrosion potential and lower passive current, the surface wettability was also enhanced with the incorporation of HA. In vitro cellular assays showed that the HDA layer stimulated cell attachment and improved the alkaline phosphate (ALP) activity. Overall, the PDA/anodized treatment provided a viable method to quickly integrate HA, and the obtained HDA layer improved both corrosion resistance and biocompatibility of the Ti surface.

Mechanical properties, electrochemical corrosion and in-vitro bioactivity of yttria stabilized zirconia reinforced hydroxyapatite coatings prepared by gas tunnel type plasma spraying.

PubMed

Yugeswaran, S; Yoganand, C P; Kobayashi, A; Paraskevopoulos, K M; Subramanian, B

2012-05-01

Yttria stabilized zirconia reinforced hydroxyapatite coatings were deposited by a gas tunnel type plasma spray torch under optimum spraying conditions. For this purpose, 10, 20 and 30 wt% of yttria stabilized zirconia (YSZ) powders were premixed individually with hydroxyapatite (HA) powder and were used as the feedstocks for the coatings. The effect of YSZ reinforcement on the phase formation and mechanical properties of the coatings such as hardness, adhesive strength and sliding wear rates was examined. The results showed that the reinforcement of YSZ in HA could significantly enhance the hardness and adhesive strength of the coatings. The potentiodynamic polarization and impedance measurements showed that the reinforced coatings exhibited superior corrosion resistance compared to the HA coating in SBF solution. Further the results of the bioactivity test conducted by immersion of coatings in SBF showed that after 10 days of immersion of the obtained coatings with all the above compositions commonly exhibited the onset of bioactive apatite formation except for HA+10%YSZ coating. The cytocompatibility was investigated by culturing the green fluorescent protein (GFP)-labeled marrow stromal cells (MSCs) on the coating surface. The cell culture results revealed that the reinforced coatings have superior cell growth than the pure HA coatings. Copyright © 2012. Published by Elsevier Ltd.

Low temperature aqueous precipitation of needle-like nanophase hydroxyapatite.

PubMed

Cox, Sophie C; Jamshidi, Parastoo; Grover, Liam M; Mallick, Kajal K

2014-01-01

The use of tissue engineered biodegradable porous scaffolds has become an important focus of the biomedical research field. The precursor materials used to form these structures play a vital role in their overall performance thus making the study and synthesis of these selected materials imperative. The authors present a comparison and characterisation of hydroxyapatite (HA), a popular calcium phosphate (CaP) biomaterial, synthesised by an aqueous precipitation (AP) method. The influence of various reaction conditions on the phase, crystallinity, particle size as well as morphology, molecular structure, potential in-vivo bioactivity and cell viability were assessed by XRD, SEM and TEM, FTIR, a simulated body fluid (SBF) test and a live/dead assay using MC3T3 osteoblast precursor cells, respectively. Naturally carbonated nanoparticles of HA with typically needle-like morphology were synthesised by the reported AP method. Initial pH was found to influence the crystallisation process and determine the CaP phase formed as well as the resultant particle and crystallite sizes. A marked change in particle morphology was also observed above pH 9. The use of toluene as a replacement solvent for water up to 60% was found to reduce the crystallinity of as-synthesised HA. This has marked influence on the effect of ethanolamine (5 wt%), which was found to improve HA crystallinity. SEM and EDS were used to confirm the growth of carbonated apatite on the surface of HA pellets immersed in SBF for up to 28 days. Cell culture results revealed viable cells on all samples where pH was controlled and maintained at 10-11 during precipitation, including those that used ethanolamine and toluene in preparation. When the initial alkali pH was not maintained non-viable cells were observed on HA substrates.

Fabrication and characterization of drug-loaded nano-hydroxyapatite/polyamide 66 scaffolds modified with carbon nanotubes and silk fibroin

PubMed Central

Yao, Meng-Zhu; Huang-Fu, Ming-Yi; Liu, Hui-Na; Wang, Xia-Rong; Sheng, Xiaoxia; Gao, Jian-Qing

2016-01-01

Nano-hydroxyapatite/polyamide 66 (nHA/PA66) porous scaffolds were fabricated by a phase inversion method. Carbon nanotubes (CNTs) and silk fibroin (SF) were used to modify the surface of the nHA/PA66 scaffolds by freeze-drying and cross-linking. Dexamethasone was absorbed to the CNTs to promote the osteogenic differentiation of bone mesenchymal stem cells (BMSCs). The cell viability of BMSCs was investigated by changing the concentration of the CNT dispersion, and the most biocompatible scaffold was selected. In addition, the morphology and mechanical property of the scaffolds were investigated. The results showed that the nHA/PA66 scaffolds modified with CNTs and SF met the requirements of bone tissue engineering scaffolds. The dexamethasone-loaded CNT/SF-nHA/PA66 composite scaffold promoted the osteogenic differentiation of BMSCs, and the drug-loaded scaffolds are expected to function as effective bone tissue engineering scaffolds. PMID:27920525

Mitigation in Multiple Effects of Graphene Oxide Toxicity in Zebrafish Embryogenesis Driven by Humic Acid.

PubMed

Chen, Yuming; Ren, Chaoxiu; Ouyang, Shaohu; Hu, Xiangang; Zhou, Qixing

2015-08-18

Graphene oxide (GO) is a widely used carbonaceous nanomaterial. To date, the influence of natural organic matter (NOM) on GO toxicity in aquatic vertebrates has not been reported. During zebrafish embryogenesis, GO induced a significant hatching delay and cardiac edema. The intensive interactions of GO with the chorion induces damage to chorion protuberances, excessive generation of (•)OH, and changes in protein secondary structure. In contrast, humic acid (HA), a ubiquitous form of NOM, significantly relieved the above adverse effects. HA reduced the interactions between GO and the chorion and mitigated chorion damage by regulating the morphology, structures, and surface negative charges of GO. HA also altered the uptake and deposition of GO and decreased the aggregation of GO in embryonic yolk cells and deep layer cells. Furthermore, HA mitigated the mitochondrial damage and oxidative stress induced by GO. This work reveals a feasible antidotal mechanism for GO in the presence of NOM and avoids overestimating the risks of GO in the natural environment.

Construction of Hyaluronic Tetrasaccharide Clusters Modified Polyamidoamine siRNA Delivery System.

PubMed

Ma, Yingcong; Sha, Meng; Cheng, Shixuan; Yao, Wang; Li, Zhongjun; Qi, Xian-Rong

2018-06-14

The CD44 protein, as a predominant receptor for hyaluronan (HA), is highly expressed on the surface of multiple tumor cells. HA, as a targeting molecule for a CD44-contained delivery system, increases intracellular drug concentration in tumor tissue. However, due to the weak binding ability of hyaluronan oligosaccharide to CD44, targeting for tumor drug delivery has been restricted. In this study, we first use a HA tetrasaccharide cluster as the target ligand to enhance the binding ability to CD44. A polyamidoamine (PAMAM) dendrimer was modified by a HA tetrasaccharide cluster as a nonviral vector for small interfering RNA (siRNA) delivery. The dendrimer/siRNA nanocomplexes increased the cellular uptake capacity of siRNA through the CD44 receptor-mediated endocytosis pathway, allowing the siRNA to successfully escape the endosome/lysosome. Compared with the control group, nanocomplexes effectively reduced the expression of GFP protein and mRNA in MDA-MB-231-GFP cells. This delivery system provides a foundation to increase the clinical applications of PAMAM nanomaterials.

Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex.

PubMed

Lee, Kwangkook; Zhong, Xiaofen; Gu, Shenyan; Kruel, Anna Magdalena; Dorner, Martin B; Perry, Kay; Rummel, Andreas; Dong, Min; Jin, Rongsheng

2014-06-20

How botulinum neurotoxins (BoNTs) cross the host intestinal epithelial barrier in foodborne botulism is poorly understood. Here, we present the crystal structure of a clostridial hemagglutinin (HA) complex of serotype BoNT/A bound to the cell adhesion protein E-cadherin at 2.4 angstroms. The HA complex recognizes E-cadherin with high specificity involving extensive intermolecular interactions and also binds to carbohydrates on the cell surface. Binding of the HA complex sequesters E-cadherin in the monomeric state, compromising the E-cadherin-mediated intercellular barrier and facilitating paracellular absorption of BoNT/A. We reconstituted the complete 14-subunit BoNT/A complex using recombinantly produced components and demonstrated that abolishing either E-cadherin- or carbohydrate-binding of the HA complex drastically reduces oral toxicity of BoNT/A complex in vivo. Together, these studies establish the molecular mechanism of how HAs contribute to the oral toxicity of BoNT/A. Copyright © 2014, American Association for the Advancement of Science.

Hydroxyapatite Coating on TiO₂ Nanotube by Sol-Gel Method for Implant Applications.

PubMed

Lim, Hyun-Pil; Park, Sang-Won; Yun, Kwi-Dug; Park, Chan; Ji, Min-Kyung; Oh, Gye-Jeong; Lee, Jong-Tak; Lee, Kwangmin

2018-02-01

The aim of this study was to determine the effect of hydroxyapatite (HA) coating on titanium dioxide (TiO2) nanotube by sol-gel process on viability of osteoblast like cell (MC3T3-E1) and bone formation in rat tibia. Specimens were divided into three groups including commercially pure titanium (control group), TiO2 nanotubes (group N), and HA coated TiO2 nanotubes (group HN). Surface characteristics were determined using field emission scanning electron microscope (FE-SEM; S-4700, Hitachi, Japan) and contact angles were measured. Cell viability was investigated in vitro after 1 day, 3 days, and 7 days of incubation. Implants (2.0 mm in diameter and 5.0 mm in length) were inserted into the tibia of rats. After 4 weeks, histomorphometric analysis was performed. Both N and HN groups showed enhanced hydrophilicity compared to control group. After 7 days of implantation, group HN showed higher cell viability with marginal significance (0.05 < P < 0.1). Bone to implant contact (BIC) ratio in the control group, group N, and group HN were 32.5%, 33.1%, and 43.8%, respectively. Results of this study showed that HA coated TiO2 nanotube using sol-gel process could be used to enhance hydrophilicity and improve osseointegration of dental implant surface.

HVOF-Sprayed Nano TiO2-HA Coatings Exhibiting Enhanced Biocompatibility

NASA Astrophysics Data System (ADS)

Lima, R. S.; Dimitrievska, S.; Bureau, M. N.; Marple, B. R.; Petit, A.; Mwale, F.; Antoniou, J.

2010-01-01

Biomedical thermal spray coatings produced via high-velocity oxy-fuel (HVOF) from nanostructured titania (n-TiO2) and 10 wt.% hydroxyapatite (HA) (n-TiO2-10wt.%HA) powders have been engineered as possible future alternatives to HA coatings deposited via air plasma spray (APS). This approach was chosen due to (i) the stability of TiO2 in the human body (i.e., no dissolution) and (ii) bond strength values on Ti-6Al-4V substrates more than two times higher than those of APS HA coatings. To explore the bioperformance of these novel materials and coatings, human mesenchymal stem cells (hMSCs) were cultured from 1 to 21 days on the surface of HVOF-sprayed n-TiO2 and n-TiO2-10 wt.%HA coatings. APS HA coatings and uncoated Ti-6Al-4V substrates were employed as controls. The profiles of the hMSCs were evaluated for (i) cellular proliferation, (ii) biochemical analysis of alkaline phosphatase (ALP) activity, (iii) cytoskeleton organization (fluorescent/confocal microscopy), and (iv) cell/substrate interaction via scanning electron microscopy (SEM). The biochemical analysis indicated that the hMSCs cultured on n-TiO2-10 wt.%HA coatings exhibited superior levels of bioactivity than hMSCs cultured on APS HA and pure n-TiO2 coatings. The cytoskeleton organization demonstrated a higher degree of cellular proliferation on the HVOF-sprayed n-TiO2-10wt.%HA coatings when compared to the control coatings. These results are considered promising for engineering improved performance in the next generation of thermally sprayed biomedical coatings.

Nano-Ag-loaded hydroxyapatite coatings on titanium surfaces by electrochemical deposition

PubMed Central

Lu, Xiong; Zhang, Bailin; Wang, Yingbo; Zhou, Xianli; Weng, Jie; Qu, Shuxin; Feng, Bo; Watari, Fumio; Ding, Yonghui; Leng, Yang

2011-01-01

Hydroxyapatite (HA) coatings on titanium (Ti) substrates have attracted much attention owing to the combination of good mechanical properties of Ti and superior biocompatibility of HA. Incorporating silver (Ag) into HA coatings is an effective method to impart the coatings with antibacterial properties. However, the uniform distribution of Ag is still a challenge and Ag particles in the coatings are easy to agglomerate, which in turn affects the applications of the coatings. In this study, we employed pulsed electrochemical deposition to co-deposit HA and Ag simultaneously, which realized the uniform distribution of Ag particles in the coatings. This method was based on the use of a well-designed electrolyte containing Ag ions, calcium ions and l-cysteine, in which cysteine acted as the coordination agent to stabilize Ag ions. The antibacterial and cell culture tests were used to evaluate the antibacterial properties and biocompatibility of HA/Ag composite coatings, respectively. The results indicated the as-prepared coatings had good antibacterial properties and biocompatibility. However, an appropriate silver content should be chosen to balance the biocompatibility and antibacterial properties. Heat treatments promoted the adhesive strength and enhanced the biocompatibility without sacrificing the antibacterial properties of the HA/Ag coatings. In summary, this study provided an alternative method to prepare bioactive surfaces with bactericidal ability for biomedical devices. PMID:20880853

Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells.

PubMed

Veiseh, Mandana; Leith, Sean J; Tolg, Cornelia; Elhayek, Sallie S; Bahrami, S Bahram; Collis, Lisa; Hamilton, Sara; McCarthy, James B; Bissell, Mina J; Turley, Eva

2015-01-01

The interaction of hyaluronan (HA) with mesenchymal progenitor cells impacts trafficking and fate after tissue colonization during wound repair and these events contribute to diseases such as cancer. How this interaction occurs is poorly understood. Using 10T½ cells as a mesenchymal progenitor model and fluorescent (F-HA) or gold-labeled HA (G-HA) polymers, we studied the role of two HA receptors, RHAMM and CD44, in HA binding and uptake in non-adherent and adherent mesenchymal progenitor (10T½) cells to mimic aspects of cell trafficking and tissue colonization. We show that fluorescent labeled HA (F-HA) binding/uptake was high in non-adherent cells but dropped over time as cells became increasingly adherent. Non-adherent cells displayed both CD44 and RHAMM but only function-blocking anti-RHAMM and not anti-CD44 antibodies significantly reduced F-HA binding/uptake. Adherent cells, which also expressed CD44 and RHAMM, primarily utilized CD44 to bind to F-HA since anti-CD44 but not anti-RHAMM antibodies blocked F-HA uptake. RHAMM overexpression in adherent 10T½ cells led to increased F-HA uptake but this increased binding remained CD44 dependent. Further studies showed that RHAMM-transfection increased CD44 mRNA and protein expression while blocking RHAMM function reduced expression. Collectively, these results suggest that cellular microenvironments in which these receptors function as HA binding proteins differ significantly, and that RHAMM plays at least two roles in F-HA binding by acting as an HA receptor in non-attached cells and by regulating CD44 expression and display in attached cells. Our findings demonstrate adhesion-dependent mechanisms governing HA binding/ uptake that may impact development of new mesenchymal cell-based therapies.

Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

PubMed Central

Veiseh, Mandana; Leith, Sean J.; Tolg, Cornelia; Elhayek, Sallie S.; Bahrami, S. Bahram; Collis, Lisa; Hamilton, Sara; McCarthy, James B.; Bissell, Mina J.; Turley, Eva

2015-01-01

The interaction of hyaluronan (HA) with mesenchymal progenitor cells impacts trafficking and fate after tissue colonization during wound repair and these events contribute to diseases such as cancer. How this interaction occurs is poorly understood. Using 10T½ cells as a mesenchymal progenitor model and fluorescent (F-HA) or gold-labeled HA (G-HA) polymers, we studied the role of two HA receptors, RHAMM and CD44, in HA binding and uptake in non-adherent and adherent mesenchymal progenitor (10T½) cells to mimic aspects of cell trafficking and tissue colonization. We show that fluorescent labeled HA (F-HA) binding/uptake was high in non-adherent cells but dropped over time as cells became increasingly adherent. Non-adherent cells displayed both CD44 and RHAMM but only function-blocking anti-RHAMM and not anti-CD44 antibodies significantly reduced F-HA binding/uptake. Adherent cells, which also expressed CD44 and RHAMM, primarily utilized CD44 to bind to F-HA since anti-CD44 but not anti-RHAMM antibodies blocked F-HA uptake. RHAMM overexpression in adherent 10T½ cells led to increased F-HA uptake but this increased binding remained CD44 dependent. Further studies showed that RHAMM-transfection increased CD44 mRNA and protein expression while blocking RHAMM function reduced expression. Collectively, these results suggest that cellular microenvironments in which these receptors function as HA binding proteins differ significantly, and that RHAMM plays at least two roles in F-HA binding by acting as an HA receptor in non-attached cells and by regulating CD44 expression and display in attached cells. Our findings demonstrate adhesion-dependent mechanisms governing HA binding/ uptake that may impact development of new mesenchymal cell-based therapies. PMID:26528478

Calcium phosphate coating on titanium using laser and plasma spray

NASA Astrophysics Data System (ADS)

Roy, Mangal

Though calcium phosphate (CaP) coated implants are commercially available, its acceptance is still not wide spread due to challenges related to weaker interfacial bonding between metal and ceramic, and low crystallinity of hydroxyapatite (HA). The objectives of this research are to improve interfacial strength, crystallinity, phase purity and bioactivity of CaP coated metallic implants for orthopaedic applications. The rationale is that forming a diffuse and gradient metal-ceramic interface will improve the interfacial strength. Moreover, reducing CaP particles exposure to high temperature during coating preparation, can lead to improvement in both crystallinity and phase purity of CaP. In this study, laser engineered net shaping (LENS(TM)) was used to coat Ti metal with CaP. LENS(TM) processing enabled generation of Ti+TCP (tricalcium phosphate) composite coating with diffused interface, that also increased the coating hardness to 1049+/-112 Hv compared to a substrate hardness of 200+/-15 Hv. In vitro bone cell-material interaction studies confirmed the bioactivity of TCP coatings. Antimicrobial properties of the TCP coatings were improved by silver (Ag) electrodeposition. Along with LENS(TM), radio frequency induction plasma spray, equipped with supersonic plasma nozzle, was used to prepare HA coatings on Ti with improved crystallinity and phase purity. The coating was made of multigrain HA particles of ˜200 nm in size, which consisted of 15--20 nm HA grains. In vitro bone cell-material interaction and in vivo rat model studies confirmed the HA coatings to be bioactive. Furthermore, incorporation of Sr2+ improved bone cell of HA coatings interaction. A combination of LENS(TM) and plasma spray was used to fabricate a compositionally graded HA coatings on Ti where the microstructure varied from pure HA at the surface to pure Ti substrate with a diffused Ti+TCP composite region in between. The plasma spray system was used to synthesize spherical HA nano powder from HA sol, where the production rate was 20 g/h, which is only 16% of the total powder produced. The effects of Sr2+ and Mg2+ doping on bone cell-CaP interaction was further studied with osteoclast cells. Mg2+ doing was found to be an effective way of controlling osteoclast differentiation.

Nanoassemblies from amphiphilic cytarabine prodrug for leukemia targeted therapy.

PubMed

Liu, Jing; Zhao, Dujuan; He, Wenxiu; Zhang, Huiyuan; Li, Zhonghao; Luan, Yuxia

2017-02-01

The anti-leukemia effect of cytarabine (Ara-C) is severely restricted by its high hydrophilic properties and rapid plasma degradation. Herein, a novel amphiphilic small molecular prodrug of Ara-C was developed by coupling a short aliphatic chain, hexanoic acid (HA) to 4-NH 2 of the parent drug. Based on the amphiphilic nature, the resulting bioconjugate (HA-Ara) could spontaneously self-assemble into stable spherical nanoassemblies (NAs) with an extremely high drug loading (∼71wt%). Moreover, folate receptor (FR)-targeting NAs with high grafting efficient folic acid - bovine serum albumin (FA-BSA) conjugate immobilized on the surface (NAs/FA-BSA) was prepared. The results of MTT assays on FR-positive K562 cells and FR-negative A549 cells demonstrated higher cytotoxicity of HA-Ara NAs than the native drug. Especially, the IC 50 values revealed that NAs/FA-BSA was 3 and 2-fold effective than non-targeted NAs after 24 and 48h treatment with K562 cells, respectively indicating FR-mediated enhanced anti-tumor efficacy. In vitro cellular uptake, larger accumulation of HA-Ara NAs were observed in comparative with the free FITC and the results further confirmed the selective uptake of NAs/FA-BSA in folate receptor enriched cancer cells. Above all, self-assembled HA-Ara NAs exhibited potential superiority for Ara-C delivery and FA-modified NAs would be an excellent candidate for targeting leukemia therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

Hyaluronic acid-functionalized polymeric nanoparticles for colon cancer-targeted combination chemotherapy

NASA Astrophysics Data System (ADS)

Xiao, Bo; Han, Moon Kwon; Viennois, Emilie; Wang, Lixin; Zhang, Mingzhen; Si, Xiaoying; Merlin, Didier

2015-10-01

Nanoparticle (NP)-based combination chemotherapy has been proposed as an effective strategy for achieving synergistic effects and targeted drug delivery for colon cancer therapy. Here, we fabricated a series of hyaluronic acid (HA)-functionalized camptothecin (CPT)/curcumin (CUR)-loaded polymeric NPs (HA-CPT/CUR-NPs) with various weight ratios of CPT to CUR (1 : 1, 2 : 1 and 4 : 1). The resultant spherical HA-CPT/CUR-NPs had a desirable particle size (around 289 nm), relative narrow size distribution, and slightly negative zeta potential. These NPs exhibited a simultaneous sustained release profile for both drugs throughout the time frame examined. Subsequent cellular uptake experiments demonstrated that the introduction of HA to the NP surface endowed NPs with colon cancer-targeting capability and markedly increased cellular uptake efficiency compared with chitosan-coated NPs. Importantly, the combined delivery of CPT and CUR in one HA-functionalized NP exerted strong synergistic effects. HA-CPT/CUR-NP (1 : 1) showed the highest antitumor activity among the three HA-CPT/CUR-NPs, resulting in an extremely low combination index. Collectively, our findings indicate that this HA-CPT/CUR-NP can be exploited as an efficient formulation for colon cancer-targeted combination chemotherapy.Nanoparticle (NP)-based combination chemotherapy has been proposed as an effective strategy for achieving synergistic effects and targeted drug delivery for colon cancer therapy. Here, we fabricated a series of hyaluronic acid (HA)-functionalized camptothecin (CPT)/curcumin (CUR)-loaded polymeric NPs (HA-CPT/CUR-NPs) with various weight ratios of CPT to CUR (1 : 1, 2 : 1 and 4 : 1). The resultant spherical HA-CPT/CUR-NPs had a desirable particle size (around 289 nm), relative narrow size distribution, and slightly negative zeta potential. These NPs exhibited a simultaneous sustained release profile for both drugs throughout the time frame examined. Subsequent cellular uptake experiments demonstrated that the introduction of HA to the NP surface endowed NPs with colon cancer-targeting capability and markedly increased cellular uptake efficiency compared with chitosan-coated NPs. Importantly, the combined delivery of CPT and CUR in one HA-functionalized NP exerted strong synergistic effects. HA-CPT/CUR-NP (1 : 1) showed the highest antitumor activity among the three HA-CPT/CUR-NPs, resulting in an extremely low combination index. Collectively, our findings indicate that this HA-CPT/CUR-NP can be exploited as an efficient formulation for colon cancer-targeted combination chemotherapy. Electronic supplementary information (ESI) available: Representative flow cytometry plots of cells incubated with or without cationic CPT/CUR-NPs (1 : 1) for 3 h; Cytotoxicity of blank chitosan-coated NPs and blank HA-functionalized NPs at different concentrations against Colon-26 cells after 48 h of co-incubation. See DOI: 10.1039/c5nr04831a

Antifungal activity of Ag:hydroxyapatite thin films synthesized by pulsed laser deposition on Ti and Ti modified by TiO2 nanotubes substrates

NASA Astrophysics Data System (ADS)

Eraković, S.; Janković, A.; Ristoscu, C.; Duta, L.; Serban, N.; Visan, A.; Mihailescu, I. N.; Stan, G. E.; Socol, M.; Iordache, O.; Dumitrescu, I.; Luculescu, C. R.; Janaćković, Dj.; Miškovic-Stanković, V.

2014-02-01

Hydroxyapatite (HA) is a widely used biomaterial for implant thin films, largely recognized for its excellent capability to chemically bond to hard tissue inducing the osteogenesis without immune response from human tissues. Nowadays, intense research efforts are focused on development of antimicrobial HA doped thin films. In particular, HA doped with Ag (Ag:HA) is expected to inhibit the attachment of microbes and contamination of metallic implant surface. We herewith report on nano-sized HA and Ag:HA thin films synthesized by pulsed laser deposition on pure Ti and Ti modified with 100 nm diameter TiO2 nanotubes (fabricated by anodization of Ti plates) substrates. The HA-based thin films were characterized by SEM, AFM, EDS, FTIR, and XRD. The cytotoxic activity was tested with HEp2 cells against controls. The antifungal efficiency of the deposited layers was tested against the Candida albicans and Aspergillus niger strains. The Ti substrates modified with TiO2 nanotubes covered with Ag:HA thin films showed the highest antifungal activity.

A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: co-expression of two influenza virus genes in insect cells.

PubMed

Weyer, U; Possee, R D

1991-12-01

A baculovirus transfer vector, pAcUW3, was developed to facilitate the insertion of two influenza virus genes, those encoding the haemagglutinin (HA) and neuraminidase (NA) membrane glycoproteins, into the Autographa californica nuclear polyhedrosis virus genome in a single cotransfection experiment. The NA gene was inserted in place of the polyhedrin coding sequences under the control of the polyhedrin promoter, whereas the HA gene was placed under the control of a copy of the p10 promoter at a site upstream of and in opposite orientation to the polyhedrin promoter. After infection of Spodoptera frugiperda cells with the recombinant virus, AcUW3HANA, both HA and NA were expressed in the very late phase of infection and were shown to be functional in appropriate assays. Immunofluorescence assays demonstrated their localization at the surface of infected insect cells. The expression of both foreign genes in the recombinant virus was found to be stable for at least 12 passages in cell culture.

3D microenvironment as essential element for osteoinduction by biomaterials.

PubMed

Habibovic, Pamela; Yuan, Huipin; van der Valk, Chantal M; Meijer, Gert; van Blitterswijk, Clemens A; de Groot, Klaas

2005-06-01

In order to unravel the mechanism of osteoinduction by biomaterials, in this study we investigated the influence of the specific surface area on osteoinductive properties of two types of calcium phosphate ceramics. Different surface areas of the ceramics were obtained by varying their sintering temperatures. Hydroxyapatite (HA) ceramic was sintered at 1150 and 1250 degrees C. Biphasic calcium phosphate (BCP) ceramic, consisting of HA and beta-tricalcium phosphate (beta-TCP), was sintered at 1100, 1150 and 1200 degrees C. Changes in sintering temperature did not influence the chemistry of the ceramics; HA remained pure after sintering at different temperatures and the weight ratio of HA and beta-TCP in the BCP was independent of the temperature as well. Similarly, macroporosity of the ceramics was unaffected by the changes of the sintering temperature. However, microporosity (pore diameter <10 microm) significantly decreased with increasing sintering temperature. In addition to the decrease of the microporosity, the crystal size increased with increasing sintering temperature. These two effects resulted in a significant decrease of the specific surface area of the ceramics with increasing sintering temperatures. Samples of HA1150, HA1250, BCP1100, BCP1150 and BCP1200 were implanted in the back muscles of Dutch milk goats and harvested at 6 and 12 weeks post implantation. After explantation, histomorphometrical analysis was performed on all implants. All implanted materials except HA1250 induced bone. However, large variations in the amounts of induced bone were observed between different materials and between individual animals. Histomorphometrical results showed that the presence of micropores within macropore walls is necessary to make a material osteoinductive. We postulate that introduction of microporosity within macropores, and consequent increase of the specific surface area, affects the interface dynamics of the ceramic in such a way that relevant cells are triggered to differentiate into the osteogenic lineage.

Targeted delivery of hyaluronic acid to the ocular surface by a polymer-peptide conjugate system for dry eye disease.

PubMed

Lee, David; Lu, Qiaozhi; Sommerfeld, Sven D; Chan, Amanda; Menon, Nikhil G; Schmidt, Tannin A; Elisseeff, Jennifer H; Singh, Anirudha

2017-06-01

Hyaluronic acid (HA) solutions effectively lubricate the ocular surface and are used for the relief of dry eye related symptoms. However, HA undergoes rapid clearance due to limited adhesion, which necessitates frequent instillation. Conversely, highly viscous artificial tear formulations with HA blur vision and interfere with blinking. Here, we developed an HA-eye drop formulation that selectively binds and retains HA for extended periods of time on the ocular surface. We synthesized a heterobifunctional polymer-peptide system with one end binding HA while the other end binding either sialic acid-containing glycosylated transmembrane molecules on the ocular surface epithelium, or type I collagen molecule within the tissue matrix. HA solution was mixed with the polymer-peptide system and tested on both ex vivo and in vivo models to determine its ability to prolong HA retention. Furthermore, rabbit ocular surface tissues treated with binding peptides and HA solutions demonstrated superior lubrication with reduced kinetic friction coefficients compared to tissues treated with conventional HA solution. The results suggest that binding peptide-based solution can keep the ocular surface enriched with HA for prolonged times as well as keep it lubricated. Therefore, this system can be further developed into a more effective treatment for dry eye patients than a standard HA eye drop. Eye drop formulations containing HA are widely used to lubricate the ocular surface and relieve dry eye related symptoms, however its low residence time remains a challenge. We designed a polymer-peptide system for the targeted delivery of HA to the ocular surface using sialic acid or type I collagen as anchors for HA immobilization. The addition of the polymer-peptide system to HA eye drop exhibited a reduced friction coefficient, and it can keep the ocular surface enriched with HA for prolonged time. This system can be further developed into a more effective treatment for dry eye than a standard HA eye drop. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Hyaluronic acid influence on platelet-induced airway smooth muscle cell proliferation.

PubMed

Svensson Holm, Ann-Charlotte B; Bengtsson, Torbjörn; Grenegård, Magnus; Lindström, Eva G

2012-03-10

Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling. Copyright © 2011 Elsevier Inc. All rights reserved.

Nano-hydroxyapatite/chitosan-starch nanocomposite as a novel bone construct: Synthesis and in vitro studies.

PubMed

Shakir, Mohammad; Jolly, Reshma; Khan, Mohd Shoeb; Iram, Noor e; Khan, Haris M

2015-09-01

A novel ternary nanocomposite system incorporating hydroxyapatite, chitosan and starch (n-HA/CS-ST) has been synthesized by co-precipitation method at room temperature, addressing the issues of biocompatibility, mechanical strength and cytotoxicity required for bone tissue engineering. The interactions, crystallite size, surface morphology and thermal stability against n-HA/CS nanocomposite have been obtained by comparing the results of FTIR, SEM, TEM, DLS, XRD and TGA/DTA. A comparative study of bioactivity and thermal stability of n-HA/CS and n-HA/CS-ST nanocomposites revealed that the incorporation of starch as templating agent enhanced these properties in n-HA/CS-ST nanocomposite. A lower swelling rate of n-HA/CS-ST relative to n-HA/CS indicates a higher mechanical strength supportive of bone tissue ingrowths. The MTT assay on murine fibroblast L929 and human osteoblasts-like MG-63 cells and in vitro bioactivity of n-HA/CS-ST matrix referred superior non-toxic nature of n-HA/CS-ST nanocomposite and greater possibility of osteointegration in vivo respectively. Furthermore n-HA/CS-ST exhibited improved antibacterial property against both Gram-positive and Gram-negative bacteria relative to n-HA/CS. Copyright © 2015. Published by Elsevier B.V.

Differentiation potential of human adipose stem cells bioprinted with hyaluronic acid/gelatin-based bioink through microextrusion and visible light-initiated crosslinking.

PubMed

Sakai, Shinji; Ohi, Hiromi; Hotta, Tomoki; Kamei, Hidenori; Taya, Masahito

2018-02-01

Bioprinting has a great potential to fabricate three-dimensional (3D) functional tissues and organs. In particular, the technique enables fabrication of 3D constructs containing stem cells while maintaining cell proliferation and differentiation abilities, which is believed to be promising in the fields of tissue engineering and regenerative medicine. We aimed to demonstrate the utility of the bioprinting technique to create hydrogel constructs consisting of hyaluronic acid (HA) and gelatin derivatives through irradiation by visible light to fabricate 3D constructs containing human adipose stem cells (hADSCs). The hydrogel was obtained from a solution of HA and gelatin derivatives possessing phenolic hydroxyl moieties in the presence of ruthenium(II) tris-bipyridyl dication and sodium ammonium persulfate. hADSCs enclosed in the bioprinted hydrogel construct elongated and proliferated in the hydrogel. In addition, their differentiation potential was confirmed by examining the expression of pluripotency marker genes and cell surface marker proteins, and differentiation to adipocytes in adipogenic differentiation medium. Our results demonstrate the great potential of the bioprinting method and the resultant hADSC-laden HA/gelatin constructs for applications in tissue engineering and regenerative medicine. © 2017 Wiley Periodicals, Inc.

Electrophoretic-deposited novel ternary silk fibroin/graphene oxide/hydroxyapatite nanocomposite coatings on titanium substrate for orthopedic applications

NASA Astrophysics Data System (ADS)

Li, Ming; Xiong, Pan; Mo, Maosong; Cheng, Yan; Zheng, Yufeng

2016-09-01

The combination of graphene oxide (GO) with robust mechanical property, silk fibroin (SF) with fascinating biological effects and hydroxyapatite (HA) with superior osteogenic activity is a competitive approach to make novel coatings for orthopedic applications. Herein, the feasibility of depositing ternary SF/GO/HA nanocomposite coatings on Ti substrate was firstly verified by exploiting electrophoretic nanotechnology, with SF being used as both a charging additive and a dispersion agent. The surface morphology, microstructure and composition, in vitro hemocompatibility and in vitro cytocompatibility of the resulting coatings were investigated by SEM, Raman, FTIR spectra and biocompatibility tests. Results demonstrated that GO, HA and SF could be co-deposited with a uniform, smooth thin-film morphology. The hemolysis rate analysis and the platelet adhesion test indicated good blood compatibility of the coatings. The human osteosarcoma MG63 cells displayed well adhesion and proliferation behaviors on the prepared coatings, with enhanced ALP activities. The present study suggested that SF/GO/HA nanocomposite coatings could be a promising candidate for the surface functionalization of biomaterials, especially as orthopedic implant coating.

Surface modification of PLGA nanoparticles by carbopol to enhance mucoadhesion and cell internalization.

PubMed

Surassmo, Suvimol; Saengkrit, Nattika; Ruktanonchai, Uracha Rungsardthong; Suktham, Kunat; Woramongkolchai, Noppawan; Wutikhun, Tuksadon; Puttipipatkhachorn, Satit

2015-06-01

Mucoadhesive poly (lactic-co-glycolic acid) (PLGA) nanoparticles having a modified shell-matrix derived from polyvinyl alcohol (PVA) and Carbopol (CP), a biodegradable polymer coating, to improve the adhesion and cell transfection properties were developed. The optimum formulations utilized a CP concentration in the range of 0.05-0.2%w/v, and were formed using modified emulsion-solvent evaporation technique. The resulting CP-PLGA nanoparticles were characterized in terms of their physical and chemical properties. The absorbed CP on the PLGA shell-matrix was found to affect the particle size and surface charge, with 0.05% CP giving rise to smooth spherical particles (0.05CP-PLGA) with the smallest size (285.90 nm), and strong negative surface charge (-25.70 mV). The introduction of CP results in an enhancement of the mucoadhesion between CP-PLGA nanoparticles and mucin particles. In vitro cell internalization studies highlighted the potential of 0.05CP-PLGA nanoparticles for transfection into SiHa cells, with uptake being time dependent. Additionally, cytotoxicity studies of CP-PLGA nanoparticles against SiHa cancer cells indicated that low concentrations of the nanoparticles were non-toxic to cells (cell viability >80%). From the various formulations studied, 0.05CP-PLGA nanoparticles proved to be the optimum model carrier having the required mucoadhesive profile and could be an alternative therapeutic efficacy carrier for targeted mucosal drug delivery systems with biodegradable polymer. Copyright © 2015 Elsevier B.V. All rights reserved.

Matrix-M™ adjuvant enhances immunogenicity of both protein- and modified vaccinia virus Ankara-based influenza vaccines in mice.

PubMed

Magnusson, Sofia E; Altenburg, Arwen F; Bengtsson, Karin Lövgren; Bosman, Fons; de Vries, Rory D; Rimmelzwaan, Guus F; Stertman, Linda

2018-04-01

Influenza viruses continuously circulate in the human population and escape recognition by virus neutralizing antibodies induced by prior infection or vaccination through accumulation of mutations in the surface proteins hemagglutinin (HA) and neuraminidase (NA). Various strategies to develop a vaccine that provides broad protection against different influenza A viruses are under investigation, including use of recombinant (r) viral vectors and adjuvants. The replication-deficient modified vaccinia virus Ankara (MVA) is a promising vaccine vector that efficiently induces B and T cell responses specific for the antigen of interest. It is assumed that live vaccine vectors do not require an adjuvant to be immunogenic as the vector already mediates recruitment and activation of immune cells. To address this topic, BALB/c mice were vaccinated with either protein- or rMVA-based HA influenza vaccines, formulated with or without the saponin-based Matrix-M™ adjuvant. Co-formulation with Matrix-M significantly increased HA vaccine immunogenicity, resulting in antigen-specific humoral and cellular immune responses comparable to those induced by unadjuvanted rMVA-HA. Of special interest, rMVA-HA immunogenicity was also enhanced by addition of Matrix-M, demonstrated by enhanced HA inhibition antibody titres and cellular immune responses. Matrix-M added to either protein- or rMVA-based HA vaccines mediated recruitment and activation of antigen-presenting cells and lymphocytes to the draining lymph node 24 and 48 h post-vaccination. Taken together, these results suggest that adjuvants can be used not only with protein-based vaccines but also in combination with rMVA to increase vaccine immunogenicity, which may be a step forward to generate new and more effective influenza vaccines.

Affinity of the interface between hydroxyapatite (0001) and titanium (0001) surfaces: a first-principles investigation.

PubMed

Sun, Jin P; Dai, Jianhong; Song, Yan; Wang, You; Yang, Rui

2014-12-10

A basic understanding of the affinity between the hydroxyapatite (HA) and α-Ti surfaces is obtained through electronic structure calculations by first-principles method. The surface energies of HA(0001), HA (011̅0), HA (101̅1), and Ti(0001) surfaces have been calculated. The HA(0001) presents the most thermodynamically stable of HA. The HA/Ti interfaces were constructed by two kinds of interface models, the single interface (denoted as SI) and the double-interface (denoted as DI). Two methods, the full relaxation and the UBER, were applied to determine the interfacial separation and the atomic arrangement in the interfacial zone. The works of adhesion of interfaces with various stoichiometric HA surfaces were evaluated. For the HA(0001)/Ti(0001) interfaces, the work of adhesion is strongly dependent on the chemical environment of the HA surface. The values are -2.33, -1.52, and -0.80 J/m(2) for the none-, single-, and double-Ca terminated HA/Ti interfaces, respectively. The influence of atomic relaxation on the work of adhesion and interface separation is discussed. Full relaxation results include -1.99 J/m(2) work of adhesion and 0.220 nm separation between HA and Ti for the DI of 1-Ca-HA/Ti interface, while they are -1.14 J/m(2) and 0.235 nm by partial relaxation. Analysis of electronic structure reveals that charge transfer between HA and Ti slabs occurs during the formation of the HA/Ti interface. The transfer generates the Ti-O or Ti-Ca bonds across the interface and drives the HA/Ti interface system to metallic characteristic. The energetically favorable interfaces are formed when the outmost layer of HA comprises more O atoms at the interface.

Biofunctionalization of PAMAM-montmorillonite decorated poly (Ɛ-caprolactone)-chitosan electrospun nanofibers for cell adhesion and electrochemical cytosensing.

PubMed

Kirbay, Fatma Ozturk; Yalcinkaya, Esra Evrim; Atik, Gozde; Evren, Gizem; Unal, Betul; Demirkol, Dilek Odaci; Timur, Suna

2018-06-30

The construction and biofunctionalization of the poly (Ɛ-caprolactone) (PCL)-chitosan (CHIT) nanofibrous mats, which included Polyamidoamine (PAMAM) dendrimer modified montmorillonite (Mt), for the cell adhesion and electrochemical cytosensing were accomplished in this report. After the intercalation of the PAMAM generation zero dendrimer into the Mt, PAMAM-Mt decorated PCL-CHIT electrospun nanofibers were formed. The addition of PAMAM caused the decrease of contact angle of PCL-CHIT nanofibers. The covalent immobilization of a tripeptide namely Arginylglycylaspartate (RGD) on both the PCL-CHIT/Mt and PCL-CHIT/PAMAM-Mt surface was carried out. U87-MG and HaCaT (negative control) cell lines were incubated on the PCL-CHIT/Mt/RGD and PCL-CHIT/PAMAM-Mt/RGD. The proliferation studies and imaging of the cells were carried out on these fibers. Finally, electrochemical measurements were performed after each modification step by differential pulse/cyclic voltammetry and electrochemical impedance spectroscopy. U87-MG cells were grown better than HaCaT cells on the PCL-CHIT/PAMAM-Mt/RGD surfaces. To the best of our knowledge, there is no study that developed electrochemical cytosensor using electrospun nanofibers as a cell adhesion platform. Copyright © 2018 Elsevier B.V. All rights reserved.

Preparation of micro-porous bioceramic containing silicon-substituted hydroxyapatite and beta-tricalcium phosphate.

PubMed

Fuh, Lih-Jyh; Huang, Ya-Jing; Chen, Wen-Cheng; Lin, Dan-Jae

2017-06-01

Dimensional instability caused by sintering shrinkage is an inevitable drawback for conventional processing of hydroxyapatite (HA). A new preparation method for biphasic calcium phosphates was developed to increase micro pores and biodegradation without significant dimensional change. Powder pressed HA discs, under 100MPa, were immersed in a colloidal mixture of tetraethoxysilane (TEOS) and ammonium hydroxide for 10min, followed by drying, and then were sintered at 900°C, 1050°C, and 1200°C, respectively. Comparing with pure HA discs, the newly prepared product sintered up to 1200°C contained silicon substituted HA, beta-tricalcium phosphate, and calcium silicate with better micro-porosity, high specific surface area, less sintering shrinkage and the strength maintained. The cytocompatibility test demonstrated a better viability for D1 mice stem cells cultured on TEOS treated HA for 14days compared to the pure HA. This simple TEOS sol-gel pretreatment has the potential to be applied to any existing manufacturing process of HA scaffold for better control of sintering shrinkage, create micropores, and increase biodegradation. Copyright © 2017 Elsevier B.V. All rights reserved.

Sol-Gel Derived Hydroxyapatite Coating on Mg-3Zn Alloy for Orthopedic Application

NASA Astrophysics Data System (ADS)

Singh, Sanjay; Manoj Kumar, R.; Kuntal, Kishor Kumar; Gupta, Pallavi; Das, Snehashish; Jayaganthan, R.; Roy, Partha; Lahiri, Debrupa

2015-04-01

In recent years, magnesium and its alloys have gained a lot of interest as orthopedic implant constituents because their biodegradability and mechanical properties are closer to that of human bone. However, one major concern with Mg in orthopedics is its high corrosion rate that results in the reduction of mechanical integrity before healing the bone tissue. The current study evaluates the sol-gel-derived hydroxyapatite (HA) coating on a selected Mg alloy (Mg-3Zn) for decreasing the corrosion rate and increasing the bioactivity of the Mg surface. The mechanical integrity of the coating is established as a function of the surface roughness of the substrate and the sintering temperature of the coating. Coating on a substrate roughness of 15-20 nm and sintering at 400°C shows the mechanical properties in similar range of bone, thus making it suitable to avoid the stress-shielding effect. The hydroxyapatite coating on the Mg alloy surface also increases corrosion resistance very significantly by 40 times. Bone cells are also found proliferating better in the HA-coated surface. All these benefits together establish the candidature of sol-gel HA-coated Mg-3Zn alloy in orthopedic application.

Cytotoxicity of solid lipid nanoparticles and nanostructured lipid carriers containing the local anesthetic dibucaine designed for topical application

NASA Astrophysics Data System (ADS)

Barbosa, R. M.; da Silva, C. M. G.; Bella, T. S.; de Araújo, D. R.; Marcato, P. D.; Durán, N.; de Paula, E.

2013-04-01

Dibucaine (DBC) is powerful long-lasting local anesthetic, but it is also considered fairly toxic to the CNS. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have attracted attention as carriers for drug delivery. The aim of this study was to develop and to evaluate the cytotoxic activity of DBC-loaded SLN and NLC against 3T3 fibroblast and HaCat keratinocyte cells. The SLN and NLC had myristyl myristate and Liponate®GC as their lipid matrices, respectively, plus a surfactant. SLN and NLC were characterized in terms in their diameter, size distribution, surface charge and DBC encapsulation efficiency. The particle size of SLN and NLC were around 234.33 and 166.62 nm, respectively. The polydispersity index was kept below 0.2 for both nanomaterials. Negative surface charges were observed for both nanoparticles, which decreased in the presence of the anesthetic. Encapsulation efficiency reached 76% and 90%, respectively, in SLN and NLC. DBC alone was found to be toxic to 3T3 and HaCat cells in culture. However, NLC and SLN loaded DBC decreased its intrinsic cytotoxic effect against 3T3 and HaCat cells. In conclusion, encapsulation of DBC in SLN and NLC decreased the in vitro toxicity of the local anesthetic, indicating the potential of these nanocarriers for clinical applications.

Hyaluronan/Tween 80-assisted synthesis of silver nanoparticles for biological application

NASA Astrophysics Data System (ADS)

Li, Hui-Jun; Zhang, An-Qi; Sui, Li; Qian, Dong-Jin; Chen, Meng

2015-02-01

Water-soluble and well-stabilized silver nanoparticles (NPs) of small size have been synthesized using hyaluronan (HA) and Tween 80 as reducing and stabilizing agents. The effect of reaction conditions on the formation process of silver NPs was studied, and an aggregative growth mechanism of the silver NPs dominated in HA/Tween 80 system at pH 12 has been proposed. The obtained Ag NPs were characterized by UV-Vis spectroscopy, transmission electron microscopy, X-ray powder diffraction, and X-ray photoelectron spectroscopy. Moreover, the stability of the HA-Tween 80-silver NPs in normal saline was also studied, and a flexible blend membrane containing chitosan, gelatin, and the HA-Tween 80-silver NPs was prepared for further biological applications. Due to the high specific surface area and improved stability of silver NPs, the chitosan-gelatin-silver membrane has shown high antibacterial activity for strains of Escherichia coli. The cell viability tests indicate that the polymer membrane is non-cytotoxic to HepG2 cells, which might be attributed to its good biocompatibility.

Synthesis and characterization of strontium-substituted hydroxyapatite nanoparticles for bone regeneration.

PubMed

Frasnelli, Matteo; Cristofaro, Francesco; Sglavo, Vincenzo M; Dirè, Sandra; Callone, Emanuela; Ceccato, Riccardo; Bruni, Giovanna; Cornaglia, Antonia Icaro; Visai, Livia

2017-02-01

The production of stable suspensions of strontium-substituted hydroxyapatite (Sr-HA) nanopowders, as Sr ions vector for bone tissue regeneration, was carried out in the present work. Sr-HA nanopowders were synthesized via aqueous precipitation methods using Sr 2+ amount from 0 to 100mol% and were characterized by several complementary techniques such as solid-state Nuclear Magnetic Resonance spectroscopy, X-ray diffraction, Infrared spectroscopy, N 2 physisorption and Transmission Electron Microscopy. The substitution of Ca 2+ with Sr 2+ in HA is always isomorphic with gradual evolution between the two limit compositions (containing 100% Ca and 100% Sr), this pointing out the homogeneity of the synthesized nanopowders and the complete solubility of strontium in HA lattice. Strontium addition is responsible for an increasing c/a ratio in the triclinic unit cell. A significant variation of the nanopowders shape and dimension is also observed, a preferential growth along the c-axis direction being evident at higher strontium loads. Modifications in the local chemical environment of phosphate and hydroxyl groups in the apatite lattice are also observed. Stable suspensions were produced by dispersing the synthesized nanopowders in bovine serum albumin. Characterization by Dynamic Light Scattering and ζ-potential determination allowed to show that Ca 2+ →Sr 2+ substitution influences the hydrodynamic diameter, which is always twice the particles size determined by TEM, the nanoparticles being always negatively charged as a result from the albumin rearrangement upon the interaction with nanoparticles surface. The biocompatibility of the suspensions was studied in terms of cell viability, apoptosis, proliferation and morphology, using osteosarcoma cell line SAOS-2. The data pointed out an increased cell proliferation for HA nanoparticles containing larger Sr 2+ load, the cells morphology remaining essentially unaffected. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of cadmium on intercellular junctions in a renal epithelial cell line grown on permeable membrane supports

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prozialeck, W.C.; Niewenhuis, R.J.

1991-03-11

Recent findings from the authors laboratories have shown that Cd{sup 2+} has relatively specific damaging effects on adhering and occluding junctions in the established porcine renal epithelial cell line, LLC-PK{sub 1}. The present studies were undertaken in order to further characterize the junction-perturbing effects of Cd{sup 2+} in polarized monolayers of LLC-PK{sub 1} cells, and to begin to identify the mechanisms underlying these effects. LLC-PK{sub 1} cells were grown to confluency on Millicell HA chambers and exposed to Cd{sup 2+} in polarized monolayers of LLC-PK{sub 1} cells, and to begin to identify the mechanisms underlying these effects. LLC-PK{sub 1} cellsmore » were grown to confluency on Millicell HA chambers an exposed to Cd{sup 2+} by adding CdCl{sub 2} to the solutions on either side of the cell monolayer. The integrity of cell-cell junctions was assessed by monitoring the transepithelial electrical resistance. The results showed that exposure to Cd{sup 2+} caused a pronounced decrease in transepithelial resistance without causing the cells to detach from the Millicell membrane. This decrease in resistance occurred more quickly and was much more pronounced when Cd{sup 2+} was added to the basolateral surface rather than the apical surface. Furthermore, the effects of Cd{sup 2+} were greatly reduced when excess Ca{sup 2+} was present in the medium. These results suggest that Cd{sup 2+} was present in the medium. These results suggest that Cd{sup 2+} may disrupt cell-cell junctions by interacting with Ca{sup 2+} binding sites or Ca{sup 2+} channels that are oriented toward the basolateral cell surface.« less

Simple in vitro generation of human leukocyte antigen-G-expressing T-regulatory cells through pharmacological hypomethylation for adoptive cellular immunotherapy against graft-versus-host disease.

PubMed

Stamou, Panagiota; Marioli, Dimitra; Patmanidi, Alexandra L; Sgourou, Argyro; Vittoraki, Angeliki; Theofani, Efthymia; Pierides, Chryso; Taraviras, Stavros; Costeas, Paul A; Spyridonidis, Alexandros

2017-04-01

Major barriers in using classical FOXP3+ regulatory T cells (Tregs) in clinical practice are their low numbers in the circulation, the lack of specific cell surface markers for efficient purification and the loss of expression of Treg signature molecules and suppressive function after in vitro expansion or in a pro-inflammatory microenviroment. A surface molecule with potent immunosuppressive function is the human leukocyte antigen-G (HLA-G), which is normally expressed in placenta protecting the "semi-allogeneic" fetus from maternal immune attack. Because HLA-G expression is strongly regulated by methylation, we asked whether hypomethylating agents (HA) may be used in vitro to induce HLA-G expression on conventional T cells and convert them to Tregs. Human peripheral blood T cells were exposed to azacytidine/decitabine and analyzed for HLA-G expression and their in vitro suppressor properties. HA treatment induces de novo expression of HLA-G on T cells through hypomethylation of the HLA-G proximal promoter. The HA-induced CD4 + HLA-G pos T cells are FOXP3 negative and have potent in vitro suppression function, which is dependent to a large extent, but not exclusively, on the HLA-G molecule. Converted HLA-G pos suppressors retain their suppressor function in the presence of tumor necrosis factor (TNF) and preserve hypomethylated the HLA-G promoter for at least 2 days after azacytidine exposure. Decitabine-treated T cells suppressed ex vivo the proliferation of T cells isolated from patients suffering from graft-versus-host disease (GVHD). We propose, in vitro generation of HLA-G-expressing T cells through pharmacological hypomethylation as a simple, Good Manufacturing Practice (GMP)-compatible and efficient strategy to produce a stable Treg subset of a defined phenotype that can be easily purified for adoptive immunotherapy. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Nanoparticulate zinc oxide as a coating material for orthopedic and dental implants.

PubMed

Memarzadeh, Kaveh; Sharili, Amir S; Huang, Jie; Rawlinson, Simon C F; Allaker, Robert P

2015-03-01

Orthopedic and dental implants are prone to infection. In this study, we describe a novel system using zinc oxide nanoparticles (nZnO) as a coating material to inhibit bacterial adhesion and promote osteoblast growth. Electrohydrodynamic atomisation (EHDA) was employed to deposit mixtures of nZnO and nanohydroxyapatite (nHA) onto the surface of glass substrates. Nano-coated substrates were exposed to Staphylococcus aureus suspended in buffered saline or bovine serum to determine antimicrobial activity. Our results indicate that 100% nZnO and 75% nZnO/25% nHA composite-coated substrates have significant antimicrobial activity. Furthermore, osteoblast function was explored by exposing cells to nZnO. UMR-106 cells exposed to nZnO supernatants showed minimal toxicity. Similarly, MG-63 cells cultured on nZnO substrates did not show release of TNF-α and IL-6 cytokines. These results were reinforced by both proliferation and differentiation studies which revealed that a substrate coated with exclusively nZnO is more efficient than composite surface coatings. Finally, electron and light microscopy, together with immunofluorescence staining, revealed that all cell types tested, including human mesenchymal cell (hMSC), were able to maintain normal cell morphology when adhered onto the surface of the nano-coated substrates. Collectively, these findings indicate that nZnO can, on its own, provide an optimal coating for future bone implants that are both antimicrobial and biocompatible. © 2014 Wiley Periodicals, Inc.

Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics.

PubMed

Tang, Zhurong; Wang, Zhe; Qing, Fangzhu; Ni, Yilu; Fan, Yujiang; Tan, Yanfei; Zhang, Xingdong

2015-03-01

Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway. © 2014 Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Svensson Holm, Ann-Charlotte B., E-mail: ann-charlotte.svensson@liu.se; Experimental Pathology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping; Bengtsson, Torbjoern

Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blockingmore » antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling.« less

Characterization of gas tunnel type plasma sprayed hydroxyapatite-nanostructure titania composite coatings

NASA Astrophysics Data System (ADS)

Yugeswaran, S.; Kobayashi, A.; Ucisik, A. Hikmet; Subramanian, B.

2015-08-01

Hydroxyapatite (HA) can be coated onto metal implants as a ceramic biocompatible coating to bridge the growth between implants and human tissue. Meanwhile many efforts have been made to improve the mechanical properties of the HA coatings without affecting its bioactivity. In the present study, nanostructure titania (TiO2) was mixed with HA powder and HA-nanostructure TiO2 composite coatings were produced by gas tunnel type plasma spraying torch under optimized spraying conditions. For this purpose, composition of 10 wt% TiO2 + 90 wt% HA, 20 wt% TiO2 + 80 wt% HA and 30 wt% TiO2 + 70 wt% HA were selected as the feedstock materials. The phase, microstructure and mechanical properties of the coatings were characterized. The obtained results validated that the increase in weight percentage of nanostructure TiO2 in HA coating significantly increased the microhardness, adhesive strength and wear resistance of the coatings. Analysis of the in vitro bioactivity and cytocompatibility of the coatings were done using conventional simulated body fluid (c-SBF) solution and cultured green fluorescent protein (GFP) labeled marrow stromal cells (MSCs) respectively. The bioactivity results revealed that the composite coating has bio-active surface with good cytocompatibility.

HA-1077 inhibits cell migration/invasion of oral squamous cell carcinoma.

PubMed

Moreira Carboni, Simone de Sales Costa; Rodrigues Lima, Nathália Alves; Pinheiro, Nanci Mendes; Tavares-Murta, Beatriz Martins; Crema, Virgínia Oliveira

2015-10-01

Oral squamous cell carcinoma (OSCC) is the most malignant lesion occurring in the head and neck. The Rho-kinases (ROCKs), effectors of Rho proteins, are involved in actin cytoskeletal organization, cell migration, and maintenance cortex. The HA-1077 inhibits the ROCKs. This study aimed to evaluate the effect of treatment with HA-1077 on cell motility in SCC-4 cells, a cell line originating from human OSCC. F-actin of SCC-4 cells treated or not with HA-1077 (1, 50 and 100 μmol/l), and also HA-1077 50 μmol/l and/or inhibitors Y-27632 30 μmol/l was stained with rhodamine-conjugated phalloidin and analyzed by confocal microscopy. Approximately 1×10 cells/well, control and treated with HA-1077 (25, 50, and 100 μmol/l) were added to the migration plate assay. In addition, 1×10 cells/well, control and treated with HA-1077 50 μmol/l, were tested by invasion assays (plate coated with Matrigel). The inhibition of ROCKs with HA-1077 and/or Y-27632 leads to morphological changes, affecting the organization of the actin. The inhibitory effect of HA-1077 (P<0.0001) was dose dependent as the number of cells migrated at 100 μmol/l was statistically different: 25 μmol/l (P<0.0001) and 50 μmol/l (P<0.01). The number of cells treated with HA-1077 50 μmol/l decreased compared with control cells that invaded through Matrigel (P<0.0001). This study shows an inhibitory effect of HA-1077 on cell migration and invasion, suggesting that the use of HA-1077 can be a potential therapy for OSCC.

Fabrication and characterization of nanoengineered biocompatible n-HA/chitosan-tamarind seed polysaccharide: Bio-inspired nanocomposites for bone tissue engineering.

PubMed

Shakir, Mohammad; Zia, Iram; Rehman, Abdur; Ullah, Rizwan

2018-05-01

In this communication we describe the fabrication of nano-hydroxyapatite/chitosan-tamarind seed polysaccharide (n-HA/CS-TSP) nanocomposite with a weight ratio of 70/20/10, 70/15/15 and 70/10/20, respectively through a co-precipitation method. A comparative assessment of the properties of n-HA/CS-TSP and n-HA/CS nanocomposites was done by FT-IR, SEM-EDX, TEM, TGA/DTA, XRD and mechanical testing. The results suggested strong chemical interactions between the three components, decreased particle size and homogeneous dispersion of n-HA particles in n-HA/CS-TSP as compared to n-HA/CS. The n-HA/CS-TSP (70/10/20) showed the most porous and rough surface, enhanced thermal stability and highest compressive strength (4.0 MPa) and modulus (81 MPa). In addition, n-HA/CS-TSP (70/10/20) exhibited greater swelling character, acceptable degradation and increased biomineralization in simulated body fluid (SBF) as compared to n-HA/CS-TSP (70/20/10, 70/15/15) and n-HA/CS nanocomposites. The superior non-toxic response with MG-63 cells and better haemocompatibility was observed with n-HA/CS-TSP (70/15/15). Thereby, n-HA/CS-TSP nanocomposites could be promising alternative biomaterials in the field of bone tissue engineering compared to the n-HA/CS nanocomposite. Copyright © 2018 Elsevier B.V. All rights reserved.

Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus × H. tuberosus

PubMed Central

Chiappetta, A.; Fambrini, M.; Petrarulo, M.; Rapparini, F.; Michelotti, V.; Bruno, L.; Greco, M.; Baraldi, R.; Salvini, M.; Pugliesi, C.; Bitonti, M. B.

2009-01-01

Background and Aims The clone EMB-2 of the interspecific hybrid Helianthus annuus × H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. Methods Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography–mass spectrometry–selected ion monitoring method and an immuno-cytochemical approach. Key Results Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. Conclusions In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone. PMID:19151043

Macrophage Biocompatibility of CoCr Wear Particles Produced under Polarization in Hyaluronic Acid Aqueous Solution

PubMed Central

Perez-Maceda, Blanca Teresa; López-Fernández, María Encarnación; Díaz, Iván; Kavanaugh, Aaron; Billi, Fabrizio; Escudero, María Lorenza; García-Alonso, María Cristina; Lozano, Rosa María

2018-01-01

Macrophages are the main cells involved in inflammatory processes and in the primary response to debris derived from wear of implanted CoCr alloys. The biocompatibility of wear particles from a high carbon CoCr alloy produced under polarization in hyaluronic acid (HA) aqueous solution was evaluated in J774A.1 mouse macrophages cultures. Polarization was applied to mimic the electrical interactions observed in living tissues. Wear tests were performed in a pin-on-disk tribometer integrating an electrochemical cell in phosphate buffer solution (PBS) and in PBS supplemented with 3 g/L HA, an average concentration that is generally found in synovial fluid, used as lubricant solution. Wear particles produced in 3 g/L HA solution showed a higher biocompatibility in J774A.1 macrophages in comparison to those elicited by particles obtained in PBS. A considerable enhancement in macrophages biocompatibility in the presence of 3 g/L of HA was further observed by the application of polarization at potentials having current densities typical of injured tissues suggesting that polarization produces an effect on the surface of the metallic material that leads to the production of wear particles that seem to be macrophage-biocompatible and less cytotoxic. The results showed the convenience of considering the influence of the electric interactions in the chemical composition of debris detached from metallic surfaces under wear corrosion to get a better understanding of the biological effects caused by the wear products. PMID:29738506

Effect of small peptide (P-15) on HJMSCs adhesion to hydroxyap-atite

NASA Astrophysics Data System (ADS)

Cheng, Wei; Tong, Xin; Hu, QinGang; Mou, YongBin; Qin, HaiYan

2016-02-01

P-15, a synthetic peptide of 15-amino acids, has been tested in clinical trials to enhance cell adhesion and promote osseointe- gration. This feature of P-15 has also inspired the development of designing new bone substitute materials. Despite the increasing applications of P-15 in bone graft alternatives, few studies focus on the mechanism of cell adhesion promoted by P-15 and the mechanical property changes of the cells interacting with P-15. In this article, we used atomic force microscope (AFM) based single cell indentation force spectroscopy to study the impact of P-15 on the stiffness and the adhesion ability of human jaw bone mesenchymal stem cells (HJMSCs) to hydroxyapatite (HA). We found that the stiffness of HJMSCs increases as the concentration of P-15 grows in short culture intervals and that the adhesion forces between HJMSCs and HA particles in both the presence and absence of P-15 are all around 30pN. Moreover, by calculating the binding energy of HJMSCs to HA particles mixed with and without P-15, we proved that P-15 could increase the adhesion energy by nearly four times. Scanning electron microscope (SEM) was also exploited to study the morphology of HJMSCs cultured in the presence and absence of P-15 on HA disc surface for a short term. Apparent morphological differences were observed between the cells cultured with and without P-15. These results explain the probable underlying adhesion mechanism of HJMSC promoted by P-15 and can serve as the bases for the design of bone graft substitute materials.

Retro-inverso d-peptide-modified hyaluronic acid/bioreducible hyperbranched poly(amido amine)/pDNA core-shell ternary nanoparticles for the dual-targeted delivery of short hairpin RNA-encoding plasmids.

PubMed

Gu, Jijin; Chen, Xinyi; Fang, Xiaoling; Sha, Xianyi

2017-07-15

The active targeting of gene carriers is a powerful strategy for improving tumour-specific delivery and therapy. Although numerous l-peptide ligands play significant roles in the active targeting of nanomedicine, retro-inverso d-peptides have been explored as targeting ligands due to their superior stability and bioactivity in vivo. In this study, retro-inverso d-peptide (RIF7)-modified hyaluronic acid (HA)/bioreducible hyperbranched poly(amido amine) (RHB)/plasmid DNA (pDNA) ternary nanoparticles were successfully developed using the layer-by-layer method for the CD44-positive tumour-specific delivery of short hairpin RNA (shRNA)-encoding pDNA through the combination of the Anxa1 (tumour vasculature) and CD44 (tumour cell-surface) receptors, which mediated the dual targeting. The potential of these newly designed nanoparticles was evaluated by examining the efficacy of their cellular uptake and transfection in cell monolayers, tumour spheroids, and malignant xenograft animal models. With negligible cytotoxicity, the spherical-shaped RIF7-HA/RHB/pDNA nanoparticles were the direct result of an electrostatic complex that had efficiently targeted CD44-positive tumour delivery, penetration, and cellular uptake in vitro. The nanoparticles showed excellent target-specific gene transfection even in the presence of serum. The in vivo therapeutic effect of RIF7-HA/RHB/pDNA-shRNA nanoparticle-mediated shRNA targeting of the Cyclin gene (shCyclin) was evaluated in tumour-bearing mice. The RIF7-HA/RHB/pDNA-shCyclin nanoparticles significantly increased the survival time of tumour-bearing mice and substantially reduced tumour growth due to their extremely specific tumour-targeting activity. These results suggested that the combination of HA and retro-inverso peptide RIF7 significantly increased the therapeutic effect of pDNA-shCyclin-loaded nanoparticles for CD44-positive tumours. Thus, RIF7-HA-mediated multi-target ternary gene vectors are an efficient and promising strategy for the delivery of pDNA-shRNA in the targeted treatment of malignant and metastatic cancers. Although l-peptide ligands play significant roles in the active targeting of nanomedicine, retro-inverso d-peptides have been explored as targeting ligands due to their superior stability and bioactivity in vivo. Retro-inverso peptide RIF7 was designed as a ligand of Anxa1 receptor. The resultant peptide, RIF7, displayed high binding efficiency within Anxa1 receptor, which is highly expressed tumour vasculature cells and some tumour cells such as B16F10 and U87MG cells. The most important feature of RIF7 is its high stability in the blood, which is suitable and promising for application in vivo. Multifunctional RIF7-HA was then synthesized by conjugating the RIF7 peptide to HA, which was used to modify the surface of RHB/pDNA nanoparticles to prepare RIF7-HA/RHB/pDNA core-shell ternary nanoparticles for the dual-targeted delivery of shRNA-encoding plasmids in vitro and in vivo. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Temperature and magnetic field responsive hyaluronic acid particles with tunable physical and chemical properties

NASA Astrophysics Data System (ADS)

Ekici, Sema; Ilgin, Pinar; Yilmaz, Selahattin; Aktas, Nahit; Sahiner, Nurettin

2011-01-01

We report the preparation and characterization of thiolated-temperature-responsive hyaluronic acid-cysteamine-N-isopropyl acrylamide (HA-CYs-NIPAm) particles and thiolated-magnetic-responsive hyaluronic acid (HA-Fe-CYs) particles. Linear hyaluronic acid (HA) crosslinked with divinyl sulfone as HA particles was prepared using a water-in-oil micro emulsion system which were then oxidized HA-O with NaIO4 to develop aldehyde groups on the particle surface. HA-O hydrogel particles were then reacted with cysteamine (CYs) which interacted with aldehydes on the HA surface to form HA particles with cysteamine (HA-CYs) functionality on the surface. HA-CYs particles were further exposed to radical polymerization with NIPAm to obtain temperature responsive HA-CYs-NIPAm hydrogel particles. To acquire magnetic field responsive HA composites, magnetic iron particles were included in HA to form HA-Fe during HA particle preparation. HA-Fe hydrogel particles were also chemically modified. The prepared HA-CYs-NIPAm demonstrated temperature dependent size variations and phase transition temperature. HA-CYs-NIPAm and HA-Fe-CYs particles can be used as drug delivery vehicles. Sulfamethoxazole (SMZ), an antibacterial drug, was used as a model drug for temperature-induced release studies from these particles.

Hyaluronic Acid-Serum Hydrogels Rapidly Restore Metabolism of Encapsulated Stem Cells and Promote Engraftment

PubMed Central

Chan, Angel T.; Karakas, Mehmet F.; Vakrou, Styliani; Afzal, Junaid; Rittenbach, Andrew; Lin, Xiaoping; Wahl, Richard L.; Pomper, Martin G.; Steenbergen, Charles J.; Tsui, Benjamin M.W.; Elisseeff, Jennifer H.; Abraham, M. Roselle

2015-01-01

Background Cell death due to anoikis, necrosis and cell egress from transplantation sites limits functional benefits of cellular cardiomyoplasty. Cell dissociation and suspension, which are a pre-requisite for most cell transplantation studies, lead to depression of cellular metabolism and anoikis, which contribute to low engraftment. Objective We tissue engineered scaffolds with the goal of rapidly restoring metabolism, promoting viability, proliferation and engraftment of encapsulated stem cells. Methods The carboxyl groups of HA were functionalized with N-hydroxysuccinimide (NHS) to yield HA succinimidyl succinate (HA-NHS) groups that react with free amine groups to form amide bonds. HA-NHS was cross-linked by serum to generate HA:Serum (HA:Ser) hydrogels. Physical properties of HA:Ser hydrogels were measured. Effect of encapsulating cardiosphere-derived cells (CDCs) in HA:Ser hydrogels on viability, proliferation, glucose uptake and metabolism was assessed in vitro. In vivo acute intra-myocardial cell retention of 18FDG-labeled CDCs encapsulated in HA:Ser hydrogels was quantified. Effect of CDC encapsulation in HA:Ser hydrogels on in vivo metabolism and engraftment at 7 days was assessed by serial, dual isotope SPECT-CT and bioluminescence imaging of CDCs expressing the Na-iodide symporter and firefly luciferase genes respectively. Effect of HA:Ser hydrogels +/− CDCs on cardiac function was assessed at 7 days & 28 days post-infarct. Results HA:Ser hydrogels are highly bio-adhesive, biodegradable, promote rapid cell adhesion, glucose uptake and restore bioenergetics of encapsulated cells within 1 h of encapsulation, both in vitro and in vivo. These metabolic scaffolds can be applied epicardially as a patch to beating hearts or injected intramyocardially. HA:Ser hydrogels markedly increase acute intramyocardial retention (~6 fold), promote in vivo viability, proliferation, engraftment of encapsulated stem cells and angiogenesis. Conclusion HA:Ser hydrogels serve as ‘synthetic stem cell niches’ that rapidly restore metabolism of encapsulated stem cells, promote stem cell engraftment and angiogenesis. These first ever, tissue engineered metabolic scaffolds hold promise for clinical translation in conjunction with CDCs and possibly other stem cell types. PMID:26378976

Effect of surface roughness of hydroxyapatite-coated titanium on the bone-implant interface shear strength.

PubMed

Hayashi, K; Inadome, T; Tsumura, H; Nakashima, Y; Sugioka, Y

1994-11-01

We have investigated the bone-implant interface shear strength of hydroxyapatite (HA)-coated Ti-6Al-4V (HA-coating A) (roughness average, Ra = 3.4 +/- 0.5 microns) and HA-coated Ti-6Al-4V with a rougher surface (HA-coating B) (Ra = 8.4 +/- 1.8 microns). There was no significant difference between HA-coating A and HA-coating B implants with respect to the bone-implant interface shear strength as determined in push-out tests using the transcortical model in adult dogs. The bone-implant interface shear strength of bead-coated porous Ti-6Al-4V was significantly greater than that of both HA-coating A and HA-coating B implants. The failure site, as determined by scanning electron microscopy, was the coating-substrate interface, not the coating-bone interface. This indicates a need to protect the HA coating from the direct shear forces. HA coating enhances early bone growth into the porous surface of the implant. Long-term fixation should depend on bone anchoring to this porous surface. Hydroxyapatite coatings must be developed which do not obstruct the pores of the surface of the implant.

The pathway to intelligent implants: osteoblast response to nano silicon-doped hydroxyapatite patterning

PubMed Central

Munir, G.; Koller, G.; Di Silvio, L.; Edirisinghe, M. J.; Bonfield, W.; Huang, J.

2011-01-01

Bioactive hydroxyapatite (HA) with addition of silicon (Si) in the crystal structure (silicon-doped hydroxyapatite (SiHA)) has become a highly attractive alternative to conventional HA in bone replacement owing to the significant improvement in the in vivo bioactivity and osteoconductivity. Nanometre-scaled SiHA (nanoSiHA), which closely resembles the size of bone mineral, has been synthesized in this study. Thus, the silicon addition provides an extra chemical cue to stimulate and enhance bone formation for new generation coatings, and the next stage in metallic implantation design is to further improve cellular adhesion and proliferation by control of cell alignment. Topography has been found to provide a powerful set of signals for cells and form contact guidance. Using the recently developed novel technique of template-assisted electrohydrodynamic atomization (TAEA), patterns of pillars and tracks of various dimensions of nanoSiHA were achieved. Modifying the parameters of TAEA, the resolution of pattern structures was controlled, enabling the topography of a substrate to be modified accordingly. Spray time, flow rate and distance between the needle and substrate were varied to improve the pattern formation of pillars and tracks. The 15 min deposition time provided the most consistent patterned topography with a distance of 50 mm and flow rate of 4 µl min−1. A titanium substrate was patterned with pillars and tracks of varying widths, line lengths and distances under the optimized TAEA processing condition. A fast bone-like apatite formation rate was found on nanoSiHA after immersion in simulated body fluid, thus demonstrating its high in vitro bioactivity. Primary human osteoblast (HOB) cells responded to SiHA patterns by stretching of the filopodia between track and pillar, attaching to the apex of the pillar pattern and stretching between two. HOB cells responded to the track pattern by elongating along and between the track, and the length of HOB cells was proportional to the gaps between track patterns, but this relationship was not observed on the pillar patterns. The study has therefore provided an insight for future design of next generation implant surfaces to control and guide cellular responses, while TAEA patterning provides a controllable technique to provide topography to medical implants. PMID:21208969

Structural and biological evaluation of lignin addition to simple and silver-doped hydroxyapatite thin films synthesized by matrix-assisted pulsed laser evaporation.

PubMed

Janković, A; Eraković, S; Ristoscu, C; Mihailescu Serban, N; Duta, L; Visan, A; Stan, G E; Popa, A C; Husanu, M A; Luculescu, C R; Srdić, V V; Janaćković, Dj; Mišković-Stanković, V; Bleotu, C; Chifiriuc, M C; Mihailescu, I N

2015-01-01

We report on thin film deposition by matrix-assisted pulsed laser evaporation of simple hydroxyapatite (HA) or silver (Ag) doped HA combined with the natural biopolymer organosolv lignin (Lig) (Ag:HA-Lig). Solid cryogenic target of aqueous dispersions of Ag:HA-Lig composite and its counterpart without silver (HA-Lig) were prepared for evaporation using a KrF* excimer laser source. The expulsed material was assembled onto TiO2/Ti substrata or silicon wafers and subjected to physical-chemical investigations. Smooth, uniform films adherent to substratum were observed. The chemical analyses confirmed the presence of the HA components, but also evidenced traces of Ag and Lig. Deposited HA was Ca deficient, which is indicative of a film with increased solubility. Recorded X-ray Diffraction patterns were characteristic for amorphous films. Lig presence in thin films was undoubtedly proved by both X-ray Photoelectron and Fourier Transform Infra-Red Spectroscopy analyses. The microbiological evaluation showed that the newly assembled surfaces exhibited an inhibitory activity both on the initial steps of biofilm forming, and on mature bacterial and fungal biofilm development. The intensity of the anti-biofilm activity was positively influenced by the presence of the Lig and/or Ag, in the case of Staphylococcus aureus, Pseudomonas aeruginosa and Candida famata biofilms. The obtained surfaces exhibited a low cytotoxicity toward human mesenchymal stem cells, being therefore promising candidates for fabricating implantable biomaterials with increased biocompatibility and resistance to microbial colonization and further biofilm development.

Calcium Phosphate Growth at Electropolished Titanium Surfaces

PubMed Central

Ajami, Elnaz; Aguey-Zinsou, Kondo-Francois

2012-01-01

This work investigated the ability of electropolished Ti surface to induce Hydroxyapatite (HA) nucleation and growth in vitro via a biomimetic method in Simulated Body Fluid (SBF). The HA induction ability of Ti surface upon electropolishing was compared to that of Ti substrates modified with common chemical methods including alkali, acidic and hydrogen peroxide treatments. Our results revealed the excellent ability of electropolished Ti surfaces in inducing the formation of bone-like HA at the Ti/SBF interface. The chemical composition, crystallinity and thickness of the HA coating obtained on the electropolished Ti surface was found to be comparable to that achieved on the surface of alkali treated Ti substrate, one of the most effective and popular chemical treatments. The surface characteristics of electropolished Ti contributing to HA growth were discussed thoroughly. PMID:24955535

The enamel protein amelotin is a promoter of hydroxyapatite mineralization.

PubMed

Abbarin, Nastaran; San Miguel, Symone; Holcroft, James; Iwasaki, Kengo; Ganss, Bernhard

2015-05-01

Amelotin (AMTN) is a recently discovered protein that is specifically expressed during the maturation stage of dental enamel formation. It is localized at the interface between the enamel surface and the apical surface of ameloblasts. AMTN knock-out mice have hypomineralized enamel, whereas transgenic mice overexpressing AMTN have a compact but disorganized enamel hydroxyapatite (HA) microstructure, indicating a possible involvement of AMTN in regulating HA mineralization directly. In this study, we demonstrated that recombinant human (rh) AMTN dissolved in a metastable buffer system, based on light scattering measurements, promotes HA precipitation. The mineral precipitates were characterized by scanning and transmission electron microscopy and electron diffraction. Colloidal gold immunolabeling of AMTN in the mineral deposits showed that protein molecules were associated with HA crystals. The binding affinity of rh-AMTN to HA was found to be comparable to that of amelogenin, the major protein of the forming enamel matrix. Overexpression of AMTN in mouse calvaria cells also increased the formation of calcium deposits in the culture medium. Overexpression of AMTN during the secretory stage of enamel formation in vivo resulted in rapid and uncontrolled enamel mineralization. Site-specific mutagenesis of the potential serine phosphorylation motif SSEEL reduced the in vitro mineral precipitation to less than 25%, revealing that this motif is important for the HA mineralizing function of the protein. A synthetic short peptide containing the SSEEL motif was only able to facilitate mineralization in its phosphorylated form ((P)S(P) SEEL), indicating that this motif is necessary but not sufficient for the mineralizing properties of AMTN. These findings demonstrate that AMTN has a direct influence on biomineralization by promoting HA mineralization and suggest a critical role for AMTN in the formation of the compact aprismatic enamel surface layer during the maturation stage of amelogenesis. © 2014 American Society for Bone and Mineral Research.

Kinetics and thermodynamics studies on the BMP-2 adsorption onto hydroxyapatite surface with different multi-morphological features.

PubMed

Lu, Zhiwei; Huangfu, Changxin; Wang, Yanying; Ge, Hongwei; Yao, Yao; Zou, Ping; Wang, Guangtu; He, Hua; Rao, Hanbing

2015-01-01

The effect of the surface topography on protein adsorption process is of great significance for designing hydroxyapatite (HA) ceramic material surfaces. In this work, three different topographies of HA materials HA-sheet, HA-rod, and HA-whisker were synthesized and testified by X-ray diffraction (XRD), Fourier transform infrared (FT-IR), Brunauer-Emmett-Teller (BET) and a field emission scanning electron microscopy (FE-SEM). We have systematically investigated the adsorption kinetics and thermodynamics of bone morphogenetic proteins (BMP-2) on the three different topography surfaces of HA, respectively. The results showed that the maximum adsorption capacities of HA-sheet, HA-rod and HA-whisker were (219.96 ± 10.18), (247.13 ± 12.35), and (354.67 ± 17.73) μg · g(-1), respectively. Kinetic parameters, rate constants, equilibrium adsorption capacities and related correlation coefficients, for each kinetic model were calculated as well as discussed. It demonstrated that the adsorption of BMP-2 onto HA could be described by the pseudo second-order equation. Adsorption of BMP-2 onto HA followed the Langmuir isotherm. It confirmed that compared with other samples HA-whisker had more adsorption sites for its high specific surface area which could provide more opportunities for protein molecules. The adsorption processes were endothermic (ΔH > 0), spontaneous (ΔG < 0) and entropy increasing (ΔS > 0). A possible adsorption mechanism has been proposed. In addition, the BMP-2 could be adsorbed to the surface which existed slight conformational changes by FT-IR. Copyright © 2015 Elsevier B.V. All rights reserved.

Combined photothermal-chemotherapy of breast cancer by near infrared light responsive hyaluronic acid-decorated nanostructured lipid carriers

NASA Astrophysics Data System (ADS)

Zheng, Shaohui; Du Nguyen, Van; Song, Seung Yoon; Han, Jiwon; Park, Jong-Oh

2017-10-01

In this study, a novel type of hyaluronic acid (HA)-decorated nanostructured lipid carrier (NLC) was prepared and investigated as a light-triggered drug release and combined photothermal-chemotherapy for cancer treatment. Polyhedral gold nanoparticles (Au NPs) with an average size of 10 nm were synthesized and co-encapsulated with doxorubicin (DOX) in the matrix of NLCs with a high drug loading efficiency (above 80%). HA decoration was achieved by the electrostatic interaction between HA and CTAB on the NLC surface. A remarkable temperature increase was observed by exposing the Au NP-loaded NLCs to an NIR laser, which heated the samples sufficiently (above 40 °C) to kill tumor cells. The entrapped DOX exhibited a sustained, stepwise NIR laser-triggered drug release pattern. The biocompatibility of the NLCs was investigated by MTT assay and the cell viability was maintained above 85%, even at high concentrations. The intracellular uptake of free DOX and entrapped DOX, observed by confocal microscopy, revealed two distinct uptake mechanisms, i.e. passive diffusion and endocytosis, respectively. In particular, internalization of the HA-Au-DOX-NLCs was more extensively enhanced than the Au-DOX-NLCs, which was attributed to HA-CD44 receptor-mediated endocytosis. Meanwhile, the internalized NLCs successfully escaped from the lysosomes, increasing the intracellular DOX. The HA-Au-DOX-NLCs IC50 value decreased from 2.3 to 0.6 μg ml-1 with NIR irradiation at 72 h, indicating the excellent synergistic antitumor effect of photothermal-chemotherapy. The photothermal ablation was further confirmed by a live/dead cell staining assay. Thus, a combined photothermal-chemotherapy approach has been proposed as a promising strategy for cancer treatment.

Sectioning studies of biomimetic collagen-hydroxyapatite coatings on Ti-6Al-4V substrates using focused ion beam

NASA Astrophysics Data System (ADS)

Hu, Changmin; Yu, Le; Wei, Mei

2018-06-01

A biomimetic bone-like collagen-hydroxyapatite (Col-HA) composite coating was formed on a surface-treated Ti-6Al-4V alloy substrate via simultaneous collagen self-assembly and hydroxyapatite nucleation. The coating process has been carried out by immersing sand-blasted, acid-etched and UV irradiated Ti-6Al-4V alloy in type I collagen-containing modified simulated body fluid (m-SBF). The surface morphology and phase composition of the coating were characterized using various techniques. More importantly, dual-beam FIB/SEMs with either gallium ion source (GFIB) or xenon plasma ion source (PFIB) were used to investigate the cross-sectional features of the biomimetic Col-HA composite coating in great details. As a result, the cross-sectional images and thin transmission electron microscopy (TEM) specimens were successfully obtained from the composite coating with no obvious damages or milling ion implantations. Both the cross-sectional SEM and TEM results have confirmed that the Col-HA coating demonstrates a similar microstructure to that of pure HA coating with homogeneously distributed elements across the whole cross section. Both coatings consist of a uniform, crack-free gradient structure with a dense layer adjacent to the interface between the Ti-6Al-4V substrate and the coating facilitating a strong bonding, while a porous structure at the coating surface aiding cell attachment.

Hyaluronic acid affects the in vitro induction effects of Synthetic PAMPS and PDMAAm hydrogels on chondrogenic differentiation of ATDC5 cells, depending on the level of concentration

PubMed Central

2013-01-01

Background It has been a common belief that articular cartilage tissue cannot regenerate in vivo. Recently, however, we have found that spontaneous hyaline cartilage regeneration can be induced in vivo by implanting a synthetic double-network (DN) hydrogel, which is composed of poly-(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) and poly-(N,N’-dimethyl acrylamide) (PDMAAm). However, the mechanism of this phenomenon has not been clarified. Recently, we have found that single-network PAMPS and PDMAAm gels can induce chondrogenic differentiation of ATDC5 cells in vitro even in a maintenance medium. In the in vivo condition, there is a strong possibility that the induction effect of the gel itself is enhanced by some molecules which exist in the joint. We have noticed that the joint fluid naturally contains hyaluronic acid (HA). The purpose of this study is to clarify in vitro effects of supplementation of HA on the differentiation effect of the PAMPS and PDMAAm gels. Methods We cultured the ATDC5 cells on the PAMPS gel, the PDMAAm gel, and the polystyrene (PS) dish surface with the maintenance medium without insulin for 7 days. HA having a molecular weight of approximately 800 kDa was supplemented into the medium so that the concentration became 0.00, 0.01, 0.10, or 1.00 mg/mL. We evaluated the cultured cells with phase-contrast microscopy and PCR analyses. Results On the PAMPS gel, supplementation with HA of 0.01 and 0.10 mg/mL significantly increased expression of type-2 collagen mRNA (p = 0.0008 and p = 0.0413) and aggrecan mRNA (p = 0.0073 and p = 0.0196) than that without HA. On the PDMAAm gel, supplementation with HA of 1.00 mg/mL significantly reduced expression of these genes in comparison with the culture without HA (p = 0.0426 and p = 0.0218). Conclusions The in vitro induction effects of the PAMPS and PDMAAm gels on chondrogenic differentiation of ATDC5 cells are significantly affected by HA, depending on the level of concentration. These results suggested that there is a high possibility that HA plays an important role in the in vivo spontaneous hyaline cartilage regeneration phenomenon induced by the PAMPS/PDMAAm DN gel. PMID:23379610

Sugar-induced conformational change found in the HA-33/HA-17 trimer of the botulinum toxin complex.

PubMed

Sagane, Yoshimasa; Hayashi, Shintaro; Matsumoto, Takashi; Miyashita, Shin-Ichiro; Inui, Ken; Miyata, Keita; Yajima, Shunsuke; Suzuki, Tomonori; Hasegawa, Kimiko; Yamano, Akihito; Nishikawa, Atsushi; Ohyama, Tohru; Watanabe, Toshihiro; Niwa, Koichi

2013-08-30

Large-sized botulinum toxin complex (L-TC) is formed by conjugation of neurotoxin, nontoxic nonhemagglutinin and hemagglutinin (HA) complex. The HA complex is formed by association of three HA-70 molecules and three HA-33/HA-17 trimers, comprised of a single HA-17 and two HA-33 proteins. The HA-33/HA-17 trimer isolated from serotype D L-TC has the ability to bind to and penetrate through the intestinal epithelial cell monolayer in a sialic acid-dependent manner, and thus it plays an important role in toxin delivery through the intestinal cell wall. In this study, we determined the solution structure of the HA-33/HA-17 trimer by using small-angle X-ray scattering (SAXS). The SAXS image of HA-33/HA-17 exhibited broadly similar appearance to the crystal image of the complex. On the other hand, in the presence of N-acetylneuraminic acid, glucose and galactose, the solution structure of the HA-33/HA-17 trimer was drastically altered compared to the structure in the absence of the sugars. Sugar-induced structural change of the HA-33/HA-17 trimer may contribute to cell binding and subsequent transport across the intestinal cell layer. Copyright © 2013 Elsevier Inc. All rights reserved.

EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer.

PubMed

Schneck, Helen; Gierke, Berthold; Uppenkamp, Frauke; Behrens, Bianca; Niederacher, Dieter; Stoecklein, Nikolas H; Templin, Markus F; Pawlak, Michael; Fehm, Tanja; Neubauer, Hans

2015-01-01

Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAMneg dual-positive (CKpos/CD45pos) cells could be traced in 28 out of 29 samples [range 1-480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAMneg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAMneg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.

Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb.

PubMed

Matsumoto, Kazu; Li, Yingcui; Jakuba, Caroline; Sugiyama, Yoshinori; Sayo, Tetsuya; Okuno, Misako; Dealy, Caroline N; Toole, Bryan P; Takeda, Junji; Yamaguchi, Yu; Kosher, Robert A

2009-08-01

The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb.

[Piezoelectric property of novel biological piezoelectric ceramic HALNK and its effect on the functional expression of rat osteoblast cells].

PubMed

Wang, Peng; Zhang, Jin-Chao; Zhang, Xiao-Zhou; Liu, Zhi-Qin; Chen, Que-Ting; Sun, Jing; Chen, Zhi-Qing

2009-09-01

To test the Piezoelectric property of novel biological piezoelectric ceramic HALNK and its effect on the proliferation and differentiation of rat osteoblast cells. The biological piezoelectric ceramic HALNK1/9 and HALNK5/5 were prepared by mixing Hydroxyapatite (HA) with lithium sodium potassium niobate (LNK) piezoelectric ceramic at a ratio of 1/9 and 5/5 (wt/wt), respectively. After poling treatment, the piezoelectric constants were measured. The osteoblast cells were then seeded on the surfaces of HALNK. The proliferation and differentiation activities of the osteoblast cells were evaluated by MTT assays, ALP activities and scanning electron microscopy examinations. Cells grown on the surfaces of HALNK showed normal morphology, and had better proliferation and differentiation activities than the control. The growth of osteoblastic cells on the surface of HALNK1/9 was significantly better than others. The surface of HALNK 1/9 possesses better piezoelectric property and osteogenesis potential than HALNK5/5.

Biomimetic Bone-like Hydroxyapatite by Mineralization on Supramolecular Porous Fiber Networks.

PubMed

Li, Bo; Kan, Lei; Zhang, Xinyue; Li, Jie; Li, Ruiting; Gui, Qinyuan; Qiu, Dengli; He, Fei; Ma, Ning; Wang, Yapei; Wei, Hao

2017-08-29

Hydroxyapatite (HA), the main inorganic component of bone tissue, is mineralized with collagen fibril scaffolds during bone formation. Inspired by the process, a self-assembled porous network architecture was designed and synthesized by using the 2-ureido-4[1H]-pyrimidone (UPy) modified glycerol molecule UPy-Gly, which was further utilized as a template for biomimetic mineralization. When incubated in simulated body fluid (SBF), the HA nucleus first formed in the holes of the template by the induction of hydroxyls on the surface, grew along the nanofibers, and fused with the template to fabricate hydroxyapatite composites (UPy-Gly/HA). Transmission electron microscopic observation demonstrates that the mineral clusters are accumulated by lamella-like nano hydroxyapatite and the elasticity modulus measured by atomic force microscopy is about 5.5 GPa, which is quite close to the natural cancellous bone tissue of human both in structure and in mechanical properties. The Cell Counting Kit 8 (CCK-8) assay of UPy-Gly and UPy-Gly/HA shows noncytotoxicity to mouse fibroblast L-929 cells. This bioinspired composite will be a promising material for potential use in bone tissue implantation and regeneration engineering.

The role of fusion activity of influenza A viruses in their biological properties.

PubMed

Jakubcová, L; Hollý, J; Varečková, E

2016-06-01

Influenza A viruses (IAVs) cause acute respiratory infections of humans, which are repeated yearly. Human IAV infections are associated with significant morbidity and mortality and therefore they represent a serious health problem. All human IAV strains are originally derived from avian IAVs, which, after their adaptation to humans, can spread in the human population and cause pandemics with more or less severe course of the disease. Presently, however, the potential of avian IAV to infect humans and to cause the disease cannot be predicted. Many studies are therefore focused on factors influencing the virulence and pathogenicity of IAV viruses in a given host. The virus-host interaction starts by virus attachment via the envelope glycoprotein hemagglutinin (HA) to the receptors on the cell surface. In addition to receptor binding, HA mediates also the fusion of viral and endosomal membranes, which follows the virus endocytosis. The fusion potential of HA trimer, primed by proteolytic cleavage, is activated by low pH in endosomes, resulting in HA refolding into the fusion-active form. The HA conformation change is predetermined by its 3-D structure, is pH-dependent, irreversible and strain-specific. The process of fusion activation of IAV hemagglutinin is crucial for virus entry into the cell and for the ability of the virus to replicate in the host. Here we discuss the known data about the characteristics of fusion activation of HA in relation to IAV virulence and pathogenicity.

A comparison study between electrospun polycaprolactone and piezoelectric poly(3-hydroxybutyrate-co-3-hydroxyvalerate) scaffolds for bone tissue engineering.

PubMed

Gorodzha, Svetlana N; Muslimov, Albert R; Syromotina, Dina S; Timin, Alexander S; Tcvetkov, Nikolai Y; Lepik, Kirill V; Petrova, Aleksandra V; Surmeneva, Maria A; Gorin, Dmitry A; Sukhorukov, Gleb B; Surmenev, Roman A

2017-12-01

In this study, bone scaffolds composed of polycaprolactone (PCL), piezoelectric poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and a combination of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and silicate containing hydroxyapatite (PHBV-SiHA) were successfully fabricated by a conventional electrospinning process. The morphological, chemical, wetting and biological properties of the scaffolds were examined. All fabricated scaffolds are composed of randomly oriented fibres with diameters from 800nm to 12μm. Fibre size increased with the addition of SiHA to PHBV scaffolds. Moreover, fibre surface roughness in the case of hybrid scaffolds was also increased. XRD, FTIR and Raman spectroscopy were used to analyse the chemical composition of the scaffolds, and contact angle measurements were performed to reveal the wetting behaviour of the synthesized materials. To determine the influence of the piezoelectric nature of PHBV in combination with SiHA nanoparticles on cell attachment and proliferation, PCL (non-piezoelectric), pure PHBV, and PHBV-SiHA scaffolds were seeded with human mesenchymal stem cells (hMSCs). In vitro study on hMSC adhesion, viability, spreading and osteogenic differentiation showed that the PHBV-SiHA scaffolds had the largest adhesion and differentiation abilities compared with other scaffolds. Moreover, the piezoelectric PHBV scaffolds have demonstrated better calcium deposition potential compared with non-piezoelectric PCL. The results of the study revealed pronounced advantages of hybrid PHBV-SiHA scaffolds to be used in bone tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

Bone tissue reactions to biomimetic ion-substituted apatite surfaces on titanium implants.

PubMed

Ballo, Ahmed M; Xia, Wei; Palmquist, Anders; Lindahl, Carl; Emanuelsson, Lena; Lausmaa, Jukka; Engqvist, Håkan; Thomsen, Peter

2012-07-07

The aim of this study was to evaluate the bone tissue response to strontium- and silicon-substituted apatite (Sr-HA and Si-HA) modified titanium (Ti) implants. Sr-HA, Si-HA and HA were grown on thermally oxidized Ti implants by a biomimetic process. Oxidized implants were used as controls. Surface properties, i.e. chemical composition, surface thickness, morphology/pore characteristics, crystal structure and roughness, were characterized with various analytical techniques. The implants were inserted in rat tibiae and block biopsies were prepared for histology, histomorphometry and scanning electron microscopy analysis. Histologically, new bone formed on all implant surfaces. The bone was deposited directly onto the Sr-HA and Si-HA implants without any intervening soft tissue. The statistical analysis showed significant higher amount of bone-implant contact (BIC) for the Si-doped HA modification (P = 0.030), whereas significant higher bone area (BA) for the Sr-doped HA modification (P = 0.034), when compared with the non-doped HA modification. The differences were most pronounced at the early time point. The healing time had a significant impact for both BA and BIC (P < 0.001). The present results show that biomimetically prepared Si-HA and Sr-HA on Ti implants provided bioactivity and promoted early bone formation.

Hydroxyapatite electrodeposition on anodized titanium nanotubes for orthopedic applications

NASA Astrophysics Data System (ADS)

Parcharoen, Yardnapar; Kajitvichyanukul, Puangrat; Sirivisoot, Sirinrath; Termsuksawad, Preecha

2014-08-01

Nanotubes modification for orthopedic implants has shown interesting biological performances (such as improving cell adhesion, cell differentiation, and enhancing osseointegration). The purpose of this study is to investigate effect of titanium dioxide (TiO2) nanotube feature on performance of hydroxyapatite-coated titanium (Ti) bone implants. TiO2 nanotubes were prepared by anodization using ammonium fluoride electrolyte (NH4F) with and without modifiers (PEG400 and Glycerol) at various potential forms, and times. After anodization, the nanotubes were subsequently annealed. TiO2 nanotubes were characterized by scanning electron microscope and X-ray diffractometer. The amorphous to anatase transformation due to annealing was observed. Smooth and highly organized TiO2 nanotubes were found when high viscous electrolyte, NH4F in glycerol, was used. Negative voltage (-4 V) during anodization was confirmed to increase nanotube thickness. Length of the TiO2 nanotubes was significantly increased by times. The TiO2 nanotube was electrodeposited with hydroxyapatite (HA) and its adhesion was estimated by adhesive tape test. The result showed that nanotubes with the tube length of 560 nm showed excellent adhesion. The coated HA were tested for biological test by live/dead cell straining. HA coated on TiO2 nanotubes showed higher cells density, higher live cells, and more spreading of MC3T3-E1 cells than that growing on titanium plate surface.

Electrochemical surface modification of titanium in dentistry.

PubMed

Kim, Kyo-Han; Ramaswamy, Narayanan

2009-01-01

Titanium and its alloys have good biocompatibility with body cells and tissues and are widely used for implant applications. However, clinical procedures place more stringent and tough requirements on the titanium surface necessitating artificial surface treatments. Among the many methods of titanium surface modification, electrochemical techniques are simple and cheap. Anodic oxidation is the anodic electrochemical technique while electrophoretic and cathodic depositions are the cathodic electrochemical techniques. By anodic oxidation it is possible to obtain desired roughness, porosity and chemical composition of the oxide. Anodic oxidation at high voltages can improve the crystallinity of the oxide. The chief advantage of this technique is doping of the coating of the bath constituents and incorporation of these elements improves the properties of the oxide. Electrophoretic deposition uses hydroxyapatite (HA) powders dispersed in a suitable solvent at a particular pH. Under these operating conditions these particles acquire positive charge and coatings are obtained on the cathodic titanium by applying an external electric field. These coatings require a post-sintering treatment to improve the coating properties. Cathodic deposition is another type of electrochemical method where HA is formed in situ from an electrolyte containing calcium and phosphate ions. It is also possible to alter structure and/or chemistry of the obtained deposit. Nano-grained HA has higher surface energy and greater biological activity and therefore emphasis is being laid to produce these coatings by cathodic deposition.

ADCC employing an NK cell line (haNK) expressing the high affinity CD16 allele with avelumab, an anti-PD-L1 antibody.

PubMed

Jochems, Caroline; Hodge, James W; Fantini, Massimo; Tsang, Kwong Y; Vandeveer, Amanda J; Gulley, James L; Schlom, Jeffrey

2017-08-01

NK-92 cells, and their derivative, designated aNK, were obtained from a patient with non-Hodgkin lymphoma. Prior clinical studies employing adoptively transferred irradiated aNK cells have provided evidence of clinical benefit and an acceptable safety profile. aNK cells have now been engineered to express IL-2 and the high affinity (ha) CD16 allele (designated haNK). Avelumab is a human IgG1 anti-PD-L1 monoclonal antibody, which has shown evidence of clinical activity in a range of human tumors. Prior in vitro studies have shown that avelumab has the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of human tumor cells when combined with NK cells. In the studies reported here, the ability of avelumab to enhance the lysis of a range of human carcinoma cells by irradiated haNK cells via the ADCC mechanism is demonstrated; this ADCC is shown to be inhibited by anti-CD16 blocking antibody and by concanamycin A, indicating the use of the granzyme/perforin pathway in tumor cell lysis. Studies also show that while NK cells have the ability to lyse aNK or haNK cells, the addition of NK cells to irradiated haNK cells does not inhibit haNK-mediated lysis of human tumor cells, with or without the addition of avelumab. Avelumab-mediated lysis of tumor cells by irradiated haNK cells is also shown to be similar to that of NK cells bearing the V/V Fc receptor high affinity allele. These studies thus provide the rationale for the clinical evaluation of the combined use of avelumab with that of irradiated adoptively transferred haNK cells. © 2017 UICC.

Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells.

PubMed

Boonkaew, Benjawan; Kempf, Margit; Kimble, Roy; Cuttle, Leila

2014-12-01

A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles (silver AMPS). This study compared the cytotoxicity of this dressing to the commercially available silver products; Acticoat™, PolyMem Silver(®) and Flamazine™ cream. Human keratinocytes (HaCaT and primary HEK) and normal human fibroblasts (NHF) were exposed to dressings incubated on Nunc™ polycarbonate inserts for 24, 48 and 72h. Four different cytotoxicity assays were performed including; Trypan Blue cell count, MTT, Celltiter-Blue™ and Toluidine Blue surface area assays. The results were expressed as relative cell viability compared to an untreated control. The cytotoxic effects of Acticoat™ and Flamazine™ cream were dependent on exposure time and cell type. After 24h exposure, Acticoat™ and Flamazine™ cream were toxic to all tested cell lines. Surprisingly, HaCaTs treated with Acticoat™ and Flamazine™ had an improved ability to survive at 48 and 72h while HEKs and NHFs had no improvement in survival with any treatment. The novel silver hydrogel and PolyMem Silver(®) showed low cytotoxicity to all tested cell lines at every time interval and these results support the possibility of using the novel silver hydrogel as a burn wound dressing. Researchers who rely on HaCaT cells as an accurate keratinocyte model should be aware that they can respond differently to primary skin cells. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

The role of phosphorylation in dentin phosphoprotein peptide absorption to hydroxyapatite surfaces: a molecular dynamics study

PubMed Central

Villarreal-Ramirez, Eduardo; Garduño-Juarez, Ramon; Gericke, Arne; Boskey, Adele

2015-01-01

Dentin phosphoprotein (DPP) is a protein expressed mainly in dentin and to a lesser extent in bone. DPP has a disordered structure, rich in glutamic acid, aspartic acid and phosphorylated serine/threonine residues. It has a high capacity for binding to calcium ions and to hydroxyapatite (HA) crystal surfaces. We used molecular dynamics (MD) simulations as a method for virtually screening interactions between DPP motifs and HA. The goal was to determine which motifs are absorbed to HA surfaces. For these simulations, we considered five peptides from the human DPP sequence. All-atom MD simulations were performed using GROMACS, the peptides were oriented parallel to the {100} HA crystal surface, the distance between the HA and the peptide was 3 nm. The system was simulated for 20 ns. Preliminary results show that for the unphosphorylated peptides, the acidic amino acids present an electrostatic attraction where their side chains are oriented towards HA. This attraction, however, is slow to facilitate bulk transport to the crystal surface. On the other hand, the phosphorylated (PP) peptides are rapidly absorbed on the surface of the HA with their centers of mass closer to the HA surface. More importantly, the root mean square fluctuation (RMSF) indicates that the average structures of the phosphorylated peptides are very inflexible and elongate, while that of the unphosphorylated peptides are flexible. Radius of gyration (Rg) analysis showed the compactness of un-phosphorylated peptides is lower than phosphorylated peptides. Phosphorylation of the DPP peptides is necessary for binding to HA surfaces. PMID:25158198

Characterization of Multiwalled Carbon Nanotube-Reinforced Hydroxyapatite Composites Consolidated by Spark Plasma Sintering

PubMed Central

Kim, Duk-Yeon; Han, Young-Hwan; Lee, Jun Hee; Kang, Inn-Kyu; Jang, Byung-Koog; Kim, Sukyoung

2014-01-01

Pure HA and 1, 3, 5, and 10 vol% multiwalled carbon nanotube- (MWNT-) reinforced hydroxyapatite (HA) were consolidated using a spark plasma sintering (SPS) technique. The relative density of pure HA increased with increasing sintering temperature, but that of the MWNT/HA composite reached almost full density at 900°C, and then decreased with further increases in sintering temperature. The relative density of the MWNT/HA composites increased with increasing MWNT content due to the excellent thermal conductivity of MWNTs. The grain size of MWNT/HA composites decreased with increasing MWNT content and increased with increasing sintering temperature. Pull-out toughening of the MWNTs of the MWNT/HA composites was observed in the fractured surface, which can be used to predict the improvement of the mechanical properties. On the other hand, the existence of undispersed or agglomerate MWNTs in the MWNT/HA composites accompanied large pores. The formation of large pores increased with increasing sintering temperature and MWNT content. The addition of MWNT in HA increased the hardness and fracture toughness by approximately 3~4 times, despite the presence of large pores produced by un-dispersed MWNTs. This provides strong evidence as to why the MWNTs are good candidates as reinforcements for strengthening the ceramic matrix. The MWNT/HA composites did not decompose during SPS sintering. The MWNT-reinforced HA composites were non-toxic and showed a good cell affinity and morphology in vitro for 1 day. PMID:24724100

Engineering of pulsed laser deposited calcium phosphate biomaterials in controlled atmospheres

NASA Astrophysics Data System (ADS)

Drukteinis, Saulius E.

Synthetic calcium phosphates (CAP) such as hydroxyapatite (HA) have been used as regenerative bone graft materials and also as thin films to improve the integration of biomedical implant devices within skeletal tissue. Pulsed laser deposition (PLD) can deposit crystalline HA with significant adhesion on titanium biomaterials. However, there are PLD processing constraints due to the complex physical and chemical interactions occurring simultaneously during PLD, which influence ablation plume formation and development. In this investigation PLD CAP films were engineered with a focus on novel decoupling of partial pressure of H2O (g) ( PH2O ) from total background pressure, in combination with substrate heat treatment and laser energy density control. Characterization of these films was performed with X-ray Diffraction, Scanning Electron Microscopy, Energy Dispersive X-ray Spectroscopy, Fourier Transform Infrared Spectroscopy, and Optical Profilometry. In vitro cellular adhesion testing was also performed using osteoblast (MC3T3) cell lines to evaluate adhesion of bone-forming cells on processed PLD CAP samples. Preferred a-axis orientation films were deposited in H2O (g) saturated atmospheres with reduced laser fluence (< 4 J/cm2). Crystalline HA/tetracalcium phosphate (TTCP) films were deposited in H2O ( g)-deficient atmospheres with higher laser fluence (> 3 J/cm 2). Varied PH2O resulted in control of biphasic HA/TTCP composition with increasing TTCP at lower PH2O . These were dense continuous films composed of micron-scale particles. Cellular adhesion assays did not demonstrate a significant difference between osteoblast adhesion density on HA films compared with biphasic HA/TTCP films. Room temperature PLD at varied PH2O combined with furnace heat treatment resulted in controlled variation in surface amplitude parameters including surface roughness (S a), root mean square (Sq), peak to valley height (St), and ten-point height ( Sz). These discontinuous films were composed of nano-scale particles and resulted in significant osteoblast adhesion compared to control samples or to PLD CAP films deposited on heated substrates. Surface amplitude parameters (Sa, Sq, St, and Sz) correlated with osteoblast adhesion. This new approach of control over H2O ( g) operating atmospheres enabled the deposition of unique PLD CAP films with potential use as thin films for biomedical implants or as regenerative bone graft materials. Keywords: hydroxyapatite, pulsed laser deposition, biomaterials.

Preparation of nano-hydroxyapatite particles with different morphology and their response to highly malignant melanoma cells in vitro

NASA Astrophysics Data System (ADS)

Li, Bo; Guo, Bo; Fan, Hongsong; Zhang, Xingdong

2008-11-01

To investigate the effects of nano-hydroxyapatite (HA) particles with different morphology on highly malignant melanoma cells, three kinds of HA particles with different morphology were synthesized and co-cultured with highly malignant melanoma cells using phosphate-buffered saline (PBS) as control. A precipitation method with or without citric acid addition as surfactant was used to produce rod-like hydroxyapatite (HA) particles with nano- and micron size, respectively, and a novel oil-in-water emulsion method was employed to prepare ellipse-like nano-HA particles. Particle morphology and size distribution of the as prepared HA powders were characterized by transmission electron microscope (TEM) and dynamic light scattering technique. The nano- and micron HA particles with different morphology were co-cultured with highly malignant melanoma cells. Immunofluorescence analysis and MTT assay were employed to evaluate morphological change of nucleolus and proliferation of tumour cells, respectively. To compare the effects of HA particles on cell response, the PBS without HA particles was used as control. The experiment results indicated that particle nanoscale effect rather than particle morphology of HA was more effective for the inhibition on highly malignant melanoma cells proliferation.

Biochemical basis of 4-hydroxyanisole induced cell toxicity towards B16-F0 melanoma cells.

PubMed

Moridani, Majid Y

2006-11-18

In the current work we investigated for the first time the biochemical basis of 4-hydroxyanisole (4-HA) induced toxicity in B16-F0 melanoma cells. It was found that dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HA induced toxicity towards B16-F0 cells whereas dithiothreitol, a thiol containing agent, and ascorbic acid (AA), a reducing agent, largely prevented 4-HA toxicity. TEMPOL and pyrogallol, free radical scavengers, did not significantly prevent 4-HA toxicity towards B16-F0 cells. GSH>AA>NADH prevented the o-quinone formation when 4-HA was metabolized by tyrosinase/O(2). 4-HA metabolism by horseradish peroxidase/H(2)O(2) was prevented more effectively by AA than NADH>GSH. We therefore concluded that quinone formation was the major pathway for 4-HA induced toxicity in B16-F0 melanoma cells whereas free radical formation played a negligible role in the 4-HA induced toxicity.

Evaluation of thermal stress in hydroxyapatite film fabricated by powder jet deposition.

PubMed

Akatsuka, Ryo; Matsumura, Ken; Noji, Miyoko; Kuriyagawa, Tsunemoto; Sasaki, Keiichi

2013-10-01

This study aimed to create a thick hydroxyapatite (HA) film on the surface of a human tooth via a powder jet deposition (PJD) device for dental handpieces, and to examine the microstructural and mechanical properties of the HA film. In particular, the effects of thermal stress on this film were evaluated. The HA film was created by blasting 3.18-μm HA particles, calcinated at 1,200°C, onto the enamel substrate at room temperature and atmospheric pressure. An HA film with an area of 3 mm × 3 mm was prepared and polished. The following HA film parameters were evaluated from the three-dimensional surface profile: surface roughness, Vickers hardness, and bonding strength before and after artificial aging induced by 500 cycles of thermal cycling (5-55°C). The HA particles in the deposited film were densely packed, and the surface of the HA film was unchanged after thermal cycling. There were also no significant differences in the hardness and the bonding strength of the HA film before and after thermal cycling. The HA film created in this study demonstrated excellent microstructural and mechanical properties, even after the application of thermal stress. © 2013 Eur J Oral Sci.

Hyaluronic acid based hydrogel system for soft tissue regeneration and drug delivery

NASA Astrophysics Data System (ADS)

Jha, Amit Kumar

We have developed hyaluronic acid (HA)-based, biomimetic hydrogel matrices that are hierarchically structured, mechanically robust and biologically active. Specifically, HA-based hydrogel particles (HGPs) with controlled sizes, defined porosity, and improved stability were synthesized using different inverse emulsion systems and crosslinking chemistries. The resultant particles either contained residual functional groups or were rendered reactive by subsequent chemical modifications. HA-based doubly crosslinked networks (DXNs) were synthesized via covalent crosslinking of HA HGPs with soluble HA macromers carrying mutually reactive functional groups. These hybrid matrices are hierarchical in nature, consisting of densely crosslinked HGPs integrated in a loosely connected secondary matrix. Their mechanical properties and degradation kinetics can be readily tuned by varying the particle size, functional group density, intra- and interparticle crosslinking. To improve the biological functions of HA HGPs, perlecan domain I (PlnDI), a basement membrane proteoglycan that has strong affinity for various heparin binding growth factors (HBGFs), was successfully conjugated to the particles through the core protein via a flexible poly(ethylene glycol) (PEG) linker. The immobilized PlnDI maintains its ability to bind bone morphogenetic proteins (BMP-2) and modulates its in vitro release. A similar, sustained release of BMP-2 was achieved by encapsulating BMP-2-loaded HGPs within a photocrosslinked HA matrix. When encapsulated in HA DXNs, primary bovine chondrocytes were able to maintain their phenotype, proliferate readily and produce abundant glycosaminoglycan. Finally, cell-adhesive HA DXNs were fabricated by encapsulating gelatin-decorated HA HGPs in a secondary HA matrix. Human MSCs were shown to adhere to the composite matrix through the focal adhesion sites clustered on particle surface. The cell-adhesive composite matrices supported hMSC proliferation and migration into the gels. Human MSCs were undifferentiated during the early time points of culture, however differentiated into osteoblast phenotype after 28 days of culture. In summary, the HA-based hydrogel matrices are hierarchically structured, mechanically robust and enzymatically stable, capable of mediating cellular functions through the spatial and temporal presentation of defined biological cues. These hydrogel systems are promising candidates for soft tissue regeneration.

Response of transformed and normal mouse cell lines to anti-melanin compounds, hyperthermia, and radiation.

PubMed

Raaphorst, G P; Azzam, E I

1992-02-01

Five cell lines (one parental, two transformed melanin producing, and two transformed non-melanin producing) were evaluated for the responses to 2- and 4-hydroxyanisole (2HA, 4HA) alone or combined with hyperthermia or radiation. All cells exhibited a non-specific toxic response to the two compounds and the effect was exposure time and concentration dependent and was greater for 4HA compared to 2HA. In addition, the two melanin-producing cell lines were more sensitive, demonstrating specific toxicity to such cell lines. The treatment with either 2HA or 4HA combined with heat and radiation resulted mostly in additive or antagonistic effects, except for one combination of 2HA plus radiation in the melanin-producing R25 cells. Thus, while these compounds may be useful in therapy for pigmented melanomas, combined treatment with radiation is not recommended.

Hydroxyapatite coating on cobalt alloys using electrophoretic deposition method for bone implant application

NASA Astrophysics Data System (ADS)

Aminatun; M, Shovita; I, Chintya K.; H, Dyah; W, Dwi

2017-05-01

Damage on bone due to osteoporosis and cancer triggered high demand for bone implant prosthesis which is a permanent implant. Thus, a prosthesis coated with hydroxyapatite (HA) is required because it is osteoconductive that can trigger the growth of osteoblast cells. The purpose of this study is to determine the optimum concentration of HA suspension in terms of the surface morphology, coating thickness, adhesion strength and corrosion rate resulting in the HA coating with the best characteristics for bone implant. Coating using electrophoretic deposition (EPD) method with concentrations of 0.02M, 0.04M, 0.06M, 0.08M, and 0.1M was performed on the voltage and time of 120V and 30 minutes respectively. The process was followed by sintering at the temperature of 900 °C for 10 minutes. The results showed that the concentration of HA suspension influences the thickness and the adhesion of layer of HA. The higher the concentration of HA-ethanol suspension the thicker the layer of HA, but its coating adhesion strength values became lower. The concentration of HA suspension of 0.04 M is the best concentration, with characteristics that meet the standards of the bone implant prosthesis. The characteristics are HA coating thickness of 199.93 ± 4.85 μm, the corrosion rate of 0.0018 mmpy and adhesion strength of 4.175 ± 0.716 MPa.

Wide-range stiffness gradient PVA/HA hydrogel to investigate stem cell differentiation behavior.

PubMed

Oh, Se Heang; An, Dan Bi; Kim, Tae Ho; Lee, Jin Ho

2016-04-15

Although stiffness-controllable substrates have been developed to investigate the effect of stiffness on cell behavior and function, the use of separate substrates with different degrees of stiffness, substrates with a narrow range stiffness gradient, toxicity of residues, different surface composition, complex fabrication procedures/devices, and low cell adhesion are still considered as hurdles of conventional techniques. In this study, a cylindrical polyvinyl alcohol (PVA)/hyaluronic acid (HA) hydrogel with a wide-range stiffness gradient (between ∼20kPa and ∼200kPa) and cell adhesiveness was prepared by a liquid nitrogen (LN2)-contacting gradual freezing-thawing method that does not use any additives or specific devices to produce the stiffness gradient hydrogel. From an in vitro cell culture using the stiffness gradient PVA/HA hydrogel, it was observed that human bone marrow mesenchymal stem cells have favorable stiffness ranges for induction of differentiation into specific cell types (∼20kPa for nerve cell, ∼40kPa for muscle cell, ∼80kPa for chondrocyte, and ∼190kPa for osteoblast). The PVA/HA hydrogel with a wide range of stiffness spectrum can be a useful tool for basic studies related with the stem cell differentiation, cell reprogramming, cell migration, and tissue regeneration in terms of substrate stiffness. It is postulated that the stiffness of the extracellular matrix influences cell behavior. To prove this concept, various techniques to prepare substrates with a stiffness gradient have been developed. However, the narrow ranges of stiffness gradient and complex fabrication procedures/devices are still remained as limitations. Herein, we develop a substrate (hydrogel) with a wide-range stiffness gradient using a gradual freezing-thawing method which does not need specific devices to produce a stiffness gradient hydrogel. From cell culture experiments using the hydrogel, it is observed that human bone marrow mesenchymal stem cells have favorable stiffness ranges for induction of differentiation into specific cell types (∼20kPa for nerve, ∼40kPa for muscle, ∼80kPa for cartilage, and ∼190kPa for bone in our hydrogel system). Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Reduction of Streptococcus mutans adherence and dental biofilm formation by surface treatment with phosphorylated polyethylene glycol.

PubMed

Shimotoyodome, Akira; Koudate, Takashi; Kobayashi, Hisataka; Nakamura, Junji; Tokimitsu, Ichiro; Hase, Tadashi; Inoue, Takashi; Matsukubo, Takashi; Takaesu, Yoshinori

2007-10-01

Initial attachment of the cariogenic Streptococcus mutans onto dental enamel is largely promoted by the adsorption of specific salivary proteins on enamel surface. Some phosphorylated salivary proteins were found to reduce S. mutans adhesion by competitively inhibiting the adsorption of S. mutans-binding salivary glycoproteins to hydroxyapatite (HA). The aim of this study was to develop antiadherence compounds for preventing dental biofilm development. We synthesized phosphorylated polyethylene glycol (PEG) derivatives and examined the possibility of surface pretreatment with them for preventing S. mutans adhesion in vitro and dental biofilm formation in vivo. Pretreatment of the HA surface with methacryloyloxydecyl phosphate (MDP)-PEG prior to saliva incubation hydrophilized the surface and thereby reduced salivary protein adsorption and saliva-promoted bacterial attachment to HA. However, when MDP-PEG was added to the saliva-pretreated HA (S-HA) surface, its inhibitory effect on bacterial binding was completely diminished. S. mutans adhesion onto S-HA was successfully reduced by treatment of the surface with pyrophosphate (PP), which desorbs salivary components from S-HA. Treatment of S-HA surfaces with MDP-PEG plus PP completely inhibited saliva-promoted S. mutans adhesion even when followed by additional saliva treatment. Finally, mouthwash with MDP-PEG plus PP prevented de novo biofilm development after thorough teeth cleaning in humans compared to either water or PP alone. We conclude that MDP-PEG plus PP has the potential for use as an antiadherence agent that prevents dental biofilm development.

Characterization, Corrosion Resistance, and Cell Response of High-Velocity Flame-Sprayed HA and HA/TiO2 Coatings on 316L SS

NASA Astrophysics Data System (ADS)

Singh, Tejinder Pal; Singh, Harpreet; Singh, Hazoor

2012-09-01

The main aim of this study is to evaluate corrosion and biocompatibility behavior of thermal spray hydroxyapatite (HA) and hydroxyapatite/titania bond (HA/TiO2)-coated 316L stainless steel (316L SS). In HA/TiO2 coatings, TiO2 was used as a bond coat between HA top coat and 316L SS substrate. The coatings were characterized by x-ray diffraction and scanning electron microscopy/energy dispersive spectroscopy, and corrosion resistance determined for the uncoated substrate and the two coatings. The biological behavior was investigated by the cell culture studies using osteosarcoma cell line KHOS-NP (R-970-5). The corrosion resistance of the steel was found to increase after the deposition of the HA and HA/TiO2 bond coatings. Both HA, as well as, HA/TiO2 coatings exhibit excellent bond strength of 49 and 47 MPa, respectively. The cell culture studies showed that HA-coated 316L SS specimens appeared more biocompatible than the uncoated and HA/TiO2-coated 316L SS specimens.

Mechanical, In Vitro Antimicrobial and Biological Properties of Plasma Sprayed Silver-Doped Hydroxyapatite Coating

PubMed Central

Roy, Mangal; Fielding, Gary A.; Beyenal, Haluk; Bandyopadhyay, Amit; Bose, Susmita

2012-01-01

Implant related infection is one of the key concerns in total joint hip arthroplasties. In order to reduce bacterial adhesion, silver (Ag) / silver oxide (Ag2O) doping was used in plasma sprayed hydroxyapatite (HA) coating on titanium substrate. HA powder was doped with 2.0, 4.0 and 6.0 wt% Ag, heat treated at 800 °C and used for plasma spray coating using a 30 kW plasma spray system, equipped with supersonic nozzle. Application of supersonic plasma nozzle significantly reduced phase decomposition and amorphous phase formation in the HA coatings as evident by X-ray diffraction (XRD) study and Fourier transformed infrared spectroscopic (FTIR) analysis. Adhesive bond strength of more than 15 MPa ensured the mechanical integrity of the coatings. Resistance against bacterial adhesion of the coatings was determined by challenging them against Pseudomonas Aeruginosa (PAO1). Live/Dead staining of the adherent bacteria on the coating surfaces indicated a significant reduction in bacterial adhesion due to the presence of Ag. In vitro cell-material interactions and alkaline phosphatase (ALP) protein expressions were evaluated by culturing human fetal osteoblast cells (hFOB). Present results suggest that the plasma sprayed HA coatings doped with an optimum amount of Ag can have excellent antimicrobial property without altering mechanical property of the Ag doped HA coatings. PMID:22313742

A Single Point Mutation Resulting in Cadherin Mislocalization Underpins Resistance against Bacillus thuringiensis Toxin in Cotton Bollworm*

PubMed Central

Xiao, Yutao; Dai, Qing; Hu, Ruqin; Pacheco, Sabino; Yang, Yongbo; Liang, Gemei; Soberón, Mario

2017-01-01

Transgenic plants that produce Bacillus thuringiensis (Bt) crystalline (Cry) toxins are cultivated worldwide to control insect pests. Resistance to B. thuringiensis toxins threatens this technology, and although different resistance mechanisms have been identified, some have not been completely elucidated. To gain new insights into these mechanisms, we performed multiple back-crossing from a 3000-fold Cry1Ac-resistant BtR strain from cotton bollworm (Helicoverpa armigera), isolating a 516-fold Cry1Ac-resistant strain (96CAD). Cry1Ac resistance in 96CAD was tightly linked to a mutant cadherin allele (mHaCad) that contained 35 amino acid substitutions compared with HaCad from a susceptible strain (96S). We observed significantly reduced levels of the mHaCad protein on the surface of the midgut epithelium in 96CAD as compared with 96S. Expression of both cadherin alleles from 96CAD and 96S in insect cells and immunofluorescence localization in insect midgut tissue sections showed that the HaCAD protein from 96S localizes on the cell membrane, whereas the mutant 96CAD-mHaCad was retained in the endoplasmic reticulum (ER). Mapping of the mutations identified a D172G substitution mainly responsible for cadherin mislocalization. Our finding of a mutation affecting membrane receptor trafficking represents an unusual and previously unrecognized B. thuringiensis resistance mechanism. PMID:28082675

Mechanical, in vitro antimicrobial, and biological properties of plasma-sprayed silver-doped hydroxyapatite coating.

PubMed

Roy, Mangal; Fielding, Gary A; Beyenal, Haluk; Bandyopadhyay, Amit; Bose, Susmita

2012-03-01

Implant-related infection is one of the key concerns in total joint hip arthroplasties. To reduce bacterial adhesion, we used silver (Ag)/silver oxide (Ag(2)O) doping in plasma sprayed hydroxyapatite (HA) coating on titanium substrate. HA powder was doped with 2.0, 4.0, and 6.0 wt % Ag, heat-treated at 800 °C and used for plasma spray coating using a 30 kW plasma spray system, equipped with supersonic nozzle. Application of supersonic plasma nozzle significantly reduced phase decomposition and amorphous phase formation in the HA coatings as evident by X-ray diffraction (XRD) study and Fourier transformed infrared spectroscopic (FTIR) analysis. Adhesive bond strength of more than 15 MPa ensured the mechanical integrity of the coatings. Resistance against bacterial adhesion of the coatings was determined by challenging them against Pseudomonas aeruginosa (PAO1). Live/dead staining of the adherent bacteria on the coating surfaces indicated a significant reduction in bacterial adhesion due to the presence of Ag. In vitro cell-material interactions and alkaline phosphatase (ALP) protein expressions were evaluated by culturing human fetal osteoblast cells (hFOB). Our results suggest that the plasma-sprayed HA coatings doped with an optimum amount of Ag can have excellent antimicrobial property without altering mechanical property of the Ag-doped HA coatings. © 2012 American Chemical Society

Synthesis and Characterization of Functional Nanofilm-Coated Live Immune Cells.

PubMed

Hwang, Jangsun; Choi, Daheui; Choi, Moonhyun; Seo, Youngmin; Son, Jaewoo; Hong, Jinkee; Choi, Jonghoon

2018-05-30

Layer-by-layer (LbL) assembly techniques have been extensively studied in cell biology because of their simplicity of preparation and versatility. The applications of the LbL platform technology using polysaccharides, silicon, and graphene have been investigated. However, the applications of the above-mentioned technology using living cells remain to be fully understood. This study demonstrates a living cell-based LbL platform using various types of living cells. In addition, it confirms that the surplus charge on the outer surface of the coated cells can be used to bind the target protein. We develop a living cell-based LbL platform technology by stacking layers of hyaluronic acid (HA) and poly-l-lysine (PLL). The HA/PLL stacking results in three bilayers with a thickness of 4 ± 1 nm on the cell surface. Furthermore, the multilayer nanofilms on the cells are completely degraded after 3 days of the application of the LbL method. We also evaluate and visualize three bilayers of the nanofilm on adherent (AML-12 cells)-, nonadherent (trypsin-treated AML-12 cells)-, and circulation type [peripheral blood mononuclear cells (PBMCs)] cells by analyzing the zeta potential, cell viability, and imaging via scanning electron microscopy and confocal microscopy. Finally, we study the cytotoxicity of the nanofilm and characteristic functions of the immune cells after the nanofilm coating. The multilayer nanofilms are not acutely cytotoxic and did not inhibit the immune response of the PBMCs against stimulant. We conclude that a two bilayer nanofilm would be ideal for further study in any cell type. The living cell-based LbL platform is expected to be useful for a variety of applications in cell biology.

Osteogenic differentiation on DLC-PDMS-h surface.

PubMed

Soininen, Antti; Kaivosoja, Emilia; Sillat, Tarvo; Virtanen, Sannakaisa; Konttinen, Yrjö T; Tiainen, Veli-Matti

2014-10-01

The hypothesis was that anti-fouling diamond-like carbon polydimethylsiloxane hybrid (DLC-PDMS-h) surface impairs early and late cellular adhesion and matrix-cell interactions. The effect of hybrid surface on cellular adhesion and cytoskeletal organization, important for osteogenesis of human mesenchymal stromal cells (hMSC), where therefore compared with plain DLC and titanium (Ti). hMSCs were induced to osteogenesis and followed over time using scanning electron microscopy (SEM), time-of-flight secondary ion mass spectrometry (ToF-SIMS), immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR), and hydroxyapatite (HA) staining. SEM at 7.5 hours showed that initial adherence and spreading of hMSC was poor on DLC-PDMS-h. At 5 days some hMSC were undergoing condensation and apoptotic fragmentation, whereas cells on DLC and Ti grew well. DAPI-actin-vinculin triple staining disclosed dwarfed cells with poorly organized actin cytoskeleton-focal complex/adhesion-growth substrate attachments on hybrid coating, whereas spread cells, organized microfilament bundles, and focal adhesions were seen on DLC and in particular on Ti. Accordingly, at day one ToF-SIMS mass peaks showed poor protein adhesion to DLC-PDMS-h compared with DLC and Ti. COL1A1, ALP, OP mRNA levels at days 0, 7, 14, 21, and/or 28 and lack of HA deposition at day 28 demonstrated delayed or failed osteogenesis on DLC-PDMS-h. Anti-fouling DLC-PDMS-h is a poor cell adhesion substrate during the early protein adsorption-dependent phase and extracellular matrix-dependent late phase. Accordingly, some hMSCs underwent anoikis-type apoptosis and failed to complete osteogenesis, due to few focal adhesions and poor cell-to-ECM contacts. DLC-PDMS-h seems to be a suitable coating for non-integrating implants/devices designed for temporary use. © 2014 Wiley Periodicals, Inc.

Adsorption and bioactivity studies of albumin onto hydroxyapatite surface.

PubMed

Mavropoulos, Elena; Costa, Andréa M; Costa, Lilian T; Achete, Carlos A; Mello, Alexandre; Granjeiro, José M; Rossi, Alexandre M

2011-03-01

Bovine serum albumin (BSA) may have an inhibitory or promoter effect on hydroxyapatite (HA) nucleation when apatite is precipitated in a medium containing the protein. In this study we evaluated the influence of BSA on the precipitation of calcium phosphate phases (CP) from simulated body fluid (SBF) when the protein was previously bounded to HA surface. The kinetics of BSA immobilization onto hydroxyapatite surface was performed in different buffers and protein concentrations in order to adjust experimental conditions in which BSA was tightly linked to HA surface for long periods in SBF solution. It was shown that for BSA concentration higher than 0.1mg/mL the adsorption to HA surface followed Langmuir-Freundlich mechanisms, which confirmed the existence of cooperative protein-protein interactions on HA surface. Fourier Transformed Infrared Attenuated Total Reflectance Microscopy (FTIRM-ATR) evidenced changes in BSA conformational state in favor of less-ordered structure. Analyses from high resolution grazing incident X-ray diffraction using synchrotron radiation (GIXRD), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) showed that a poorly crystalline calcium phosphate was precipitated on the surface of HA discs coated with BSA, after the immersion in SBF for 4 days. The new bioactive layer had morphological characteristics similar to the one formed on the HA surface without protein. It was identified as a carbonated apatite with preferential crystal growth along apatite 002 direction. The GIXRD results also revealed that BSA layer bound to the surface inhibited the HA dissolution leading to a reduction on the formation of new calcium phosphate phase. 2010 Elsevier B.V. All rights reserved.

Bone integration capability of nanopolymorphic crystalline hydroxyapatite coated on titanium implants

PubMed Central

Yamada, Masahiro; Ueno, Takeshi; Tsukimura, Naoki; Ikeda, Takayuki; Nakagawa, Kaori; Hori, Norio; Suzuki, Takeo; Ogawa, Takahiro

2012-01-01

The mechanism by which hydroxyapatite (HA)-coated titanium promotes bone–implant integration is largely unknown. Furthermore, refining the fabrication of nano-structured HA to the level applicable to the mass production process for titanium implants is challenging. This study reports successful creation of nanopolymorphic crystalline HA on microroughened titanium surfaces using a combination of flame spray and low-temperature calcination and tests its biological capability to enhance bone–implant integration. Sandblasted microroughened titanium implants and sandblasted + HA-coated titanium implants were subjected to biomechanical and histomorphometric analyses in a rat model. The HA was 55% crystallized and consisted of nanoscale needle-like architectures developed in various diameters, lengths, and orientations, which resulted in a 70% increase in surface area compared to noncoated microroughened surfaces. The HA was free from impurity contaminants, with a calcium/phosphorus ratio of 1.66 being equivalent to that of stoichiometric HA. As compared to microroughened implants, HA-coated implants increased the strength of bone–implant integration consistently at both early and late stages of healing. HA-coated implants showed an increased percentage of bone–implant contact and bone volume within 50 μm proximity of the implant surface, as well as a remarkably reduced percentage of soft tissue intervention between bone and the implant surface. In contrast, bone volume outside the 50 μm border was lower around HA-coated implants. Thus, this study demonstrated that the addition of pure nanopolymorphic crystalline HA to microroughened titanium not only accelerates but also enhances the level of bone–implant integration and identified the specific tissue morphogenesis parameters modulated by HA coating. In particular, the nanocrystalline HA was proven to be drastic in increasing osteoconductivity and inhibiting soft tissue infiltration, but the effect was limited to the immediate microenvironment surrounding the implant. PMID:22359461

Intravirion cohesion of matrix protein M1 with ribonucleocapsid is a prerequisite of influenza virus infectivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhirnov, O.P., E-mail: zhirnov@inbox.ru; Manykin, A.A.; Rossman, J.S.

Influenza virus has two major structural modules, an external lipid envelope and an internal ribonucleocapsid containing the genomic RNA in the form of the ribonucleoprotein (RNP) complex, both of which are interlinked by the matrix protein M1. Here we studied M1-RNP cohesion within virus exposed to acidic pH in vitro. The effect of acidification was dependent on the cleavage of the surface glycoprotein HA. Acidic pH caused a loss of intravirion RNP-M1 cohesion and activated RNP polymerase activity in virus with cleaved HA (HA1/2) but not in the uncleaved (HA0) virus. The in vitro acidified HA1/2 virus rapidly lost infectivitymore » whereas the HA0 one retained infectivity, following activation by trypsin, suggesting that premature activation and release of the RNP is detrimental to viral infectivity. Rimantadine, an inhibitor of the M2 ion channel, was found to protect the HA1/2 virus interior against acidic disintegration, confirming that M2-dependent proton translocation is essential for the intravirion RNP release and suggesting that the M2 ion channel is only active in virions with cleaved HA. Acidic treatment of both HA0 and HA1/2 influenza viruses induces formation of spikeless bleb-like protrusion of ~25 nm in diameter on the surface of the virion, though only the HA1/2 virus was permeable to protons and permitted RNP release. It is likely that this bleb corresponds to the M2-enriched and M1-depleted focus arising from pinching off of the virus during the completion of budding. Cooperatively, the data suggest that the influenza virus has an asymmetric structure where the M1-mediated organization of the RNP inside the virion is a prerequisite for infectious entry into target cell. - Highlights: • The influenza A virus has a novel asymmetric internal structure. • The structure is largely maintained by M1-RNP cohesion within the virion. • This asymmetry plays an important role during viral entry, facilitating virus uncoating and the initiation of a productive infection.« less

Investigation of boundary conditions for biomimetic HA deposition on titanium oxide surfaces.

PubMed

Lindgren, M; Astrand, M; Wiklund, U; Engqvist, H

2009-07-01

To improve the clinical outcome of metal implants, i.e. earlier loading and reduction of the incidence of revision surgery, better bone bonding ability is wanted. One method to achieve this is to change the surface chemistry to give a surface that facilitates bone bonding in vivo, i.e. a bioactive surface. Crystalline titanium oxide has recently been proven to be bioactive in vitro and is an interesting option to the more common hydroxylapatite (HA) coatings on implants. A materials possible in vitro bioactivity is tested through soaking in simulated body fluid and studies of possible HA formation on the surface. For bioactive materials, the formed HA layer can also be used as a coating. The aim of the current paper is to investigate some boundary conditions for HA formation on crystalline titanium oxide surfaces regarding influence from coating thickness, soaking time and soaking temperature. The influence from soaking time and temperature on the HA growth were investigated on oxidised Ti samples, (24 h at 800 degrees C) resulting in a rutile surface structure. The oxidised samples were tested at three temperatures (4, 37 and 65 degrees C) and four times (1 h, 1 day, 1 week and 4 weeks). The influence from titanium coating thickness on the HA growth was investigated via depositing thin films of crystalline titanium dioxide on Ti plates using a reactive magnetron sputtering process. Four different PVD runs with coating thicknesses between 19 and 74 nm were tested. The soaking temperature had an effect on the HA formation and growth on both rutile surfaces and native oxide on Ti substrates. Higher temperatures lead to easier formation of HA. It was even possible, at 65 degrees C, to grow HA on native titanium oxide from soaking in PBS. The coating quality was better for HA formed at 65 degrees C compared to 37 degrees C. All PVD-coatings showed HA growth after 1 week in PBS at 37 degrees C, thus even very thin coatings of crystalline titanium oxide coatings are bioactive.

Tumor-associated mesenchymal stem-like cells provide extracellular signaling cue for invasiveness of glioblastoma cells

PubMed Central

Yoo, Ki-Chun; Lee, Ji-Hyun; Kim, In-Gyu; Kim, Min-Jung; Chang, Jong Hee; Kang, Seok-Gu; Lee, Su-Jae

2017-01-01

Hyaluronic acid (HA) is abundant in tumor microenvironment and closely associated with invasiveness of glioblastoma (GBM) cells. However, the cellular mechanism underlying HA-rich microenvironment in GBM remains unexplored. Here, we show that tumor-associated mesenchymal stem-like cells (tMSLCs) contribute to abundance of hyaluronic acid (HA) in tumor microenvironment through HA synthase-2 (HAS2) induction, and thereby enhances invasiveness of GBM cells. In an autocrine manner, C5a secreted by tMSLCs activated ERK MAPK for HAS2 induction in tMSLCs. Importantly, HA acted as a signaling ligand of its cognate receptor RHAMM for intracellular signaling activation underlying invasiveness of GBM cells. Taken together, our study suggests that tMSLCs contribute to HA-rich proinvasive ECM microenvironment in GBM. PMID:27903965

Hydroxyapatite grafted carbon nanotubes and graphene nanosheets: Promising bone implant materials

NASA Astrophysics Data System (ADS)

Oyefusi, Adebola; Olanipekun, Opeyemi; Neelgund, Gururaj M.; Peterson, Deforest; Stone, Julia M.; Williams, Ebonee; Carson, Laura; Regisford, Gloria; Oki, Aderemi

2014-11-01

In the present study, hydroxyapatite (HA) was successfully grafted to carboxylated carbon nanotubes (CNTs) and graphene nanosheets. The HA grafted CNTs and HA-graphene nanosheets were characterized using FT-IR, TGA, SEM and X-ray diffraction. The HA grafted CNTs and graphene nanosheets (CNTs-HA and Gr-HA) were further used to examine the proliferation and differentiation rate of temperature-sensitive human fetal osteoblastic cell line (hFOB 1.19). Total protein assays and western blot analysis of osteocalcin expression were used as indicators of cell proliferation and differentiation. Results indicated that hFOB 1.19 cells proliferate and differentiate well in treatment media containing CNTs-HA and graphene-HA. Both CNTs-HA and graphene-HA could be promising nanomaterials for use as scaffolds in bone tissue engineering.

Hydroxyapatite degradation and biocompatibility

NASA Astrophysics Data System (ADS)

Wang, Haibo

Hydroxyapatite (HA) is widely used as a bioactive ceramics since it forms a chemical bonding to bone. The disadvantage of this material is its poor mechanical properties. HA can be degraded in body, which is the reason for its bioactivity, but too fast degradation rate could cause negative effects, such as macrophage present, particle generation, and even implant clinical failure. HA degradation rate will be greatly changed under many conditions: purity, HA form (i.e. bulk form, porous form, coating, or HA/polymer composites), microstructure, implant site, body conditions, etc. Although much work has been done in HA properties and application areas, the HA degradation behavior and mechanism under these different conditions are still not clear. In this research, three aspects of HA degradation have been studied: (1) Two very common impurities---Tri-Calcium Phosphate (TCP) and Calcium Oxide and their influences on HA degradation in vitro and in vivo, (2) influence of HA/polymer composite form on HA degradation, (3) HA material particle generation and related mechanism. From the in vitro and in vivo tests on bulk HA disks with various Ca/P ratios, HA degradation can clearly be found. The degradation level is different in different Ca/P ratio samples as well as in different test environments. In same test environment, non-stoichiometric HA samples have higher degradation rate than stoichiometric HA. HA/PMMA composite design successfully intensifies HA degradation both in vitro and in vivo. Grain boundary damage can be found on in vivo test samples, which has not been clearly seen on bulk HA degraded surface. HA particle generation is found in in vitro and in vivo HA/PMMA composite surface and in vivo bulk HA surface. Sintering temperature and time does affect HA grain size, and this affect HA degradation rate. Intergranular fracture is found in a several micron zone close to the Ca/P ratio 1.62 and 1.67 sample degraded surfaces. At Ca/P ratio greater than 1.667, after HA degradation in water, solution pH increases because of CaO presence.

Surface characteristics of a novel hydroxyapatite-coated dental implant

PubMed Central

Jung, Ui-Won; Hwang, Ji-Wan; Choi, Da-Yae; Hu, Kyung-Seok; Kwon, Mi-Kyung; Choi, Seong-Ho

2012-01-01

Purpose This study evaluated the surface characteristics and bond strength produced using a novel technique for coating hydroxyapatite (HA) onto titanium implants. Methods HA was coated on the titanium implant surface using a super-high-speed (SHS) blasting method with highly purified HA. The coating was performed at a low temperature, unlike conventional HA coating methods. Coating thickness was measured. The novel HA-coated disc was fabricated. X-ray diffraction analysis was performed directly on the disc to evaluate crystallinity. Four novel HA-coated discs and four resorbable blast medium (RBM) discs were prepared. Their surface roughnesses and areas were measured. Five puretitanium, RBM-treated, and novel HA-coated discs were prepared. Contact angle was measured. Two-way analysis of variance and the post-hoc Scheffe's test were used to analyze differences between the groups, with those with a probability of P<0.05 considered to be statistically significant. To evaluate exfoliation of the coating layer, 7 sites on the mandibles from 7 mongrel dogs were used. Other sites were used for another research project. In total, seven novel HA-coated implants were placed 2 months after extraction of premolars according to the manufacturer's instructions. The dogs were sacrificed 8 weeks after implant surgery. Implants were removed using a ratchet driver. The surface of the retrieved implants was evaluated microscopically. Results A uniform HA coating layer was formed on the titanium implants with no deformation of the RBM titanium surface microtexture when an SHS blasting method was used. Conclusions These HA-coated implants exhibited increased roughness, crystallinity, and wettability when compared with RBM implants. PMID:22586524

Scaffolds for bone regeneration made of hydroxyapatite microspheres in a collagen matrix.

PubMed

Cholas, Rahmatullah; Kunjalukkal Padmanabhan, Sanosh; Gervaso, Francesca; Udayan, Gayatri; Monaco, Graziana; Sannino, Alessandro; Licciulli, Antonio

2016-06-01

Biomimetic scaffolds with a structural and chemical composition similar to native bone tissue may be promising for bone tissue regeneration. In the present work hydroxyapatite mesoporous microspheres (mHA) were incorporated into collagen scaffolds containing an ordered interconnected macroporosity. The mHA were obtained by spray drying of a nano hydroxyapatite slurry prepared by the precipitation technique. X-ray diffraction (XRD) analysis revealed that the microspheres were composed only of hydroxyapatite (HA) phase, and energy-dispersive x-ray spectroscopy (EDS) analysis revealed the Ca/P ratio to be 1.69 which is near the value for pure HA. The obtained microspheres had an average diameter of 6 μm, a specific surface area of 40 m(2)/g as measured by Brunauer-Emmett-Teller (BET) analysis, and Barrett-Joyner-Halenda (BJH) analysis showed a mesoporous structure with an average pore diameter of 16 nm. Collagen/HA-microsphere (Col/mHA) composite scaffolds were prepared by freeze-drying followed by dehydrothermal crosslinking. SEM observations of Col/mHA scaffolds revealed HA microspheres embedded within a porous collagen matrix with a pore size ranging from a few microns up to 200 μm, which was also confirmed by histological staining of sections of paraffin embedded scaffolds. The compressive modulus of the composite scaffold at low and high strain values was 1.7 and 2.8 times, respectively, that of pure collagen scaffolds. Cell proliferation measured by the MTT assay showed more than a 3-fold increase in cell number within the scaffolds after 15 days of culture for both pure collagen scaffolds and Col/mHA composite scaffolds. Attractive properties of this composite scaffold include the potential to load the microspheres for drug delivery and the controllability of the pore structure at various length scales. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Ningbo, E-mail: curl-zhao@163.com; Wang, Xin, E-mail: 394041230@qq.com; Qin, Lei, E-mail: qinlei30@126.com

Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased bymore » increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation. - Highlights: • Effect of hyaluronan on cell proliferation and differentiation is evaluated in vitro. • Hyaluronan of low molecular weight increases cell proliferation. • Hyaluronan of high molecular weight promotes cell osteogenic differentiation. • Molecular weight and concentration of hyaluronan show interactive effect.« less

Selective pre-priming of HA-specific CD4 T cells restores immunological reactivity to HA on heterosubtypic influenza infection.

PubMed

Alam, Shabnam; Chan, Cory; Qiu, Xing; Shannon, Ian; White, Chantelle L; Sant, Andrea J; Nayak, Jennifer L

2017-01-01

A hallmark of the immune response to influenza is repeated encounters with proteins containing both genetically conserved and variable components. Therefore, the B and T cell repertoire is continually being remodeled, with competition between memory and naïve lymphocytes. Our previous work using a mouse model of secondary heterosubtypic influenza infection has shown that this competition results in a focusing of CD4 T cell response specificity towards internal virion proteins with a selective decrease in CD4 T cell reactivity to the novel HA epitopes. Strikingly, this shift in CD4 T cell specificity was associated with a diminished anti-HA antibody response. Here, we sought to determine whether the loss in HA-specific reactivity that occurs as a consequence of immunological memory could be reversed by selectively priming HA-specific CD4 T cells prior to secondary infection. Using a peptide-based priming strategy, we found that selective expansion of the anti-HA CD4 T cell memory repertoire enhanced HA-specific antibody production upon heterosubtypic infection. These results suggest that the potentially deleterious consequences of repeated exposure to conserved influenza internal virion proteins could be reversed by vaccination strategies that selectively arm the HA-specific CD4 T cell compartment. This could be a potentially useful pre-pandemic vaccination strategy to promote accelerated neutralizing antibody production on challenge with a pandemic influenza strain that contains few conserved HA epitopes.

Interactions of hyaluronan grafted on protein surfaces studied using a quartz crystal microbalance and a surface force balance.

PubMed

Jiang, Lei; Han, Juan; Yang, Limin; Ma, Hongchao; Huang, Bo

2015-10-07

Vocal folds are complex and multilayer-structured where the main layer is widely composed of hyaluronan (HA). The viscoelasticity of HA is key to voice production in the vocal fold as it affects the initiation and maintenance of phonation. In this study a simple layer-structured surface model was set up to mimic the structure of the vocal folds. The interactions between two opposing surfaces bearing HA were measured and characterised to analyse HA's response to the normal and shear compression at a stress level similar to that in the vocal fold. From the measurements of the quartz crystal microbalance, atomic force microscopy and the surface force balance, the osmotic pressure, normal interactions, elasticity change, volume fraction, refractive index and friction of both HA and the supporting protein layer were obtained. These findings may shed light on the physical mechanism of HA function in the vocal fold and the specific role of HA as an important component in the effective treatment of the vocal fold disease.

B and T lymphocyte attenuator mediates inhibition of tumor-reactive CD8+ T cells in patients after allogeneic stem cell transplantation.

PubMed

Hobo, Willemijn; Norde, Wieger J; Schaap, Nicolaas; Fredrix, Hanny; Maas, Frans; Schellens, Karen; Falkenburg, J H Frederik; Korman, Alan J; Olive, Daniel; van der Voort, Robbert; Dolstra, Harry

2012-07-01

Allogeneic stem cell transplantation (allo-SCT) can cure hematological malignancies by inducing alloreactive T cell responses targeting minor histocompatibility antigens (MiHA) expressed on malignant cells. Despite induction of robust MiHA-specific T cell responses and long-term persistence of alloreactive memory T cells specific for the tumor, often these T cells fail to respond efficiently to tumor relapse. Previously, we demonstrated the involvement of the coinhibitory receptor programmed death-1 (PD-1) in suppressing MiHA-specific CD8(+) T cell immunity. In this study, we investigated whether B and T lymphocyte attenuator (BTLA) plays a similar role in functional impairment of MiHA-specific T cells after allo-SCT. In addition to PD-1, we observed higher BTLA expression on MiHA-specific CD8(+) T cells compared with that of the total population of CD8(+) effector-memory T cells. In addition, BTLA's ligand, herpes virus entry mediator (HVEM), was found constitutively expressed by myeloid leukemia, B cell lymphoma, and multiple myeloma cells. Interference with the BTLA-HVEM pathway, using a BTLA blocking Ab, augmented proliferation of BTLA(+)PD-1(+) MiHA-specific CD8(+) T cells by HVEM-expressing dendritic cells. Notably, we demonstrated that blocking of BTLA or PD-1 enhanced ex vivo proliferation of MiHA-specific CD8(+) T cells in respectively 7 and 9 of 11 allo-SCT patients. Notably, in 3 of 11 patients, the effect of BTLA blockade was more prominent than that of PD-1 blockade. Furthermore, these expanded MiHA-specific CD8(+) T cells competently produced effector cytokines and degranulated upon Ag reencounter. Together, these results demonstrate that BTLA-HVEM interactions impair MiHA-specific T cell functionality, providing a rationale for interfering with BTLA signaling in post-stem cell transplantation therapies.

Electrophoretic deposition of graphene oxide reinforced chitosan-hydroxyapatite nanocomposite coatings on Ti substrate.

PubMed

Shi, Y Y; Li, M; Liu, Q; Jia, Z J; Xu, X C; Cheng, Y; Zheng, Y F

2016-03-01

Electrophoretic deposition (EPD) is a facile and feasible technique to prepare functional nanocomposite coatings for application in orthopedic-related implants. In this work, a ternary graphene oxide-chitosan-hydroxyapatite (GO-CS-HA) composite coating on Ti substrate was successfully fabricated by EPD. Coating microstructure and morphologies were investigated by scanning electron microscopy, contact angle test, Raman spectroscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis. It was found GO-CS surface were uniformly decorated by HA nanoparticles. The potentiodynamic polarization test in simulated body fluid indicated that the GO-CS-HA coatings could provide effective protection of Ti substrate from corrosion. This ternary composite coating also exhibited good biocompatibility during incubation with MG63 cells. In addition, the nanocomposite coatings could decrease the attachment of Staphylococcus aureus.

Preparation of bone-implants by coating hydroxyapatite nanoparticles on self-formed titanium dioxide thin-layers on titanium metal surfaces.

PubMed

Wijesinghe, W P S L; Mantilaka, M M M G P G; Chathuranga Senarathna, K G; Herath, H M T U; Premachandra, T N; Ranasinghe, C S K; Rajapakse, R P V J; Rajapakse, R M G; Edirisinghe, Mohan; Mahalingam, S; Bandara, I M C C D; Singh, Sanjleena

2016-06-01

Preparation of hydroxyapatite coated custom-made metallic bone-implants is very important for the replacement of injured bones of the body. Furthermore, these bone-implants are more stable under the corrosive environment of the body and biocompatible than bone-implants made up of pure metals and metal alloys. Herein, we describe a novel, simple and low-cost technique to prepare biocompatible hydroxyapatite coated titanium metal (TiM) implants through growth of self-formed TiO2 thin-layer (SFTL) on TiM via a heat treatment process. SFTL acts as a surface binder of HA nanoparticles in order to produce HA coated implants. Colloidal HA nanorods prepared by a novel surfactant-assisted synthesis method, have been coated on SFTL via atomized spray pyrolysis (ASP) technique. The corrosion behavior of the bare and surface-modified TiM (SMTiM) in a simulated body fluid (SBF) medium is also studied. The highest corrosion rate is found to be for the bare TiM plate, but the corrosion rate has been reduced with the heat-treatment of TiM due to the formation of SFTL. The lowest corrosion rate is recorded for the implant prepared by heat treatment of TiM at 700 °C. The HA-coating further assists in the passivation of the TiM in the SBF medium. Both SMTiM and HA coated SMTiM are noncytotoxic against osteoblast-like (HOS) cells and are in high-bioactivity. The overall production process of bone-implant described in this paper is in high economic value. Copyright © 2016 Elsevier B.V. All rights reserved.

Hyaluronan Enhances Bone Marrow Cell Therapy for Myocardial Repair After Infarction

PubMed Central

Chen, Chien-Hsi; Wang, Shoei-Shen; Wei, Erika IH; Chu, Ting-Yu; Hsieh, Patrick CH

2013-01-01

Hyaluronan (HA) has been shown to play an important role during early heart development and promote angiogenesis under various physiological and pathological conditions. In recent years, stem cell therapy, which may reduce cardiomyocyte apoptosis, increase neovascularization, and prevent cardiac fibrosis, has emerged as a promising approach to treat myocardial infarction (MI). However, effective delivery of stem cells for cardiac therapy remains a major challenge. In this study, we tested whether transplanting a combination of HA and allogeneic bone marrow mononuclear cells (MNCs) promotes cell therapy efficacy and thus improves cardiac performance after MI in rats. We showed that HA provided a favorable microenvironment for cell adhesion, proliferation, and vascular differentiation in MNC culture. Following MI in rats, compared with the injection of HA alone or MNC alone, injection of both HA and MNCs significantly reduced inflammatory cell infiltration, cardiomyocyte apoptosis, and infarct size and also improved cell retention, angiogenesis, and arteriogenesis, and thus the overall cardiac performance. Ultimately, HA/MNC treatment improved vasculature engraftment of transplanted cells in the infarcted region. Together, our results indicate that combining the biocompatible material HA with bone marrow stem cells exerts a therapeutic effect on heart repair and may further provide potential treatment for ischemic diseases. PMID:23295948

Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb

PubMed Central

Matsumoto, Kazu; Li, Yingcui; Jakuba, Caroline; Sugiyama, Yoshinori; Sayo, Tetsuya; Okuno, Misako; Dealy, Caroline N.; Toole, Bryan P.; Takeda, Junji; Yamaguchi, Yu; Kosher, Robert A.

2009-01-01

Summary The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb. PMID:19633173

Nano-decoration of the Hemagglutinating Virus of Japan envelope (HVJ-E) using a layer-by-layer assembly technique.

PubMed

Okada, Takaharu; Uto, Koichiro; Sasai, Masao; Lee, Chun Man; Ebara, Mitsuhiro; Aoyagi, Takao

2013-06-18

In this study, we created a nanoscale layer of hyaluronic acid (HA) on the inactivated Hemagglutinating Virus of Japan envelope (HVJ-E) via a layer-by-layer (LbL) assembly technique for CD-44 targeted delivery. HVJ-E was selected as the template virus because it has shown a tumor-suppressing ability by eliciting inflammatory cytokine production in dendritic cells. Although it has been required to increase the tumor-targeting ability and reduce nonspecific binding because HVJ-E fuses with virtually all cells and induces hemagglutination in the bloodstream, complete modifications of single-envelope-type viruses with HA have been difficult. Therefore, we studied the surface ζ potential of HVJ-E at different pH values and carefully examined the deposition conditions for the first layer using three cationic polymers: poly-L-lysine (PLL), chitosan (CH), and glycol chitosan (GC). GC-coated HVJ-E particles showed the highest disperse ability under physiological pH and salt conditions without aggregation. An HA layer was then prepared via alternating deposition of HA and GC. The successive decoration of multilayers on HVJ-E has been confirmed by dynamic light scattering (DLS), ζ potentials, and transmission electron microscopy (TEM). An enzymatic degradation assay revealed that only the outermost HA layer was selectively degraded by hyaluronidase. However, entire layers were destabilized at lower pH. Therefore, the HA/GC-coated HVJ-E describe here can be thought of as a potential bomb for cancer immunotherapy because of the ability of targeting CD44 as well as the explosion of nanodecorated HA/GC layers at endosomal pH while preventing nonspecific binding at physiological pH and salt conditions such as in the bloodstream or normal tissues.

Sphere-shaped nano-hydroxyapatite/chitosan/gelatin 3D porous scaffolds increase proliferation and osteogenic differentiation of human induced pluripotent stem cells from gingival fibroblasts.

PubMed

Ji, Jun; Tong, Xin; Huang, Xiaofeng; Wang, Tiancong; Lin, Zitong; Cao, Yazhou; Zhang, Junfeng; Dong, Lei; Qin, Haiyan; Hu, Qingang

2015-07-08

Hydroxyapatite (HA) is an important component of human bone and bone tissue engineering scaffolds. A plethora of bone tissue engineering scaffolds have been synthesized so far, including nano-HA/chitosan/gelatin (nHA/CG) scaffolds; and for seeding cells, stem cells, especially induced pluripotent stem cells (iPSCs), have been a promising cell source for bone tissue engineering recently. However, the influence of different HA nano-particle morphologies on the osteogenic differentiation of human iPSCs (hiPSCs) from human gingival fibroblasts (hGFs) is unknown. The purpose of this study was to investigate the osteogenic differentiation of hiPSCs from hGFs seeded on nHA/CG scaffolds with 2 shapes (rod and sphere) of nHA particles. Firstly, hGFs isolated from discarded normal gingival tissues were reprogrammed into hiPSCs. Secondly, hiPSCs were seeded on rod-like nHA/CG (rod-nHA/CG) and sphere-shaped nHA/CG (sphere-nHA/CG) scaffolds respectively and then cell/scaffold complexes were cultured in vitro. Scanning electron microscope, hematoxyline and eosin (HE) staining, Masson's staining, and quantitative real-time polymerase chain reaction techniques were used to examine hiPSC morphology, proliferation, and differentiation on rod-nHA/CG and sphere-nHA/CG scaffolds. Finally, hiPSCs composited with 2 kinds of nHA/CG were transplanted in vivo in a subcutaneous implantation model for 12 weeks; pure scaffolds were also transplanted as a blank control. HE, Masson's, and immunohistochemistry staining were applied to detect new bone regeneration ability. The results showed that sphere-nHA/CG significantly increased hiPSCs from hGF proliferation and osteogenic differentiation in vitro. hiPSCs and sphere-nHA/CG composities generated large bone, whereas hiPSCs and rod-nHA/CG composities produced tiny bone in vivo. Moreover, pure scaffolds without cells almost produced no bone. In conclusion, our work provided a potential innovative bone tissue engineering approach using clinically discarded gingival tissues and sphere-nHA/CG scaffolds.

Magnesium- and strontium-co-substituted hydroxyapatite: the effects of doped-ions on the structure and chemico-physical properties.

PubMed

Aina, Valentina; Lusvardi, Gigliola; Annaz, Basil; Gibson, Iain R; Imrie, Flora E; Malavasi, Gianluca; Menabue, Ledi; Cerrato, Giuseppina; Martra, Gianmario

2012-12-01

The present study is aimed at investigating the contribution of two biologically important cations, Mg(2+) and Sr(2+), when substituted into the structure of hydroxyapatite (Ca(10)(PO(4))(6)(OH)(2),HA). The substituted samples were synthesized by an aqueous precipitation method that involved the addition of Mg(2+)- and Sr(2+)-containing precursors to partially replace Ca(2+) ions in the apatite structure. Eight substituted HA samples with different concentrations of single (only Mg(2+)) or combined (Mg(2+) and Sr(2+)) substitution of cations have been investigated and the results compared with those of pure HA. The obtained materials were characterized by X-ray powder diffraction, specific surface area and porosity measurements (N(2) adsorption at 77 K), FT-IR and Raman spectroscopies and scanning electron microscopy. The results indicate that the co-substitution gives rise to the formation of HA and β-TCP structure types, with a variation of their cell parameters and of the crystallinity degree of HA with varying levels of substitution. An evaluation of the amount of substituents allows us to design and prepare BCP composite materials with a desired HA/β-TCP ratio.

Ha-ras(val12) induces HSP70b transcription via the HSE/HSF1 system, but HSP70b expression is suppressed in Ha-ras(val12)-transformed cells.

PubMed

Stanhill, A; Levin, V; Hendel, A; Shachar, I; Kazanov, D; Arber, N; Kaminski, N; Engelberg, D

2006-03-09

Heat shock proteins (Hsps) are overexpressed in many tumors, but are downregulated in some tumors. To check for a direct effect of Ha-Ras(val12) on HSP70 transcription, we transiently expressed the oncoprotein in Rat1 fibroblasts and monitored its effect on HSP70b promoter-driven reporter gene. We show that expression of Ha-Ras(val12) induced this promoter. Promoter analysis via systematic deletions and point mutations revealed that Ha-Ras(val12) induces HSP70b transcription via heat shock elements (HSEs). Also, Ha-Ras(val12) induction of HSE-mediated transcription was dramatically reduced in HSF1-/- cells. Yet, residual effect of Ha-Ras(val12) that was still measured in HSF1-/- cells suggests that some of the Ha-Ras(val12) effect is Hsf1-independent. When HSF1-/- cells, stably expressing Ha-Ras(val12), were grown on soft agar only small colonies were formed suggesting a role for heat shock factor 1 (Hsf1) in Ha-Ras(val12)-mediated transformation. Although Ha-ras(Val12) seems to be an inducer of HSP70's expression, we found that in Ha-ras(Val12-)transformed fibroblasts expression of this gene is suppressed. This suppression is correlated with higher sensitivity of Ha-ras(val12)-transformed cells to heat shock. We suggest that Ha-ras(Val12) is involved in Hsf1 activation, thereby inducing the cellular protective response. Cells that repress this response are perhaps those that acquire the capability to further proliferate and become transformed clones.

The optimal SAM surface functional group for producing a biomimetic HA coating on Ti.

PubMed

Liu, D P; Majewski, P; O'Neill, B K; Ngothai, Y; Colby, C B

2006-06-15

Commercial interest is growing in biomimetic methods that employ self assembled mono-layers (SAMs) to produce biocompatible HA coatings on Ti-based orthopedic implants. Recently, separate studies have considered HA formation for various SAM surface functional groups. However, these have often neglected to verify crystallinity of the HA coating, which is essential for optimal bioactivity. Furthermore, differing experimental and analytical methods make performance comparisons difficult. This article investigates and evaluates HA formation for four of the most promising surface functional groups: --OH, --SO(3)H, --PO(4)H(2) and --COOH. All of them successfully formed a HA coating at Ca/P ratios between 1.49 and 1.62. However, only the --SO(3)H and --COOH end groups produced a predominantly crystalline HA. Furthermore, the --COOH end group yielded the thickest layer and possessed crystalline characteristics very similar to that of the human bone. The --COOH end group appears to provide the optimal SAM surface interface for nucleation and growth of biomimetic crystalline HA. Intriguingly, this finding may lend support to explanations elsewhere of why human bone sialoprotein is such a potent nucleator of HA and is attributed to the protein's glutamic acid-rich sequences.

Novel Development of Phosphate Treated Porous Hydroxyapatite.

PubMed

Doi, Kazuya; Abe, Yasuhiko; Kobatake, Reiko; Okazaki, Yohei; Oki, Yoshifumi; Naito, Yoshihito; Prananingrum, Widyasri; Tsuga, Kazuhiro

2017-12-08

Phosphoric acid-etching treatment to the hydroxyapatite (HA) surface can modify the solubility calcium structure. The aim of the present study was to develop phosphate treated porous HA, and the characteristic structures and stimulation abilities of bone formation were evaluated to determine its suitability as a new type of bone graft material. Although the phosphoric acid-etching treatment did not alter the three-dimensional structure, a micrometer-scale rough surface topography was created on the porous HA surface. Compared to porous HA, the porosity of phosphate treated porous HA was slightly higher and the mechanical strength was lower. Two weeks after placement of the cylindrical porous or phosphate treated porous HA in a rabbit femur, newly formed bone was detected in both groups. At the central portion of the bone defect area, substantial bone formation was detected in the phosphate treated porous HA group, with a significantly higher bone formation ratio than detected in the porous HA group. These results indicate that phosphate treated porous HA has a superior surface topography and bone formation abilities in vivo owing to the capacity for both osteoconduction and stimulation abilities of bone formation conferred by phosphoric acid etching.

Novel Development of Phosphate Treated Porous Hydroxyapatite

PubMed Central

Doi, Kazuya; Abe, Yasuhiko; Kobatake, Reiko; Okazaki, Yohei; Oki, Yoshifumi; Naito, Yoshihito; Prananingrum, Widyasri; Tsuga, Kazuhiro

2017-01-01

Phosphoric acid-etching treatment to the hydroxyapatite (HA) surface can modify the solubility calcium structure. The aim of the present study was to develop phosphate treated porous HA, and the characteristic structures and stimulation abilities of bone formation were evaluated to determine its suitability as a new type of bone graft material. Although the phosphoric acid-etching treatment did not alter the three-dimensional structure, a micrometer-scale rough surface topography was created on the porous HA surface. Compared to porous HA, the porosity of phosphate treated porous HA was slightly higher and the mechanical strength was lower. Two weeks after placement of the cylindrical porous or phosphate treated porous HA in a rabbit femur, newly formed bone was detected in both groups. At the central portion of the bone defect area, substantial bone formation was detected in the phosphate treated porous HA group, with a significantly higher bone formation ratio than detected in the porous HA group. These results indicate that phosphate treated porous HA has a superior surface topography and bone formation abilities in vivo owing to the capacity for both osteoconduction and stimulation abilities of bone formation conferred by phosphoric acid etching. PMID:29292788

Bone-like apatite layer formation on hydroxyapatite prepared by spark plasma sintering (SPS).

PubMed

Gu, Y W; Khor, K A; Cheang, P

2004-08-01

Hydroxyapatite (HA) compacts with high density and superior mechanical properties were fabricated by spark plasma sintering (SPS) using spray-dried HA powders as feedstock. The formation of bone-like apatite layer on SPS consolidated HA compacts were investigated by soaking the HA compacts in simulated body fluid (SBF) for various periods (maximum of 28 days). The structural changes in HA post-SBF were analyzed with scanning electron microscopy, grazing incidence X-ray diffraction and X-ray photoelectron spectroscopy. It was found that a layer consisting microcrystalline carbonate-containing hydroxyapatite was formed on the surface of HA compacts after soaking for 24h. The formation mechanism of apatite on the surface of HA compacts after soaking in SBF was attributed to the ion exchange between HA compacts and the SBF solution. The increase in ionic concentration of calcium and phosphorus as well as the increase in pH after SBF immersion resulted in an increase in ionic activity product of apatite in the solution, and provided a specific surface with a low interface energy that is conducive to the nucleation of apatite on the surface of HA compacts.

Structural characteristics of alkaline phosphatase from the moderately halophilic bacterium Halomonas sp. 593

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arai, Shigeki; Yonezawa, Yasushi; Ishibashi, Matsujiro

2014-03-01

In order to clarify the structural basis of the halophilic characteristics of an alkaline phosphatase derived from the moderate halophile Halomonas sp. 593 (HaAP), the tertiary structure of HaAP was determined to 2.1 Å resolution by X-ray crystallography. The structural properties of surface negative charge and core hydrophobicity were shown to be intermediate between those characteristic of halophiles and non-halophiles, and may explain the unique functional adaptation to a wide range of salt concentrations. Alkaline phosphatase (AP) from the moderate halophilic bacterium Halomonas sp. 593 (HaAP) catalyzes the hydrolysis of phosphomonoesters over a wide salt-concentration range (1–4 M NaCl). Inmore » order to clarify the structural basis of its halophilic characteristics and its wide-range adaptation to salt concentration, the tertiary structure of HaAP was determined by X-ray crystallography to 2.1 Å resolution. The unit cell of HaAP contained one dimer unit corresponding to the biological unit. The monomer structure of HaAP contains a domain comprised of an 11-stranded β-sheet core with 19 surrounding α-helices similar to those of APs from other species, and a unique ‘crown’ domain containing an extended ‘arm’ structure that participates in formation of a hydrophobic cluster at the entrance to the substrate-binding site. The HaAP structure also displays a unique distribution of negatively charged residues and hydrophobic residues in comparison to other known AP structures. AP from Vibrio sp. G15-21 (VAP; a slight halophile) has the highest similarity in sequence (70.0% identity) and structure (C{sup α} r.m.s.d. of 0.82 Å for the monomer) to HaAP. The surface of the HaAP dimer is substantially more acidic than that of the VAP dimer (144 exposed Asp/Glu residues versus 114, respectively), and thus may enable the solubility of HaAP under high-salt conditions. Conversely, the monomer unit of HaAP formed a substantially larger hydrophobic interior comprising 329 C atoms from completely buried residues, whereas that of VAP comprised 264 C atoms, which may maintain the stability of HaAP under low-salt conditions. These characteristics of HaAP may be responsible for its unique functional adaptation permitting activity over a wide range of salt concentrations.« less

Structural characterization of nanocrystalline hydroxyapatite and adhesion of pre-osteoblast cells

NASA Astrophysics Data System (ADS)

Zhu, Xiaolong; Eibl, Oliver; Berthold, Christoph; Scheideler, Lutz; Geis-Gerstorfer, Jürgen

2006-06-01

Nanocrystalline hydroxyapatite (Nano HA), a prototype of minerals of bones and teeth, attracts increasing interest in medicine and dentistry. Different parameters for synthesis and post-treatment were investigated to determine their effects on crystallinity of nano HA, and in vitro cell responses to nano HA were studied. XRD and TEM analyses indicate that the crystallinity of nano HA synthesized by a chemical method was within the range of 15-50 nm, which is adapted to natural minerals of hard tissues. Increasing the ageing temperature significantly increased the crystallinity of nano HA, while lengthening the ageing time or varying the post-ageing drying process did not have any influence on its crystallinity. Nano HA annealed between 300 and 900 °C showed a small increase in crystallinity with increasing annealing temperature due to the long-range ordering effect. Cell attachment and spreading on nano HA were lower than those on pure titanium, and decreased as the crystallinity of nano HA increased. However, cells on nano HA demonstrated well-developed filopodia and lamelliopodia, which facilitate migration of the cells on it. This may benefit osteogenesis at the interface between bone and nano HA in vivo.

Effect of hyaluronic acid in bone formation and its applications in dentistry.

PubMed

Zhao, Ningbo; Wang, Xin; Qin, Lei; Zhai, Min; Yuan, Jing; Chen, Ji; Li, Dehua

2016-06-01

Hyaluronic acid (HA), the simplest glycosaminoglycan, participates in several important biological procedures, including mediation of cellular signaling, regulation of cell adhesion and proliferation, and manipulation of cell differentiation. The effect of HA on cell proliferation and differentiation depends on its molecular weight (MW) and concentration. Moreover, the properties of high viscosity, elasticity, highly negative charge, biocompatibility, biodegradability, and nonimmunogenicity make HA attractive in tissue engineering and disease treatment. This review comprises an overview of the effect of HA on cell proliferation and differentiation in vitro, the role of HA in bone regeneration in vivo, and the clinical applications of HA in dentistry, focusing on the mechanism underlining the effect of MW and concentration of HA on cell proliferation and osteogenic differentiation. It is expected that practical progress of HA both in laboratory-based experiments and clinical applications will be achieved in the next few years. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1560-1569, 2016. © 2016 Wiley Periodicals, Inc.

[Developing a canine vocal fold scar model by CO₂ laser and studying the LOX, HSP70 and HA expression in its extra celluar matrix].

PubMed

Liang, G T; Duan, B Y; Zhang, Y Y; Luo, S W; Lu, L; Yang, L P; Wang, B R

2017-01-20

Objective: Building a canine vocal fold scar model and analyzing the changes in morphology, histopathology and related factors of extra cellular matrix (ECM) of vocal cord healing at different time periods. Method: Five experimental dogs were randomly divided into the control group(one dog) and the experimental group (four dogs). No special treatment was done on the control group, and the experimental group was given CO₂ laser through laryngoscope with minimally invasive surgery on bilateral vocal cords. Observation of the morphological changes of injured vocal cords was made at five different time, pre-operation and 6 h, 3 w, 8 w and 12 w post-operation. HE staining, immunofluorescence, were used respectively to observe the histopathological and ultrastructural changes of each layer of vocal cord. Observation were made on the changing pattern of lysyl oxidase (LOX), heat shock proteins70 (HSP70), and the integrated optical density index (IOD) of Hyaluronic acid (HA) in vocal cord tissues. Result: ①Laryngoscope shows there were mild congestion and edema and inflammatory exudation on the wound surface of vocal cord 3w post-operation. On the wound surface of vocal cord 8 w post-operation, the congestion and edema disappear, the local contraction sink, and no adhesion and granulation form. 12 w post-operation, the surface of the vocal cord was smooth, there were local contractures, vocal fold scar form, and hoarseness in the bilateral vocal cords was obvious. ②HE staining shows 6 h post-operation there were a large number of inflammatory cell infiltration, red blood cell leakage, and cell congestion and edema on the wound surface of vocal cord. 3 w post-operation, there were fibroblast proliferation, angiogenesis, and a large number of fibrous tissues disorderly arranged on the wound surface of vocal cord. 8w post-operation, each layer of fibrous tissues were in hyperplasia and the blood vessels was thicken after on the wound surface of vocal cord, 12 w post-operation, a large number of collagens increases and were in group or fasciculation disorder. There were many irregular gaps in fibers, and blood vessels and glands become rare or disappear. ③The immunofluorescence showed LOX, HSP70 and HA were all localized in the cellular cytoplasm and nucleus. The expression levels were different at different postoperative time, and they were mainly relatively strong expressed in the inflammatory cells, vascular endothelial cells and the glands. ④The comparison of IOD values: The IOD values of LOX were different at different time periods ( P < 0.05). There were no significant differences in IOD of HSP70 between pre-operation and 12 w post-operation, but there are significant differences among other groups ( P < 0.05). There were no significant differences in IOD of HA between pre-operation and 12 w post-operation, but there were significant differences among other groups ( P < 0.01). ⑤The expression of LOX decreased 6h post-operation and increased 3-12 w post-operation. The expression of HSP70 post-operation reached the peak 6 h post-operation and decreased 3-12 w post-operation. The expression of HA decreased 6 h post-operation, increased to the peak 8 w post-operation, and decreased 8-12 w post-operation.⑥The transmission electron microscope showed 3 w to 8 w post-operation, in the intrinsic layer of the vocal cord, the fibroblasts were extremely active, the cells swelled, and the organelles were abundant.12 w post-operation, there were a small number of fibroblasts in the active state, and larger gaps between the fibers and fewer and thinner elastic fibers. Conclusion: CO₂ laser ablation of vocal cords under the Micro Post-Laryngoscope could establish reliable animal model of vocal fold scar, and 12 w reticular the vocal fold scars basically form. LOX, HSP70, HA play different roles at early, middle, and late stages in the vocal fold scar formation, and they can be used as a more sensitive index for vocal fold scar formation. Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.

Osteogenesis of human adipose-derived stem cells on hydroxyapatite-mineralized poly(lactic acid) nanofiber sheets.

PubMed

Kung, Fu-Chen; Lin, Chi-Chang; Lai, Wen-Fu T

2014-12-01

Electrospun fiber sheets with various orientations (random, partially aligned, and aligned) and smooth and roughened casted membranes were prepared. Hydroxyapatite (HA) crystals were in situ formed on these material surfaces via immersion in 10× simulated body fluid solution. The size and morphology of the resulting fibers were examined using scanning electron microscopy. The average diameter of the fibers ranged from 225±25 to 1050±150 nm depending on the electrospinning parameters. Biological experiment results show that human adipose-derived stem cells exhibit different adhesion and osteogenic differentiation on the three types of fiber. The cell proliferation and osteogenic differentiation were best on the aligned fibers. Similar results were found for phosphorylated focal adhesion kinase expression. Electrospun poly(lactic acid) aligned fibers mineralized with HA crystals provide a good environment for cell growth and osteogenic differentiation and thus have great potential in the tissue engineering field. Copyright © 2014 Elsevier B.V. All rights reserved.

Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion

PubMed Central

Mekhedov, Elena; Waters, Hang; Sodt, Alexander; Schwartz, Cindi; Nair, Vinod; Blank, Paul S.

2017-01-01

ABSTRACT The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion. IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion. PMID:28794042

Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion.

PubMed

Chlanda, Petr; Mekhedov, Elena; Waters, Hang; Sodt, Alexander; Schwartz, Cindi; Nair, Vinod; Blank, Paul S; Zimmerberg, Joshua

2017-11-01

The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion. IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion. Copyright © 2017 American Society for Microbiology.

Innovative micro-textured hydroxyapatite and poly(l-lactic)-acid polymer composite film as a flexible, corrosion resistant, biocompatible, and bioactive coating for Mg implants.

PubMed

Kim, Sae-Mi; Kang, Min-Ho; Kim, Hyoun-Ee; Lim, Ho-Kyung; Byun, Soo-Hwan; Lee, Jong-Ho; Lee, Sung-Mi

2017-12-01

The utility of a novel ceramic/polymer-composite coating with a micro-textured microstructure that would significantly enhance the functions of biodegradable Mg implants is demonstrated here. To accomplish this, bioactive hydroxyapatite (HA) micro-dots can be created by immersing a Mg implant with a micro-patterned photoresist surface in an aqueous solution containing calcium and phosphate ions. The HA micro-dots can then be surrounded by a flexible poly(l-lactic)-acid (PLLA) polymer using spin coating to form a HA/PLLA micro-textured coating layer. The HA/PLLA micro-textured coating layer showed an excellent corrosion resistance when it was immersed in a simulated body fluid (SBF) solution and good biocompatibility, which was assessed by in vitro cell tests. In addition, the HA/PLLA micro-textured coating layer had high deformation ability, where no apparent changes in the coating layer were observed even after a 5% elongation, which would be unobtainable using HA and PLLA coating layers; furthermore, this allowed the mechanically-strained Mg implant with the HA/PLLA micro-textured coating layer to preserve its excellent corrosion resistance and biocompatibility in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

Silicon-hydroxyapatite bioactive coatings (Si-HA) from diatomaceous earth and silica. Study of adhesion and proliferation of osteoblast-like cells.

PubMed

López-Alvarez, M; Solla, E L; González, P; Serra, J; León, B; Marques, A P; Reis, R L

2009-05-01

The aim of this study consisted on investigating the influence of silicon substituted hydroxyapatite (Si-HA) coatings over the human osteoblast-like cell line (SaOS-2) behaviour. Diatomaceous earth and silica, together with commercial hydroxyapatite were respectively the silicon and HA sources used to produce the Si-HA coatings. HA coatings with 0 wt% of silicon were used as control of the experiment. Pulsed laser deposition (PLD) was the selected technique to deposit the coatings. The Si-HA thin films were characterized by Fourier Transformed Infrared Spectroscopy (FTIR) demonstrating the efficient transfer of Si to the HA structure. The in vitro cell culture was established to assess the cell attachment, proliferation and osteoblastic activity respectively by, Scanning Electron Microscopy (SEM), DNA and alkaline phosphatase (ALP) quantification. The SEM analysis demonstrated a similar adhesion behaviour of the cells on the tested materials and the maintenance of the typical osteoblastic morphology along the time of culture. The Si-HA coatings did not evidence any type of cytotoxic behaviour when compared with HA coatings. Moreover, both the proliferation rate and osteoblastic activity results showed a slightly better performance on the Si-HA coatings from diatoms than on the Si-HA from silica.

Hydroxyapatite grafted carbon nanotubes and graphene nanosheets: promising bone implant materials.

PubMed

Oyefusi, Adebola; Olanipekun, Opeyemi; Neelgund, Gururaj M; Peterson, Deforest; Stone, Julia M; Williams, Ebonee; Carson, Laura; Regisford, Gloria; Oki, Aderemi

2014-11-11

In the present study, hydroxyapatite (HA) was successfully grafted to carboxylated carbon nanotubes (CNTs) and graphene nanosheets. The HA grafted CNTs and HA-graphene nanosheets were characterized using FT-IR, TGA, SEM and X-ray diffraction. The HA grafted CNTs and graphene nanosheets (CNTs-HA and Gr-HA) were further used to examine the proliferation and differentiation rate of temperature-sensitive human fetal osteoblastic cell line (hFOB 1.19). Total protein assays and western blot analysis of osteocalcin expression were used as indicators of cell proliferation and differentiation. Results indicated that hFOB 1.19 cells proliferate and differentiate well in treatment media containing CNTs-HA and graphene-HA. Both CNTs-HA and graphene-HA could be promising nanomaterials for use as scaffolds in bone tissue engineering. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell death induced by hydroxyapatite on L929 fibroblast cells.

PubMed

Inayat-Hussain, S H; Rajab, N F; Roslie, H; Hussin, A A; Ali, A M; Annuar, B O

2004-05-01

Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.

Structural characterization of viral epitopes recognized by broadly cross-reactive antibodies.

PubMed

Lee, Peter S; Wilson, Ian A

2015-01-01

Influenza hemagglutinin (HA) is the major surface glycoprotein on influenza viruses and mediates viral attachment and subsequent fusion with host cells. The HA is the major target of the immune response, but due to its high level of variability, as evidenced by substantial antigenic diversity, it had been historically considered to elicit only a narrow, strain-specific antibody response. However, a recent explosion in the discovery of broadly neutralizing antibodies (bnAbs) to influenza virus has identified two major supersites of vulnerability on the HA through structural characterization of HA-antibody complexes. These commonly targeted epitopes are involved with receptor binding as well as the fusion machinery and, hence, are functionally conserved and less prone to mutation. These bnAbs can neutralize viruses by blocking infection or the spread of infection by preventing progeny release. Structural analyses of these bnAbs show they exhibit striking similarities and trends in recognition of the HA and use recurring recognition motifs, despite substantial differences in their germline genes. This information can be utilized in design of novel therapeutics as well as in immunogens for improved vaccines with greater breadth and efficacy.

A graphitic hollow carbon nitride nanosphere as a novel photochemical internalization agent for targeted and stimuli-responsive cancer therapy

NASA Astrophysics Data System (ADS)

Liu, Chaoqun; Chen, Zhaowei; Wang, Zhenzhen; Li, Wei; Ju, Enguo; Yan, Zhengqing; Liu, Zhen; Ren, Jinsong; Qu, Xiaogang

2016-06-01

As a novel technique, photochemical internalization (PCI) has been employed as a new approach to overcome endo/lysosomal restriction, which is one of the main difficulties in both drug and gene delivery. However, the complicated synthesis procedure (usually requiring the self-assembly of polymers, photosensitizers and cargos) and payload specificity greatly limit its further application. In this paper, we employ a highly fluorescent graphitic hollow carbon nitride nanosphere (GHCNS) to simultaneously serve as a PCI photosensitizer, an imaging agent and a drug carrier. The surface modification of GHCNS with multifunctional polysaccharide hyaluronic acid (HA) endows the system with colloidal stability, biocompatibility and cancer cell targeting ability. After CD44 receptor-mediated endocytosis, the nanosystem is embedded in endo/lysosomal vesicles and HA could be specially degraded by hyaluronidase (Hyal), inducing open pores. In the following, with visible light illumination, GHCNS could produce ROS that effectively induced lipid peroxidation and caused endo/lysosomal membrane break, accelerating the cytoplasmic release of the drug in the targeted and irradiated cells. As a result, significantly increased therapeutic potency and specificity against cancer cells could be achieved.As a novel technique, photochemical internalization (PCI) has been employed as a new approach to overcome endo/lysosomal restriction, which is one of the main difficulties in both drug and gene delivery. However, the complicated synthesis procedure (usually requiring the self-assembly of polymers, photosensitizers and cargos) and payload specificity greatly limit its further application. In this paper, we employ a highly fluorescent graphitic hollow carbon nitride nanosphere (GHCNS) to simultaneously serve as a PCI photosensitizer, an imaging agent and a drug carrier. The surface modification of GHCNS with multifunctional polysaccharide hyaluronic acid (HA) endows the system with colloidal stability, biocompatibility and cancer cell targeting ability. After CD44 receptor-mediated endocytosis, the nanosystem is embedded in endo/lysosomal vesicles and HA could be specially degraded by hyaluronidase (Hyal), inducing open pores. In the following, with visible light illumination, GHCNS could produce ROS that effectively induced lipid peroxidation and caused endo/lysosomal membrane break, accelerating the cytoplasmic release of the drug in the targeted and irradiated cells. As a result, significantly increased therapeutic potency and specificity against cancer cells could be achieved. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07719b

Repair of rabbit cartilage defect based on the fusion of rabbit bone marrow stromal cells and Nano-HA/PLLA composite material.

PubMed

Zhu, Weimin; Guo, Daiqi; Peng, Liangquan; Chen, Yun Fang; Cui, Jiaming; Xiong, Jianyi; Lu, Wei; Duan, Li; Chen, Kang; Zeng, Yanjun; Wang, Daping

2017-02-01

Objective To assess the effect of the fusion of rabbit bone marrow stromal cells (rBMSCs) and Nano-hydroxyapatite/poly (l-lactic acid) (Nano-HA/PLLA) in repairing the rabbit knee joint with full-thickness cartilage defect. Method The rBMSCs were isolated and cultured in vitro, and the third generation of rBMSCs was co-cultured with the Nano-HA/PLLA to construct the tissue-engineered cartilage (TEC). Eighteen New Zealand white rabbits were selected and randomly divided into three groups, namely, TEC group, Nano-HA/PLLA group, and control group. A cartilage defect model with the diameter of 4.5 mm and depth of 5 mm was constructed on the articular surface of medial malleolus of rabbit femur. General observation, histological observation, and Wakitani's histological scoring were conducted in the 12th and 24th week postoperatively. Results The results of TEC group indicated that new cartilage tissue was formed on the defect site and subchondral bone achieved physiological integration basically. Histological and immunohistochemical analyses indicated the generation of massive extracellular matrix. In contrast, limited regeneration and reconstruction of cartilage was achieved in the Nano-HA/PLLA group and control group, with a significant difference from the TEC group (p < 0.05). Moreover, the effect of cartilage repair was positively correlated with time. Conclusion The porous Nano-HA/PLLA combined with BMSCs promoted the repair of weight-bearing bone of adult rabbit's knee joint with cartilage defect.

Surface acoustic wave actuated cell sorting (SAWACS).

PubMed

Franke, T; Braunmüller, S; Schmid, L; Wixforth, A; Weitz, D A

2010-03-21

We describe a novel microfluidic cell sorter which operates in continuous flow at high sorting rates. The device is based on a surface acoustic wave cell-sorting scheme and combines many advantages of fluorescence activated cell sorting (FACS) and fluorescence activated droplet sorting (FADS) in microfluidic channels. It is fully integrated on a PDMS device, and allows fast electronic control of cell diversion. We direct cells by acoustic streaming excited by a surface acoustic wave which deflects the fluid independently of the contrast in material properties of deflected objects and the continuous phase; thus the device underlying principle works without additional enhancement of the sorting by prior labelling of the cells with responsive markers such as magnetic or polarizable beads. Single cells are sorted directly from bulk media at rates as fast as several kHz without prior encapsulation into liquid droplet compartments as in traditional FACS. We have successfully directed HaCaT cells (human keratinocytes), fibroblasts from mice and MV3 melanoma cells. The low shear forces of this sorting method ensure that cells survive after sorting.

Effect of humic acid preloading on phosphate adsorption onto zirconium-modified zeolite.

PubMed

Lin, Jianwei; Zhang, Zhe; Zhan, Yanhui

2017-05-01

A zirconium-modified zeolite (ZrMZ) was prepared, and then, humic acid (HA) was immobilized on the ZrMZ surface to prepare HA-loaded ZrMZ (HA-ZrMZ). The obtained ZrMZ and HA-ZrMZ were characterized by energy dispersive X-ray spectroscopy, elemental analyzer, N 2 adsorption/desorption isotherms, pH at the point of zero charge, and X-ray photoelectron spectroscopy. The adsorption characteristics of phosphate on ZrMZ and HA-ZrMZ were comparatively investigated in batch mode. The adsorption mechanism of phosphate on ZrMZ and HA-ZrMZ was investigated by ionic strength effect and 31 P nuclear magnetic resonance. The mechanism for phosphate adsorption onto ZrMZ was the formation of inner-sphere phosphate complexes at the solid/solution interface. The preloading of HA on ZrMZ reduced the phosphate adsorption capacity, and the more the HA loading amount, the lower the phosphate adsorption capacity. However, the preloading of HA on ZrMZ did not change the phosphate adsorption mechanism; i.e., the formation of inner-sphere phosphate surface complexes was still responsible for the adsorption of phosphate on HA-ZrMZ. The decreased phosphate adsorption capacity for ZrMZ after HA coating could be attributed to the fact that the coating of HA on ZrMZ reduced the amount of binding active sites available for phosphate adsorption, changed the adsorbent surface charges, and reduced the specific surface areas and pore volumes of ZrMZ.

Low-molecular-weight hyaluronan (LMW-HA) accelerates lymph node metastasis of melanoma cells by inducing disruption of lymphatic intercellular adhesion.

PubMed

Du, Yan; Cao, Manlin; Liu, Yiwen; He, Yiqing; Yang, Cuixia; Wu, Man; Zhang, Guoliang; Gao, Feng

2016-01-01

Endothelial integrity defects initiate lymphatic metastasis of tumor cells. Low-molecular-weight hyaluronan (LMW-HA) derived from plasma and interstitial fluid was reported to be associated with tumor lymphatic metastasis. In addition, LMW-HA was proved to disrupt lymphatic vessel endothelium integrity, thus promoting lymphatic metastasis of tumor cells. Until now, there are few reports on how LMW-HA modulates lymphatic endothelial cells adhesion junctions and affects cancer cells metastasizing into lymph vessels. The aim of our study is to unravel the novel mechanism of LMW-HA in mediating tumor lymphatic metastasis. Here, we employed a melanoma metastasis model to investigate whether LMW-HA facilitates tumor cells transferring from foci to remote lymph nodes by disrupting the lymphatic endothelial integrity. Our data indicate that LMW-HA significantly induces metastasis of melanoma cells to lymph nodes and accelerates interstitial-lymphatic flow in vivo . Further experiments show that increased migration of melanoma cells across human dermal lymphatic endothelial cell (HDLEC) monolayers is accompanied by impaired lymphatic endothelial barrier function and increased permeability. The mechanism study reveals that VE-cadherin-β-catenin pathway and relevant signals are involved in modulating the interactions between endothelial cells and that a significant inhibition of lymphatic endothelium disruption is observed when antibodies to the LMW-HA receptor (LYVE-1) are present. Thus, our findings demonstrate a disruptive effect of LMW-HA on lymphatic endothelium continuity which leads to a promotion on melanoma lymphatic metastasis and also suggest a cellular signaling mechanism associated with VE-cadherin-mediated lymphatic intercellular junctions.

Cytotoxicity of curcumin silica nanoparticle complexes conjugated with hyaluronic acid on colon cancer cells.

PubMed

Singh, Surya Prakash; Sharma, Mrinalini; Gupta, Pradeep Kumar

2015-03-01

We report results of our investigations on the cytotoxic efficacy of Organically modified silica nanoparticle (SiNp)-curcumin complex conjugated with hyaluronic acid (HA) (HA-SiNp-cur) and HA free SiNp-cur complex in human colon carcinoma (colo-205) cells. Curcumin was loaded in SiNp and resulting complexes were conjugated with HA, which has a strong affinity for cancer cells expressing CD44. After conjugation with HA, the average size of the SiNp-cur nanoparticles increased from 45 nm to 70 nm, and zeta potential changed to -33 mV from -26 mV. Compared to free curcumin and SiNp-cur, curcumin in HA-SiNp was more stable. The uptake and cytotoxicity of curcumin delivered through HA-SiNp-cur was significantly higher in monolayer and spheroids as compared to free curcumin and HA free SiNp-cur. Concomitantly, HA-SiNp-cur complex treatment resulted in higher inhibition of growth and migration of cells in spheroids. Further, incubation of colo-205 cancer cells with an excess of HA impaired the uptake of HA-SiNp-cur confirming the involvement of receptor mediated endocytosis in the uptake of HA conjugated nanocomplex. Time dependent increase in the fluorescence of curcumin observed in the release media when HA-SiNp-cur was incubated with hyaluronidase suggests involvement of enzyme in release of curcumin from nanoparticle. Copyright © 2014 Elsevier B.V. All rights reserved.

Silver/hydroxyapatite composite coatings on porous titanium surfaces by sol-gel method.

PubMed

Qu, Jie; Lu, Xiong; Li, Dan; Ding, Yonghui; Leng, Yang; Weng, Jie; Qu, Shuxin; Feng, Bo; Watari, Fumio

2011-04-01

Hydroxyapatite (HA) coatings loaded with nanosilver particles is an attractive method to impart the HA coating with antibacterial properties. Producing Ag/HA coatings on porous Ti substrates have been an arduous job since commonly used line-of-sight techniques are not able to deposit uniform coatings on the inner pore surfaces of the porous Ti. In this study, porous Ti scaffolds with high porosity and interconnected structures were prepared by polymer impregnating method. A sol-gel process was used to produce uniform Ag/HA composite coatings on the surfaces of porous Ti substrates. Ca(NO(3) )(2) ·4H(2) O and P(2) O(5) in an ethyl alcohol based system was selected to prepare the sol, which ensured the homogeneous distribution of Ag in the sol. The characterization revealed that silver particles uniformly distributed in the coatings without agglomeration. High antibacterial ratio (>95%), against E. coli and S. albus was expressed by the silver-containing coatings (Ag/HA 0.8 and 1.6 wt %). The biocompatibility of the Ag/HA 0.8 surfaces was as good as that of pure HA surface, as revealed by culturing osteoblasts on them. The results indicated that Ag/HA 0.8 had the good balance between the biocompatibility and antibacterial properties of the coatings. Copyright © 2011 Wiley Periodicals, Inc.

DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay.

PubMed

Rajab, N F; Yaakob, T A; Ong, B Y; Hamid, M; Ali, A M; Annuar, B O; Inayat-Hussain, S H

2004-05-01

Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.

Hyaluronic acid doped-poly(3,4-ethylenedioxythiophene)/chitosan/gelatin (PEDOT-HA/Cs/Gel) porous conductive scaffold for nerve regeneration.

PubMed

Wang, Shuping; Guan, Shui; Zhu, Zhibo; Li, Wenfang; Liu, Tianqing; Ma, Xuehu

2017-02-01

Conducting polymer, as a "smart" biomaterial, has been increasingly used to construct tissue engineered scaffold for nerve tissue regeneration. In this study, a novel porous conductive scaffold was prepared by incorporating conductive hyaluronic acid (HA) doped-poly(3,4-ethylenedioxythiophene) (PEDOT-HA) nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. The physicochemical characteristics of Cs/Gel scaffold with 0-10wt% PEDOT-HA were analyzed and the results indicated that the incorporation of PEDOT-HA into scaffold increased the electrical and mechanical properties while decreasing the porosity and water absorption. Moreover, in vitro biodegradation of scaffold displayed a declining trend with the PEDOT-HA content increased. About the biocompatibility of conductive scaffold, neuron-like rat phaeochromocytoma (PC12) cells were cultured in scaffold to evaluate cell adhesion and growth. 8% PEDOT-HA/Cs/Gel scaffold had a higher cell adhesive efficiency and cell viability than the other conductive scaffolds. Furthermore, cells in the scaffold with 8wt% PEDOT-HA expressed higher synapse growth gene of GAP43 and SYP compared with Cs/Gel control group. These results suggest that 8%PEDOT-HA/Cs/Gel scaffold is an attractive cell culture conductive substrate which could support cell adhesion, survival, proliferation, and synapse growth for the application in nerve tissue regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression and distribution of hyaluronic acid and CD44 in unphonated human vocal fold mucosa.

PubMed

Sato, Kiminori; Umeno, Hirohito; Nakashima, Tadashi; Nonaka, Satoshi; Harabuchi, Yasuaki

2009-11-01

The tension caused by phonation (vocal fold vibration) is hypothesized to stimulate vocal fold stellate cells (VFSCs) in the maculae flavae (MFe) to accelerate production of extracellular matrices. The distribution of hyaluronic acid (HA) and expression of CD44 (a cell surface receptor for HA) were examined in human vocal fold mucosae (VFMe) that had remained unphonated since birth. Five specimens of VFMe (3 adults, 2 children) that had remained unphonated since birth were investigated with Alcian blue staining, hyaluronidase digestion, and immunohistochemistry for CD44. The VFMe containing MFe were hypoplastic and rudimentary. The VFMe did not have a vocal ligament, Reinke's space, or a layered structure, and the lamina propria appeared as a uniform structure. In the children, HA was distributed in the VFMe containing MFe. In the adults, HA had decreased in the VFMe containing MFe. In both groups, the VFSCs in the MFe and the fibroblasts in the lamina propria expressed little CD44. This study supports the hypothesis that the tensions caused by vocal fold vibration stimulate the VFSCs in the MFe to accelerate production of extracellular matrices and form the layered structure. Phonation after birth is one of the important factors in the growth and development of the human VFMe.

The minor histocompatibility antigen HA-3 arises from differential proteasome-mediated cleavage of the lymphoid blast crisis (Lbc) oncoprotein.

PubMed

Spierings, Eric; Brickner, Anthony G; Caldwell, Jennifer A; Zegveld, Suzanne; Tatsis, Nia; Blokland, Els; Pool, Jos; Pierce, Richard A; Mollah, Sahana; Shabanowitz, Jeffrey; Eisenlohr, Laurence C; van Veelen, Peter; Ossendorp, Ferry; Hunt, Donald F; Goulmy, Els; Engelhard, Victor H

2003-07-15

Minor histocompatibility (H) antigens crucially affect the outcome of human leukocyte antigen (HLA)-identical allogeneic stem cell transplantation (SCT). To understand the basis of alloimmune responses against minor H antigens, identification of minor H peptides and their antigenicity-determining mechanisms is essential. Here we report the identification of HA-3 and its encoding gene. The HA-3 peptide, VTEPGTAQY (HA-3T), is encoded by the lymphoid blast crisis (Lbc) oncogene. We thus show for the first time that a leukemia-associated oncogene can give rise to immunogenic T-cell epitopes that may have participated in antihost and antileukemic alloimmune responses. Genotypic analysis of HA-3- individuals revealed the allelic counterpart VMEPGTAQY (HA-3M). Despite the lack of T-cell recognition of HA-3- cells, the Thr-->Met substitution had only a modest effect on peptide binding to HLA-A1 and a minimal impact on recognition by T cells when added exogenously to target cells. This substitution did not influence transporter associated with antigen processing (TAP) transport, but, in contrast to the HA-3T peptide, HA-3M is destroyed by proteasome-mediated digestion. Thus, the immunogenicity of minor H antigens can result from proteasome-mediated destruction of the negative allelic peptide.

Humic acid inhibits HBV-induced autophagosome formation and induces apoptosis in HBV-transfected Hep G2 cells

PubMed Central

Pant, Kishor; Yadav, Ajay K.; Gupta, Parul; Rathore, Abhishek Singh; Nayak, Baibaswata; Venugopal, Senthil K.

2016-01-01

Hepatitis B Virus (HBV) utilizes several mechanisms to survive in the host cells and one of the main pathways being autophagosome formation. Humic acid (HA), one of the major components of Mineral pitch, is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. We hypothesized that HA could induce cell death and inhibit HBV-induced autophagy in hepatic cells. Incubation of Hep G2.2.1.5 cells (HepG2 cells stably expressing HBV) with HA (100 μM) inhibited both cell proliferation and autophagosome formation significantly, while apoptosis induction was enhanced. Western blot results showed that HA incubation resulted in decreased levels of beclin-1, SIRT-1 and c-myc, while caspase-3 and β-catenin expression were up-regulated. Western blot results showed that HA significantly inhibited the expression of HBx (3-fold with 50 μM and 5-fold with 100 μM) compared to control cells. When HA was incubated with HBx-transfected Hep G2 cells, HBx-induced autophagosome formation and beclin-1 levels were decreased. These data showed that HA induced apoptosis and inhibited HBV-induced autophagosome formation and proliferation in hepatoma cells. PMID:27708347

Efficacy of a New Ocular Surface Modulator in Restoring Epithelial Changes in an In Vitro Model of Dry Eye Syndrome.

PubMed

Barabino, Stefano; De Servi, Barbara; Aragona, Salvatore; Manenti, Demetrio; Meloni, Marisa

2017-03-01

So far tear substitutes have demonstrated a limited role in restoring ocular surface damage in dry eye syndrome (DES). The aim of this study was to assess the efficacy of a new ocular surface modulator in an in vitro model of human corneal epithelium (HCE) damaged by severe osmotic stress mirroring the features of dry eye conditions. A reconstructed HCE model challenged by the introduction of sorbitol in the culture medium for 16 h was used to induce an inflammatory pathway and to impair the tight junctions integrity determining a severe modification of the superficial layer ultrastructure. At the end of the overnight stress period in the treated HCE series, 30 μl of the ocular surface modulator (T-LysYal, Sildeha, Switzerland) and of hyaluronic acid (HA) in the control HCE series were applied for 24 h. The following parameters were quantified: scanning electron microscopy (SEM), trans-epithelial electrical resistance (TEER), immunofluorescence analysis of integrin β1 (ITG-β1), mRNA expression of Cyclin D-1 (CCND1), and ITG-β1. In the positive control after the osmotic stress the HCE surface damage was visible at the ultrastructural level with loss of cell-cell interconnections, intercellular matrix destruction, and TEER reduction. After 24 h of treatment with T-LysYal, HCE showed a significant improvement of the ultrastructural morphological organization and increased expression of ITG-β1 at the tissue level when compared to positive and control series. A significant increase of mRNA expression for ITG-β1 and CCND1 was shown in the HA-treated cells compared to T-LysYal. TEER measurement showed a significant reduction in all groups after 16 h without modifications after the treatment period. This study has shown the possibility of a new class of agents denominated ocular surface modulators to restore corneal cells damaged by dry eye conditions. Further in vivo studies are certainly necessary to confirm these results.

Effect of cell-seeded hydroxyapatite scaffolds on rabbit radius bone regeneration.

PubMed

Rathbone, C R; Guda, T; Singleton, B M; Oh, D S; Appleford, M R; Ong, J L; Wenke, J C

2014-05-01

Highly porous hydroxyapatite (HA) scaffolds were developed as bone graft substitutes using a template coating process, characterized, and seeded with bone marrow-derived mesenchymal stem cells (BMSCs). To test the hypothesis that cell-seeded HA scaffolds improve bone regeneration, HA scaffolds without cell seeding (HA-empty), HA scaffolds with 1.5 × 10(4) BMSCs (HA-low), and HA scaffolds with 1.5 × 10(6) BMSCs (HA-high) were implanted in a 10-mm rabbit radius segmental defect model for 4 and 8 weeks. Three different fluorochromes were administered at 2, 4, and 6 weeks after implantation to identify differences in temporal bone growth patterns. It was observed from fluorescence histomorphometry analyses that an increased rate of bone infiltration occurred from 0 to 2 weeks (p < 0.05) of implantation for the HA-high group (2.9 ± 0.5 mm) as compared with HA-empty (1.8 ± 0.8 mm) and HA-low (1.3 ± 0.2 mm) groups. No significant differences in bone formation within the scaffold or callus formation was observed between all groups after 4 weeks, with a significant increase in bone regenerated for all groups from 4 to 8 weeks (28.4% across groups). Although there was no difference in bone formation within scaffolds, callus formation was significantly higher in HA-empty scaffolds (100.9 ± 14.1 mm(3) ) when compared with HA-low (57.8 ± 7.3 mm(3) ; p ≤ 0.003) and HA-high (69.2 ± 10.4 mm(3) ; p ≤ 0.02) after 8 weeks. These data highlight the need for a better understanding of the parameters critical to the success of cell-seeded HA scaffolds for bone regeneration. Copyright © 2013 Wiley Periodicals, Inc.

Modeling the interaction and toxicity of Cu-Cd mixture to wheat roots affected by humic acids, in terms of cell membrane surface characteristics.

PubMed

Wang, Yi-Min; Zhou, Dong-Mei; Yuan, Xu-Yin; Zhang, Xiao-Hui; Li, Yi

2018-05-01

Responses of wheat (Triticum aestivum L.) seedling roots to the mixtures of copper (Cu), cadmium (Cd) and humic acids (HA) were investigated using the solution culture experiments, focusing on the interaction patterns between multiple metals and their influences on root proton release. A concentration-addition multiplication (CA) model was introduced into the modeling analysis. In comparison with metal ion activities in bulk-phase solutions, the incorporation of ion activities at the root cell membrane surfaces (CMs) (denoted as {Cu 2+ } 0 and {Cd 2+ } 0 ) into the CA model could significantly improve their correlation with RRE (relative root elongation) from 0.819 to 0.927. Modeling analysis indicated that the co-existence of {Cu 2+ } 0 significantly enhanced the rhizotoxicity of {Cd 2+ } 0 , while no significant effect of {Cd 2+ } 0 on the {Cu 2+ } 0 rhizotoxicity. 10 mg/L HA stimulated the root elongation even under metal stress. Although high concentration of metal ions inhibited the root proton release rate (ΔH + ), both the low concentration of metal ions and HA treatments increased the values of ΔH + . In HA-Cu-Cd mixtures, actions of metal ions on ΔH + values were varied intricately among treatments but well modeled by the CA model. We concluded from the CA models that the electrostatic effect is vitally important for explaining the effect of {Cu 2+ } 0 on the rhizotoxicity of {Cd 2+ } 0 , while it plays no unique role in understanding the influence of {Cd 2+ } 0 on the rhizotoxicity of {Cu 2+ } 0. Thus our study provide a novel way for modeling multiple metals behaviors in the environment and understanding the mechanisms of ion interactions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enterokinase Enhances Influenza A Virus Infection by Activating Trypsinogen in Human Cell Lines

PubMed Central

Hayashi, Hideki; Kubo, Yoshinao; Izumida, Mai; Takahashi, Etsuhisa; Kido, Hiroshi; Sato, Ko; Yamaya, Mutsuo; Nishimura, Hidekazu; Nakayama, Kou; Matsuyama, Toshifumi

2018-01-01

Cleavage and activation of hemagglutinin (HA) by trypsin-like proteases in influenza A virus (IAV) are essential prerequisites for its successful infection and spread. In host cells, some transmembrane serine proteases such as TMPRSS2, TMPRSS4 and HAT, along with plasmin in the bloodstream, have been reported to cleave the HA precursor (HA0) molecule into its active forms, HA1 and HA2. Some trypsinogens can also enhance IAV proliferation in some cell types (e.g., rat cardiomyoblasts). However, the precise activation mechanism for this process is unclear, because the expression level of the physiological activator of the trypsinogens, the TMPRSS15 enterokinase, is expected to be very low in such cells, with the exception of duodenal cells. Here, we show that at least two variant enterokinases are expressed in various human cell lines, including A549 lung-derived cells. The exogenous expression of these enterokinases was able to enhance the proliferation of IAV in 293T human kidney cells, but the proliferation was reduced by knocking down the endogenous enterokinase in A549 cells. The enterokinase was able to enhance HA processing in the cells, which activated trypsinogen in vitro and in the IAV-infected cells also. Therefore, we conclude that enterokinase plays a role in IAV infection and proliferation by activating trypsinogen to process viral HA in human cell lines. PMID:29629340

Assessing the dynamic biofilm removal of sulfonated phenolics using CP-OCT

NASA Astrophysics Data System (ADS)

Englund, K.; Nikrad, J.; Jones, R.

2017-02-01

Examining the physical mechanisms related to biofilm removal of sulfonated phenolics (SP) is difficult using conventional microscopy techniques. A custom flow cell system integrated with a real time cross polarization optical coherence tomography system investigated the dynamic speed of biofilm removal when oral multi-species biofilms are exposed to SP under sheer stress. The Near infrared 1310-nm CP-OCT system non-destructively imaged fluid immersed oral biofilms at nearly 30 frames/s. This dynamic imaging was able to determine the cohesive and adhesion related disruption of SP on oral biofilms adhering to tooth like surfaces. For multi-species biofilms that are initially grown without the presence of sucrose, the disruption of biofilms on saliva coated hydroxyapatite (HA) is dominated as a adhesive failure at the HA-biofilm interface. For multi-species biofilms that are grown in the presence of sucrose, the disruption is dominated by cohesive disruption followed by adhesive failure. This novel CP-OCT flow cell assay has the potential to examine rapid interactions between anti-biofilm agents and tooth like surfaces.

Combining stable insect cell lines with baculovirus-mediated expression for multi-HA influenza VLP production.

PubMed

Sequeira, Daniela P; Correia, Ricardo; Carrondo, Manuel J T; Roldão, António; Teixeira, Ana P; Alves, Paula M

2018-05-24

Safer and broadly protective vaccines are needed to cope with the continuous evolution of circulating influenza virus strains and promising approaches based on the expression of multiple hemagglutinins (HA) in a virus-like particle (VLP) have been proposed. However, expression of multiple genes in the same vector can lead to its instability due to tandem repetition of similar sequences. By combining stable with transient expression systems we can rationally distribute the number of genes to be expressed per platform and thus mitigate this risk. In this work, we developed a modular system comprising stable and baculovirus-mediated expression in insect cells for production of multi-HA influenza enveloped VLPs. First, a stable insect High Five cell population expressing two different HA proteins from subtype H3 was established. Infection of this cell population with a baculovirus vector encoding three other HA proteins from H3 subtype proved to be as competitive as traditional co-infection approaches in producing a pentavalent H3 VLP. Aiming at increasing HA expression, the stable insect cell population was infected at increasingly higher cell concentrations (CCI). However, cultures infected at CCI of 3×10 6 cells/mL showed lower HA titers per cell in comparison to standard CCI of 2×10 6 cells/mL, a phenomenon named "cell density effect". To lessen the negative impact of this phenomenon, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth. Noteworthy, cultures supplemented and infected at a CCI of 4×10 6 cells/mL showed comparable HA titers per cell to those of CCI of 2×10 6 cells/mL, thus leading to an increase of up to 4-fold in HA titers per mL. Scalability of the modular strategy herein proposed was successfully demonstrated in 2L stirred tank bioreactors with comparable HA protein levels observed between bioreactor and shake flasks cultures. Overall, this work demonstrates the suitability of combining stable with baculovirus-mediated expression in insect cells as an efficient platform for production of multi-HA influenza VLPs, surpassing the drawbacks of traditional co-infection strategies and/or the use of larger, unstable vectors. Copyright © 2017 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lui, E.; Moya, Monica L.

This study seeks to validate the reproducibility of previous bioprinting work at Lawrence Livermore National Laboratory (LLNL) on a new Aerotech motion controller system and to modify an existing bioink, fibrin, by adding varying percent volumes of hyaluronic acid (HA). Endothelial and fibroblast cells bioprinted in fibrin gels using the Aerotech system were confirmed to be more than 77 percent viable after one day, and all bioprinted samples retained sterility after one week of culture. To characterize cell behavior in fibrin with HA addition, static co-­culture gels with varying percent volumes of HA were cultured in vitro for one week.more » Resulting confocal microscope images showed increased cell network formation in all concentrations of HA compared to the control (no HA), and rheological tests mimicking static gel compositions displayed positive correlations between gelation time, gel stiffness (G’), and hyaluronic acid concentration. Although the current data is insufficient to quantitatively associate HA concentration with the level of cell vascularization, future work will aim to develop a targeted HA concentration in fibrin for maximum cell network formation, to optimize the printing process parameters for this new bioink composition, and to analyze cell viability in bioprinted fibrin-­HA structures.« less

Controllable preparation of a nano-hydroxyapatite coating on carbon fibers by electrochemical deposition and chemical treatment.

PubMed

Wang, Xudong; Zhao, Xueni; Wang, Wanying; Zhang, Jing; Zhang, Li; He, Fuzhen; Yang, Jianjun

2016-06-01

A nano-hydroxyapatite (HA) coating with appropriate thickness and morphology similar to that of human bone tissue was directly prepared onto the surfaces of carbon fibers (CFs). A mixed solution of nitric acid, hydrochloric acid, sulfuric acid, and hydrogen peroxide (NHSH) was used in the preparation process. The coating was fabricated by combining NHSH treatment and electrochemical deposition (ECD). NHSH treatment is easy to operate, produces rapid reaction, and highly effective. This method was first used to induce the nucleation and growth of HA crystals on the CF surfaces. Numerous O-containing functional groups, such as hydroxyl (-OH) and carboxyl (-COOH) groups, were grafted onto the CF surfaces by NHSH treatment (NHSH-CFs); as such, the amounts of these groups on the functionalized CFs increased by nearly 8- and 12-fold, respectively, compared with those on untreated CFs. After treatment, the NHSH-CFs not only acquired larger specific surface areas but retained surfaces free from serious corrosion or breakage. Hence, NHSH-CFs are ideal depositional substrates of HA coating during ECD. ECD was successfully used to prepare a nano-rod-like HA coating on the NHSH-CF surfaces. The elemental composition, structure, and morphology of the HA coating were effectively controlled by adjusting various technological parameters, such as the current density, deposition time, and temperature. The average central diameter of HA crystals and the coating density increased with increasing deposition time. The average central diameter of most HA crystals on the NHSH-CFs varied from approximately 60 nm to 210 nm as the deposition time increased from 60 min to 180 min. Further studies on a possible deposition mechanism revealed that numerous O-containing functional groups on the NHSH-CF surfaces could associate with electrolyte ions (Ca(2+)) to form special chemical bonds. These bonds can induce HA coating deposition and improve the interfacial bonding strength between the HA coating and NHCH-CFs. The results of this study and the proposed preparation of uniform and dense nano-HA coating provide theoretical and practical guidance for future investigations of active HA coatings on fiber materials for medical products and implants. This work also lays the foundation for the wider use of HA-coated CFs/HA composite implants in clinical application. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell damage caused by vaginal Candida albicans isolates from women with different symptomatologies.

PubMed

Faria, Daniella Renata; Sakita, Karina Mayumi; Akimoto-Gunther, Luciene Setsuko; Kioshima, Érika Seki; Svidzinski, Terezinha Inez Estivalet; Bonfim-Mendonça, Patrícia de Souza

2017-08-01

The present study aimed to characterize cell damage caused by vaginal Candida albicans isolates from women with different symptomatologies. It was evaluated 12 clinical isolates of C. albicans from vaginal samples: 4 from asymptomatic women (AS), 4 from women with a single episode of vulvovaginal candidiasis (VVC) and 4 from women with recurrent vulvovaginal candidiasis (RVVC). We evaluated the ability of C. albicans to adhere to human cervical cancer cells (SiHa), the yeast-SiHa cell interactions and cell damage. All of the clinical isolates presented a high adhesion capacity on SiHa cells. However, clinical isolates from symptomatic women (VVC and RVVC) had higher filamentation after contact (24 h) with SiHa cells and a greater capacity to cause cell damage (>80 %). Clinical isolates from symptomatic women had greater potential to invade SiHa cells, suggesting that they are more pathogenic than AS isolates.

Elastin-like Protein-Hyaluronic acid (ELP-HA) Hydrogels with Decoupled Mechanical and Biochemical cues for Cartilage Regeneration

PubMed Central

Zhu, Danqing; Wang, Huiyuan; Trinh, Pavin; Heilshorn, Sarah C.; Yang, Fan

2018-01-01

Hyaluronic acid (HA) is a major component of cartilage extracellular matrix and is an attractive material for use as 3D injectable matrices for cartilage regeneration. While previous studies have shown the promise of HA-based hydrogels to support cell-based cartilage formation, varying HA concentration generally led to simultaneous changes in both biochemical cues and stiffness. How cells respond to the change of biochemical content of HA remains largely unknown. Here we report an adaptable elastin-like protein-hyaluronic acid (ELP-HA) hydrogel platform using dynamic covalent chemistry, which allows varyiation of HA concentration without affecting matrix stiffness. ELP-HA hydrogels were created through dynamic hydrazone bonds via the reaction between hydrazine-modified ELP (ELP-HYD) and aldehyde-modified HA (HA-ALD). By tuning the stoichiometric ratio of aldehyde groups to hydrazine groups while maintaining ELP-HYD concentration constant, hydrogels with variable HA concentration (1.5%, 3%, or 5%) (w/v) were fabricated with comparable stiffness. To evaluate the effects of HA concentration on cell-based cartilage regeneration, chondrocytes were encapsulated within ELP-HA hydrogels with varying HA concentration. Increasing HA concentration led to a dose-dependent increase in cartilage-marker gene expression and enhanced sGAG deposition while minimizing undesirable fibrocartilage phenotype. The use of adaptable protein hydrogels formed via dynamic covalent chemistry may be broadly applicable as 3D scaffolds with decoupled niche properties to guide other desirable cell fates and tissue repair. PMID:28268018

Elastin-like protein-hyaluronic acid (ELP-HA) hydrogels with decoupled mechanical and biochemical cues for cartilage regeneration.

PubMed

Zhu, Danqing; Wang, Huiyuan; Trinh, Pavin; Heilshorn, Sarah C; Yang, Fan

2017-05-01

Hyaluronic acid (HA) is a major component of cartilage extracellular matrix and is an attractive material for use as 3D injectable matrices for cartilage regeneration. While previous studies have shown the promise of HA-based hydrogels to support cell-based cartilage formation, varying HA concentration generally led to simultaneous changes in both biochemical cues and stiffness. How cells respond to the change of biochemical content of HA remains largely unknown. Here we report an adaptable elastin-like protein-hyaluronic acid (ELP-HA) hydrogel platform using dynamic covalent chemistry, which allows variation of HA concentration without affecting matrix stiffness. ELP-HA hydrogels were created through dynamic hydrazone bonds via the reaction between hydrazine-modified ELP (ELP-HYD) and aldehyde-modified HA (HA-ALD). By tuning the stoichiometric ratio of aldehyde groups to hydrazine groups while maintaining ELP-HYD concentration constant, hydrogels with variable HA concentration (1.5%, 3%, or 5%) (w/v) were fabricated with comparable stiffness. To evaluate the effects of HA concentration on cell-based cartilage regeneration, chondrocytes were encapsulated within ELP-HA hydrogels with varying HA concentration. Increasing HA concentration led to a dose-dependent increase in cartilage-marker gene expression and enhanced sGAG deposition while minimizing undesirable fibrocartilage phenotype. The use of adaptable protein hydrogels formed via dynamic covalent chemistry may be broadly applicable as 3D scaffolds with decoupled niche properties to guide other desirable cell fates and tissue repair. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vitro and in vivo performance of bioactive Ti6Al4V/TiC/HA implants fabricated by a rapid microwave sintering technique.

PubMed

Choy, Man Tik; Tang, Chak Yin; Chen, Ling; Wong, Chi Tak; Tsui, Chi Pong

2014-09-01

Failure of the bone-implant interface in a joint prosthesis is a main cause of implant loosening. The introduction of a bioactive substance, hydroxyapatite (HA), to a metallic bone-implant may enhance its fixation on human bone by encouraging direct bone bonding. Ti6Al4V/TiC/HA composites with a reproducible porous structure (porosity of 27% and pore size of 6-89 μm) were successfully fabricated by a rapid microwave sintering technique. This method allows the biocomposites to be fabricated in a short period of time under ambient conditions. Ti6Al4V/TiC/HA composites exhibited a compressive strength of 93 MPa, compressive modulus of 2.9 GPa and microhardness of 556 HV which are close to those of the human cortical bone. The in vitro preosteoblast MC3T3-E1 cells cultured on the Ti6Al4V/TiC/HA composite showed that the composite surface could provide a biocompatible environment for cell adhesion, proliferation and differentiation without any cytotoxic effects. This is among the first attempts to study the in vivo performance of load-bearing Ti6Al4V/TiC and Ti6Al4V/TiC/HA composites in a live rabbit. The results indicated that the Ti6Al4V/TiC/HA composite had a better bone-implant interface compared with the Ti6Al4V/TiC implant. Based on the microstructural features, the mechanical properties, and the in vitro and in vivo test results from this study, the Ti6Al4V/TiC/HA composites have the potential to be employed in load-bearing orthopedic applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Serum IgG titres, but not avidity, correlates with neutralizing antibody response after H5N1 vaccination.

PubMed

Pedersen, Gabriel Kristian; Höschler, Katja; Øie Solbak, Sara Marie; Bredholt, Geir; Pathirana, Rishi Delan; Afsar, Aram; Breakwell, Lucy; Nøstbakken, Jane Kristin; Raae, Arnt Johan; Brokstad, Karl Albert; Sjursen, Haakon; Zambon, Maria; Cox, Rebecca Jane

2014-07-31

Influenza H5N1 virus constitutes a pandemic threat and development of effective H5N1 vaccines is a global priority. Anti-influenza antibodies directed towards the haemagglutinin (HA) define a correlate of protection. Both antibody concentration and avidity may be important for virus neutralization and resolving influenza disease. We conducted a phase I clinical trial of a virosomal H5N1 vaccine adjuvanted with the immunostimulating complex Matrix M™. Sixty adults were intramuscularly immunized with two vaccine doses (21 days apart) of 30 μg HA alone or 1.5, 7.5 or 30 μg HA adjuvanted with Matrix M™. Serum H5 HA1-specific antibodies and virus neutralization were determined at days 0, 21, 42, 180 and 360 and long-term memory B cells at day 360 post-vaccination. The binding of the HA specific antibodies was measured by avidity NaSCN-elution ELISA and surface plasmon resonance (SPR). The H5 HA1-specific IgG response peaked after the second dose (day 42), was dominated by IgG1 and IgG3 and was highest in the adjuvanted vaccine groups. IgG titres correlated significantly with virus neutralization at all time points (Spearman r≥0.66, p<0.0001). By elution ELISA, serum antibody avidity was highest at days 180 and 360 post vaccination and did not correlate with virus neutralization. Long-lasting H5 HA1-specific memory B cells produced high IgG antibody avidity similar to serum IgG. Maturation of serum antibody avidity continued up to day 360 after influenza H5N1 vaccination. Virus neutralization correlated with serum H5 HA1-specific IgG antibody concentrations and not antibody avidity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Adsorption of hyaluronic acid on solid supports: role of pH and surface chemistry in thin film self-assembly.

PubMed

Choi, Jae-Hyeok; Kim, Seong-Oh; Linardy, Eric; Dreaden, Erik C; Zhdanov, Vladimir P; Hammond, Paula T; Cho, Nam-Joon

2015-06-15

Owing to its biocompatibility, resistance to biofouling, and desirable physicochemical and biological properties, hyaluronic acid (HA) has been widely used to modify the surface of various materials. The role of various physicochemical factors in HA adsorption remains, however, to be clarified. Herein, we employed quartz crystal microbalance with dissipation (QCM-D) in order to investigate HA adsorption at different pH conditions onto three substrates-silicon oxide, amine-terminated self-assembled monolayer (SAM) on gold, and carboxylic acid-terminated SAM on gold. The QCM-D experiments indicated specific pH conditions where either strong or weak HA adsorption occurs. The morphology of the adsorbed HA layers was investigated by atomic force microscopy (AFM), and we identified that strong HA adsorption produced a complete, homogenous and smooth HA layer, while weak HA adsorption resulted in rough and inhomogeneous HA layers. The observed specifics of the kinetics of HA adsorption, including a short initial linear phase and subsequent long non-linear phase, were described by using a mean-field kinetic model taking HA diffusion limitations and reconfiguration in the adsorbed state into account. The findings extend the physicochemical background of design strategies for improving the use of passive HA adsorption for surface modification applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Fabrication and in vivo evaluation of an osteoblast-conditioned nano-hydroxyapatite/gelatin composite scaffold for bone tissue regeneration.

PubMed

Samadikuchaksaraei, Ali; Gholipourmalekabadi, Mazaher; Erfani Ezadyar, Elham; Azami, Mahmoud; Mozafari, Masoud; Johari, Behrooz; Kargozar, Saeid; Jameie, Seyed Behnamedin; Korourian, Alireza; Seifalian, Alexander M

2016-08-01

In this study, the effects of osteoblast-conditioning on mechanical behavior, biocompatibility, biodegradation and osteoinductive properties of a nano-hydroxyapatite/gelatin (HA/GEL) nanocomposite scaffold was investigated. The scaffold was fabricated using the layer solvent casting combined with the freeze-drying and lamination techniques. The scaffolds were conditioned by culture of osteoblasts on their surface and their elimination by a repeated freeze-thawing process. The potential of the osteoblast-conditioned HA/GEL (HA/GEL/OC) scaffold to support cell adhesion and growth and its cytotoxicity was assessed in vitro using rat mesenchymal stem cells. For in vivo studies, the HA/GEL/OC nanocomposite was implanted in the critical size bone defect created on rat calvarium and studied after 7, 30 and 90 days. The results showed that mechanical and in vitro biological properties of the scaffold were not affected by the process of conditioning. However, in vivo studies demonstrated that osteoblast-conditioning enhanced biocompatibility and osteoinductivity and of the nanocomposite scaffold. The osteoblast conditioning also accelerated collagen content during the bone healing. In the experimental group that received the HA/GEL/OC and MSCs, the newly formed bone occupied almost the entire defect (93.4 ± 3.3%) within 3 months. In conclusion, this study indicates that osteoblast-conditioning is a viable strategy for the development of bone tissue engineering scaffolds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2001-2010, 2016. © 2016 Wiley Periodicals, Inc.

Surface zwitterionization: Effective method for preventing oral bacterial biofilm formation on hydroxyapatite surfaces

NASA Astrophysics Data System (ADS)

Lee, Myoungjin; Kim, Heejin; Seo, Jiae; Kang, Minji; Kang, Sunah; Jang, Joomyung; Lee, Yan; Seo, Ji-Hun

2018-01-01

In this study, we conducted surface zwitterionization of hydroxyapatite (HA) surfaces by immersing them in the zwitterionic polymer solutions to provide anti-bacterial properties to the HA surface. Three different monomers containing various zwitterionic groups, i.e., phosphorylcholine (PC), sulfobetaine (SB), and carboxybetaine (CB), were copolymerized with the methacrylic monomer containing a Ca2+-binding moiety, using the free radical polymerization method. As a control, functionalization of the copolymer containing the Ca2+-binding moiety was synthesized using a hydroxy group. The stable immobilization of the zwitterionic functional groups was confirmed by water contact angle analysis and X-ray photoelectron spectroscopy (XPS) measurement conducted after the sonication process. The zwitterionized HA surface showed significantly decreased protein adsorption, whereas the hydroxyl group-coated HA surface showed limited efficacy. The anti-bacterial adhesion property was confirmed by conducting Streptococcus mutans (S. mutans) adhesion tests for 6 h and 24 h. When furanone C-30, a representative anti-quorum sensing molecule for S. mutans, was used, only a small amount of bacteria adhered after 6 h and the population did not increase after 24 h. In contrast, zwitterionized HA surfaces showed almost no bacterial adhesion after 6 h and the effect was retained for 24 h, resulting in the lowest level of oral bacterial adhesion. These results confirm that surface zwitterionization is a promising method to effectively prevent oral bacterial adhesion on HA-based materials.

The role of amino acids in hydroxyapatite mineralization

PubMed Central

2016-01-01

Polar and charged amino acids (AAs) are heavily expressed in non-collagenous proteins (NCPs), and are involved in hydroxyapatite (HA) mineralization in bone. Here, we review what is known on the effect of single AAs on HA precipitation. Negatively charged AAs, such as aspartic acid, glutamic acid (Glu) and phosphoserine are largely expressed in NCPs and play a critical role in controlling HA nucleation and growth. Positively charged ones such as arginine (Arg) or lysine (Lys) are heavily involved in HA nucleation within extracellular matrix proteins such as collagen. Glu, Arg and Lys intake can also increase bone mineral density by stimulating growth hormone production. In vitro studies suggest that the role of AAs in controlling HA precipitation is affected by their mobility. While dissolved AAs are able to inhibit HA precipitation and growth by chelating Ca2+ and PO43− ions or binding to nuclei of calcium phosphate and preventing their further growth, AAs bound to surfaces can promote HA precipitation by attracting Ca2+ and PO43− ions and increasing the local supersaturation. Overall, the effect of AAs on HA precipitation is worth being investigated more, especially under conditions closer to the physiological ones, where the presence of other factors such as collagen, mineralization inhibitors, and cells heavily influences HA precipitation. A deeper understanding of the role of AAs in HA mineralization will increase our fundamental knowledge related to bone formation, and could lead to new therapies to improve bone regeneration in damaged tissues or cure pathological diseases caused by excessive mineralization in tissues such as cartilage, blood vessels and cardiac valves. PMID:27707904

Impact of non-thermal plasma surface modification on porous calcium hydroxyapatite ceramics for bone regeneration

PubMed Central

Moriguchi, Yu; Lee, Dae-Sung; Thamina, Khair; Masuda, Kazuto; Itsuki, Dai; Yoshikawa, Hideki; Hamaguchi, Satoshi; Myoui, Akira

2018-01-01

In the physiochemical sciences, plasma is used to describe an ionized gas. Previous studies have implicated plasma surface treatment in the enhancement of hydrophilicity of implanted musculoskeletal reconstructive materials. Hydroxyapatite (HA) ceramics, widely used in bone tissue regeneration, have made great advancements to skeletal surgery. In the present study, we investigate the impact of low-pressure plasma on the interconnected porous calcium hydroxyapatite (IP-CHA) both in vitro and in vivo. Our results indicate that dielectric barrier discharge (DBD) plasma, when used with oxygen, can augment the hydrophilicity of non-porous HA surfaces and the osteoconductivity of the IP-CHA disc via increased water penetration of inner porous structures, as demonstrated through microfocus computed tomography (μCT) assay. In vivo implantation of plasma-treated IP-CHA displayed superior bone ingrowth than untreated IP-CHA. Though plasma-treated IP-CHA did not alter osteoblast cell proliferation, it accelerated osteogenic differentiation of seeded marrow mesenchymal stem cells. In vitro X-ray photoelectron spectroscopy (XPS) revealed that this plasma treatment increases levels of oxygen, rather than nitrogen, on the plasma-treated IP-CHA surface. These findings suggest that plasma treatment, an easy and simple processing, can significantly improve the osteoconductive potential of commonly used artificial bones such as IP-CHA. Further optimization of plasma treatment and longer-term follow-up of in vivo application are required toward its clinical application. PMID:29538457

Microstructural disintegration in dense hydroxyapatite and hydroxyapatite-coated metal implants

NASA Astrophysics Data System (ADS)

Seo, Dong Seok; Lee, Jong Kook

2007-08-01

Hydroxyapatite (HA) has been widely used as a coating for orthopedic metal implants. An important concern regarding HA coating is its degradation of the biological milieu. In this study, the microstructure of a retrieved HA-coated acetabular cup implanted for four years after total hip arthroplasty (THA) was investigated by field emission scanning electron microscopy. In order to understand the underlying mechanism, of degradation and exfoliation of the HA coating, degradation of phase-pure and dense HA ceramics was also observed by in vitro and in vivo testing. The surface morphology and fracture surfaces of HA ceramics revealed that the dissolution starting at the surface extended inwards resulting in particle loosening and microstructural-level degradation. The dissolution features of HA ceramics were similar to the case of HA coating. It was found that extensive dissolution of the coating occurred and most of the coating disappeared. The majority of the remaining graints were fractured by the intergranular mode, suggesting that grain boundaries should be predominantly dissolved. These observations may explain the mechanism through which the biological stability of the HA coated layer becomes unexpectedly poor.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liuyun, Jiang, E-mail: jlytxg@163.com; Chengdong, Xiong; Lixin, Jiang

Graphical abstract: In this manuscript, we initiated a systematic study to investigate the effect of HA on thermal properties, inner structure, reduction of mechanical strength, surface morphology and the surface deposit of n-HA/PLGA composite with respect to the soaking time. The results showed that n-HA played an important role in improving the degradation behavior of n-HA/PLGA composite, which can accelerate the degradation of n-HA/PLGA composite and endow it with bioactivity, after n-HA was detached from PLGA during the degradation, so that n-HA/PLGA composite may have a more promising prospect of the clinical application than pure PLGA as bone fracture internalmore » fixation materials, and the results would be of reference significance to predict the in vivo degradation and biological properties. - Highlights: • Effect of n-HA on degradation behavior of n-HA/PLGA composite was investigated. • Degradation behaviors of n-HA/PLGA and PLGA were carried out in SBF for 6 months. • Viscosity, thermal properties, inner structure and bending strength were tested. • n-HA can accelerate the degradation and endows it with bioactivity. - Abstract: To investigate the effect of hydroxyapatite(HA) on the degradation behavior of hydroxyapatite/poly(lactic-co-glycolic) acid (HA/PLGA) nanocomposite, the degradation experiment of n-HA/PLGA composite and pure PLGA were carried out by soaking in simulated body fluid(SBF) at 37 °C for 1, 2, 4 and 6 months. The change of intrinsic viscosity, thermal properties, inner structure, bending strength reduction, surface morphology and the surface deposit of n-HA/PLGA composite and pure PLGA with respect to the soaking time were investigated by means of UbbeloHde Viscometer, differential scanning calorimeter (DSC), scanning electron microscope(SEM), electromechanical universal tester, a conventional camera and X-ray diffraction (XRD). The results showed that n-HA played an important role in improving the degradation behavior of n-HA/PLGA composite, which can accelerate the degradation PLGA and endow it with bioactivity, after n-HA was detached from PLGA during the degradation, so that n-HA/PLGA composite may have a more promising prospect of the clinical application than pure PLGA as bone fracture internal fixation materials.« less

No influence of exogenous hyaluronan on the behavior of human cancer cells or endothelial cell capillary formation.

PubMed

Seino, Satoshi; Takeshita, Fumitaka; Asari, Akira; Masuda, Yasunobu; Kunou, Masaaki; Ochiya, Takahiro

2014-07-01

Hyaluronan (HA), a type of glycosaminoglycan used to construct the extracellular matrix, is involved in the proliferation and motility of cells, including cancer cells. The aim of this study was to determine whether exogenous HA has an influence on cancer in vitro and in vivo. High-molecular-weight HA (900 kDa) and low-molecular-weight HA (10 kDa) were added to several types of cancer cell lines in vitro, and proliferation and invasion were assessed. The effect of HA on capillary formation by human umbilical vein endothelial cells was also analyzed. The results showed that both types of HA had no apparent effect on cellular proliferation, invasion, or capillary formation. In an animal study, the 2 types of HA were orally administered to tumor-bearing mice at a dosage of 200 mg/kg/d for 4 wk. Analysis using an in vivo imaging system revealed that tumor proliferation and metastasis were not greatly altered by HA administration. Furthermore, CD31 immunohistochemical staining revealed no obvious change in tumor microvessels. Taken together, these results demonstrate that exogenously administered HA has little effect on cancer. This study may support the safety of various forms of HA administration, including oral intake. Orally administered hyaluronan was recently found to have beneficial effects. However, the effect of exogenous hyaluronan on cancer remains unclear. Our findings support the safety of orally administered hyaluronan and its use as a functional food ingredient. © 2014 Institute of Food Technologists®

Construction of surface HA/TiO2 coating on porous titanium scaffolds and its preliminary biological evaluation.

PubMed

Chen, Hongjie; Wang, Chunli; Yang, Xiao; Xiao, Zhanwen; Zhu, Xiangdong; Zhang, Kai; Fan, Yujiang; Zhang, Xingdong

2017-01-01

A simple approach to fabricating hydroxyxapatite/titanium dioxide (HA/TiO 2 ) coating on porous titanium (Ti) scaffolds was developed in the present study. Surface TiO 2 layer was firstly formed on porous Ti scaffolds with multi-scale pores by acid-alkali (AA) treatment. The outer HA layer was then formed on the TiO 2 layer by subsequent pulse electrochemical deposition (ED) technique. All the three main process parameters, i.e. deposition times, current density and mass transfer mode affected the properties of the HA coating notably. Under the conditions of 90 deposition cycles, -10mA/cm 2 of pulse current density and stirring, a thin layer of homogeneous and nanorod-like HA sediments was formed on the substrate surface of porous Ti scaffolds. The results of protein adsorption and cellular experiments showed that compared to the single TiO 2 surface, the HA/TiO 2 surface allowed more adsorption of serum proteins and further enhanced the alkaline phosphatase (ALP) activity of MC3T3-E1 osteoblasts. Copyright © 2016 Elsevier B.V. All rights reserved.

β-Cyclodextrin grafted polypyrrole magnetic nanocomposites toward the targeted delivery and controlled release of doxorubicin

NASA Astrophysics Data System (ADS)

Hong, Shasha; Li, Zengbo; Li, Chenzhong; Dong, Chuan; Shuang, Shaomin

2018-01-01

The Fe3O4@PPy-HA-β-CD nanocomposites as the novel nanocarrier were prepared by grafting ethylenediamine derivative of​ β-​CD to the surface of polypyrrole-coated magnetic nanoparticles (Fe3O4@PPy) via using hyaluronan (HA) as the intermediate linker. HA was also the efficient target ligand for CD44. The as-prepared drug carrier was characterized by TEM, TGA, XRD, and VSM and used for the delivery of doxorubicin hydrochloride (DOX) with the high loading content of 447 mg/g. The multilayer Freundlich isotherm model was found to be a good fit for the loading of the drug carrier for DOX. Significant NIR-triggered release of DOX was observed in a weak acidic pH. And the release data in vitro was well described using the Retiger-Pepper kinetic model. Furthermore, MTT assay and confocal microscopy against Hep-G2 cells clearly illustrated that the drug carrier had no associated cytotoxicity and could easily enter the cells. The release and accumulation of DOX were observed in the cell nuclei. Thus, the DOX-loaded drug carrier killed the cancer cells efficaciously and minimized adverse side effects due to its target effect. These results suggested the as-prepared drug carrier would be of great potential for the controlled release and targeted delivery of DOX.

Anti-Fas conjugated hyaluronic acid microsphere gels for neural stem cell delivery.

PubMed

Shendi, Dalia; Albrecht, Dirk R; Jain, Anjana

2017-02-01

Central nervous system (CNS) injuries and diseases result in neuronal damage and loss of function. Transplantation of neural stem cells (NSCs) has been shown to improve locomotor function after transplantation. However, due to the immune and inflammatory response at the injury site, the survival rate of the engrafted cells is low. Engrafted cell viability has been shown to increase when transplanted within a hydrogel. Hyaluronic acid (HA) hydrogels have natural anti-inflammatory properties and the backbone can be modified to introduce bioactive agents, such as anti-Fas, which we have previously shown to promote NSC survival while suppressing immune cell activity in bulk hydrogels in vitro. Although bulk HA hydrogels have shown to promote stem cell survival, microsphere gels for NSC encapsulation and delivery may have additional advantages. In this study, a flow-focusing microfluidic device was used to fabricate either vinyl sulfone-modified HA (VS-HA) or anti-Fas-conjugated HA (anti-Fas HA) microsphere gels encapsulated with NSCs. The majority of encapsulated NSCs remained viable for at least 24 h in the VS-HA and anti-Fas HA microsphere gels. Moreover, T-cells cultured in suspension with the anti-Fas HA microsphere gels had reduced viability after contact with the microsphere gels compared to the media control and soluble anti-Fas conditions. This approach can be adapted to encapsulate various cell types for therapeutic strategies in other physiological systems in order to increase survival by reducing the immune response. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 608-618, 2017. © 2016 Wiley Periodicals, Inc.

Modifying alginate with early embryonic extracellular matrix, laminin, and hyaluronic acid for adipose tissue engineering.

PubMed

Chen, Yo-Shen; Chen, Yen-Yu; Hsueh, Yu-Sheng; Tai, Hao-Chih; Lin, Feng-Huei

2016-03-01

Extracellular matrix provides both mechanistic and chemical cues that can influence cellular behaviors such as adhesion, migration, proliferation, and differentiation. In this study, a new material, HA-L-Alg, was synthesized by linking developmentally essential ECM constituents hyaluronic acid (HA) and laminin(L) to alginate (Alg). The fabrication of HA-L-Alg was confirmed by FTIR spectroscopy, and it was used to form 3D cell-carrying beads. HA-L-Alg beads had a steady rate of degradation and retained 63.25% of mass after 9 weeks. HA-L-Alg beads showed biocompatibility comparable to beads formed by Alg-only with no obvious cytotoxic effect on the embedded 3T3-L1 preadipocytes. HA-L-Alg encapsulated 3T3-L1 cells were found to have a higher proliferation rate over those in Alg-only beads. These cells also showed better differentiation capacity after 2 weeks of adipogenic induction within HA-L-Alg beads. These results support that HA-L-Alg facilitated cell survival and proliferation, as well as stimulated and maintained cell differentiation. Our results suggest that HA-L-Alg has a great clinical potential to be used as stem cell carrier for adipose tissue engineering. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 669-677, 2016. © 2015 Wiley Periodicals, Inc.

Heterogeneous Electrochemical Immunoassay of Hippuric Acid on the Electrodeposited Organic Films

PubMed Central

Choi, Young-Bong; Kim, Nam-Hyuk; Kim, Seung-Hoi; Tae, Gun-Sik; Kim, Hyug-Han

2014-01-01

By directly coordinating hippuric acid (HA) to the ferrate (Fe) as an electron transfer mediator, we synthesized a Fe-HA complex, which shows a good electrochemical signal and thus enables the electrochemical immunoanalysis for HA. We electrodeposited organic films containing imidazole groups on the electrode surface and then bonded Ni ion (positive charge) to induce immobilization of Fe-HA (negative charge) through the electrostatic interaction. The heterogeneous competitive immunoassay system relies on the interaction between immobilized Fe-HA antigen conjugate and free HA antigen to its antibody (anti-HA). The electric signal becomes weaker due to the hindered electron transfer reaction when a large-sized HA antibody is bound onto the Fe-HA. However, in the presence of HA, the electric signal increases because free HA competitively reacts with the HA antibody prior to actual reaction and thus prevents the HA antibody from interacting with Fe-HA at the electrode surface. This competition reaction enabled an electrochemical quantitative analysis of HA concentration with a detection limit of 0.5 μg mL−1, and thus allowed us to develop a simple and rapid electrochemical immunosensor. PMID:25313491

Mesoporous silica nanoparticles functionalized with hyaluronic acid. Effect of the biopolymer chain length on cell internalization.

PubMed

Nairi, Valentina; Magnolia, Silvia; Piludu, Marco; Nieddu, Mariella; Caria, Cristian Antonio; Sogos, Valeria; Vallet-Regì, Maria; Monduzzi, Maura; Salis, Andrea

2018-02-12

Mesoporous silica nanoparticles (MSNs) were functionalized with amino groups (MSN-NH 2 ) and then with hyaluronic acid, a biocompatible biopolymer which can be recognized by CD44 receptors in tumor cells, to obtain a targeting drug delivery system. To this purpose, three hyaluronic acid samples differing for the molecular weight, namely HA S (8-15 kDa), HA M (30-50 kDa) and HA L (90-130 kDa), were used. The MSN-HA S , MSN-HA M , and MSN-HA L materials were characterized through zeta potential and dynamic light scattering measurements at pH = 7.4 and T = 37 °C to simulate physiological conditions. While zeta potential showed an increasing negative value with the increase of the HA chain length, an anomalous value of the hydrodynamic diameter was observed for MSN-HA L , which was smaller than that of MSN-HA S and MSN-HA M samples. The cellular uptake of MSN-HA samples on HeLa cells at 37 °C was studied by optical and electron microscopy. HA chain length affected significantly the cellular uptake that occurred at a higher extent for MSN-NH 2 and MSN-HA S than for MSN-HA M and MSN-HA L samples. Cellular uptake experiments carried out at 4 °C showed that the internalization process was inhibited for MSN-HA samples but not for MSN-NH 2 . This suggests the occurrence of two different mechanisms of internalization. For MSN-NH 2 the uptake is mainly driven by the attractive electrostatic interaction with membrane phospholipids, while MSN-HA internalization involves CD44 receptors overexpressed in HeLa cells. Copyright © 2018 Elsevier B.V. All rights reserved.

PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines.

PubMed

Katona, Éva; Juhász, Tamás; Somogyi, Csilla Szűcs; Hajdú, Tibor; Szász, Csaba; Rácz, Kálmán; Kókai, Endre; Gergely, Pál; Zákány, Róza

2016-03-01

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment.

PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines

PubMed Central

KATONA, ÉVA; JUHÁSZ, TAMÁS; SOMOGYI, CSILLA SZŰCS; HAJDÚ, TIBOR; SZÁSZ, CSABA; RÁCZ, KÁLMÁN; KÓKAI, ENDRE; GERGELY, PÁL; ZÁKÁNY, RÓZA

2016-01-01

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment. PMID:26717964

Elasticity, biodegradability and cell adhesive properties of chitosan/hyaluronan multilayer films

NASA Astrophysics Data System (ADS)

Schneider, Aurore; Richert, Ludovic; Francius, Gregory; Voegel, Jean-Claude; Picart, Catherine

2007-03-01

In the bioengineering field, a recent and promising approach to modifying biomaterial surfaces is the layer-by-layer (LbL) technique used to build thin polyelectrolyte multilayer films. In this work, we focused on polyelectrolyte multilayer films made of two polysaccharides, chitosan (CHI) and hyaluronan (HA), and on the control of their physico-chemical and cell adhesive properties by chemical cross-linking. CHI/HA films were cross-linked using a water soluble carbodiimide and observed by confocal laser scanning microscopy (CLSM) with a fluorescently labeled CHI. Film thicknesses were similar for native and cross-linked films. The film nanometer roughness was measured by atomic force microscopy and was found to be higher for cross-linked films. Cross-linking the films also leads to a drastic change in film stiffness. The elastic modulus of the films (Young's modulus) as measured by AFM nano-indentation was about tenfold increased for cross-linked films as compared to native ones. From a biological point of view, cross-liked films are more resistant to enzymatic degradation by hyaluronidase. Furthermore, the increase in film stiffness has a favorable effect on the adhesion and spreading of chondrosarcoma cells. Thus, the CHI/HA cross-linked films could be used for various applications due to their adhesive properties and to their mechanical properties (including stability in enzymatic media).

Domination of volumetric toughening by silver nanoparticles over interfacial strengthening of carbon nanotubes in bactericidal hydroxyapatite biocomposite.

PubMed

Herkendell, Katharina; Shukla, Vishnu Raj; Patel, Anup Kumar; Balani, Kantesh

2014-01-01

In order to address the problem of bacterial infections in bone-substitution surgery, it is essential that bone replacement biomaterials are equipped with bactericidal components. This research aims to optimize the content of silver (Ag), a well-known antibacterial metal, in a multiwalled carbon nanotube (CNT) reinforced hydroxyapatite (HA) composite, to yield a bioceramic which can be used as an antibacterial and tough surface of bone replacement prosthesis. The bactericidal properties evaluated using Escherichia coli and Staphylococcus epidermidis indicate that CNT reinforcement supports growth of Gram negative E. coli bacteria (~8.5% more adhesion than pure HA); but showed a strong decrease of Gram positive S. epidermidis bacteria (~diminished to 66%) compared to that of pure HA. Small amounts of silver (2-5wt.%) already show a severe bactericidal effect when compared to that of HA-CNT (by 30% and ~60% respectively). MTT assay confirmed enhanced biocompatibility of L929 cells on HA-4wt.% CNT (~121%), HA-4wt.% CNT-1wt.% Ag (~124%) sample and HA-4wt.% CNT-2wt.% Ag (~100%) when compared to that of pure HA. The samples with higher silver content showed decreased biocompatibility (77% for HA-4wt.% CNT-5wt.% Ag sample and 73% for HA-4wt.% CNT-10wt.% Ag). Though reinforcement of 4wt.% CNT has shown an increase of fracture toughness by ~62%, silver reinforcement has shown enhancement of up to 244% (i.e. 3.43 times). Accordingly, isolation of toughening contribution indicates that volumetric toughening by silver dominates over interfacial strengthening contributed by CNTs towards enhanced fracture toughness of potential HA-Ag-CNT biocomposites. © 2013.

Differential osteogenic activity of osteoprogenitor cells on HA and TCP/HA scaffold of tissue engineered bone.

PubMed

Ng, Angela M H; Tan, K K; Phang, M Y; Aziyati, O; Tan, G H; Isa, M R; Aminuddin, B S; Naseem, M; Fauziah, O; Ruszymah, B H I

2008-05-01

Biomaterial, an essential component of tissue engineering, serves as a scaffold for cell attachment, proliferation, and differentiation; provides the three dimensional (3D) structure and, in some applications, the mechanical strength required for the engineered tissue. Both synthetic and naturally occurring calcium phosphate based biomaterial have been used as bone fillers or bone extenders in orthopedic and reconstructive surgeries. This study aims to evaluate two popular calcium phosphate based biomaterial i.e., hydroxyapatite (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) granules as scaffold materials in bone tissue engineering. In our strategy for constructing tissue engineered bone, human osteoprogenitor cells derived from periosteum were incorporated with human plasma-derived fibrin and seeded onto HA or TCP/HA forming 3D tissue constructs and further maintained in osteogenic medium for 4 weeks to induce osteogenic differentiation. Constructs were subsequently implanted intramuscularly in nude mice for 8 weeks after which mice were euthanized and constructs harvested for evaluation. The differential cell response to the biomaterial (HA or TCP/HA) adopted as scaffold was illustrated by the histology of undecalcified constructs and evaluation using SEM and TEM. Both HA and TCP/HA constructs showed evidence of cell proliferation, calcium deposition, and collagen bundle formation albeit lesser in the former. Our findings demonstrated that TCP/HA is superior between the two in early bone formation and hence is the scaffold material of choice in bone tissue engineering. Copyright 2007 Wiley Periodicals, Inc.

Correlation between surface properties and wettability of multi-scale structured biocompatible surfaces

NASA Astrophysics Data System (ADS)

Gorodzha, S. N.; Surmeneva, M. A.; Prymak, O.; Wittmar, A.; Ulbricht, M.; Epple, M.; Teresov, A.; Koval, N.; Surmenev, R. A.

2015-11-01

The influence of surface properties of radio-frequency (RF) magnetron deposited hydroxyapatite (HA) and Si-containing HA coatings on wettability was studied. The composition and morphology of the coatings fabricated on titanium (Ti) were characterized using atomic force microscopy (AFM) and X-ray diffraction (XRD). The surface wettability was studied using contact angle analysis. Different geometric parameters of acid-etched (AE) and pulse electron beam (PEB)-treated Ti substrates and silicate content in the HA films resulted in the different morphology of the coatings at micro- and nano- length scales. Water contact angles for the HA coated Ti samples were evaluated as a combined effect of micro roughness of the substrate and nano-roughness of the HA films resulting in higher water contact angles compared with acid-etched (AE) or pulse electron beam (PEB) treated Ti substrates.

A Modified ELISA Accurately Measures Secretion of High Molecular Weight Hyaluronan (HA) by Graves' Disease Orbital Cells

PubMed Central

Krieger, Christine C.

2014-01-01

Excess production of hyaluronan (hyaluronic acid [HA]) in the retro-orbital space is a major component of Graves' ophthalmopathy, and regulation of HA production by orbital cells is a major research area. In most previous studies, HA was measured by ELISAs that used HA-binding proteins for detection and rooster comb HA as standards. We show that the binding efficiency of HA-binding protein in the ELISA is a function of HA polymer size. Using gel electrophoresis, we show that HA secreted from orbital cells is primarily comprised of polymers more than 500 000. We modified a commercially available ELISA by using 1 million molecular weight HA as standard to accurately measure HA of this size. We demonstrated that IL-1β-stimulated HA secretion is at least 2-fold greater than previously reported, and activation of the TSH receptor by an activating antibody M22 from a patient with Graves' disease led to more than 3-fold increase in HA production in both fibroblasts/preadipocytes and adipocytes. These effects were not consistently detected with the commercial ELISA using rooster comb HA as standard and suggest that fibroblasts/preadipocytes may play a more prominent role in HA remodeling in Graves' ophthalmopathy than previously appreciated. PMID:24302624

Process and kinetics of bonelike apatite formation on sintered hydroxyapatite in a simulated body fluid.

PubMed

Kim, Hyun-Min; Himeno, Teruyuki; Kokubo, Tadashi; Nakamura, Takashi

2005-07-01

The surfaces of two hydroxyapatites (HA), which have been sintered at different temperatures of 800 and 1200 degrees C, was investigated as a function of soaking time in simulated body fluid (SBF) using transmission electron microscopy (TEM) attached with energy-dispersive spectrometry (EDX) and laser electrophoresis spectroscopy. The TEM-EDX indicated that after soaking in SBF, both the HAs form bonelike apatite by undergoing the same surface structural change, i.e., formations of a Ca-rich amorphous or nano-crystalline calcium phosphate (ACP) and a Ca-poor ACP, which eventually crystallized into bonelike apatite. Zeta potential characterized by the electrophoresis indicated that during exposure to SBF, the HA surfaces reveal negative surface charge, thereby interacting with the positive calcium ions in the fluid to form the Ca-rich ACP, which gains positive surface charge. The Ca-rich ACP on the HAs then interacts with the negative phosphate ions in the fluid to form the Ca-poor ACP, which stabilizes by being crystallized into bonelike apatite with a low solubility in the SBF. The exposure times for formations of these phases of the Ca-rich ACP, the Ca-poor ACP as well as the apatite were, however, all late on HA sintered at 1200 degrees C, compared with the HA sintered at 800 degrees C. This phenomenon was attributed to a lower initial negative surface charge of the HA sintered at 800 degrees C than of that one sintered at 1200 degrees C, owing to poverty in surface hydroxyl and phosphate groups which are responsible for the surface negativity of the HA. These indicate that sintered temperature of HA might influence not in terms of the process but in terms of the rate of formation of biologically active bonelike apatite on its surface, through which the HA integrates with living bone.

Zinc-substituted hydroxyapatite: a biomaterial with enhanced bioactivity and antibacterial properties.

PubMed

Thian, E S; Konishi, T; Kawanobe, Y; Lim, P N; Choong, C; Ho, B; Aizawa, M

2013-02-01

Hydroxyapatite (HA) is a synthetic biomaterial and has been found to promote new bone formation when implanted in a bone defect site. However, its use is often limited due to its slow osteointegration rate and low antibacterial activity, particularly where HA has to be used for long term biomedical applications. This work will describe the synthesis and detailed characterization of zinc-substituted HA (ZnHA) as an alternative biomaterial to HA. ZnHA containing 1.6 wt% Zn was synthesized via a co-precipitation reaction between calcium hydroxide, orthophosphoric acid and zinc nitrate hexahydrate. Single-phase ZnHA particles with a rod-like morphology measuring ~50 nm in length and ~15 nm in width, were obtained and characterized using transmission electron microscopy and X-ray diffraction. The substitution of Zn into HA resulted in a decrease in both the a- and c-axes of the unit cell parameters, thereby causing the HA crystal structure to alter. In vitro cell culture work showed that ZnHA possessed enhanced bioactivity since an increase in the growth of human adipose-derived mesenchymal stem cells along with the bone cell differentiation markers, were observed. In addition, antibacterial work demonstrated that ZnHA exhibited antimicrobial capability since there was a significant decrease in the number of viable Staphylococcus aureus bacteria after in contact with ZnHA.

Stimulation of TLRs by LMW-HA induces self-defense mechanisms in vaginal epithelium.

PubMed

Dusio, Giuseppina F; Cardani, Diego; Zanobbio, Laura; Mantovani, Martina; Luchini, Patrizia; Battini, Lorenzo; Galli, Valentina; Diana, Angela; Balsari, Andrea; Rumio, Cristiano

2011-07-01

The innate immune system is present throughout the female reproductive tract and functions in synchrony with the adaptive immune system to provide protection in a way that enhances the chances for fetal survival, while protecting against potential pathogens. Recent data show that activation of Toll-like receptor (TLR)2 and 4 by low-molecular weight hyaluronic acid (LMW-HA) in the epidermis induces secretion of the antimicrobial peptide β-defensin 2. In the present work, we show that LMW-HA induces vaginal epithelial cells to release different antimicrobial peptides, via activation of TLR2 and TLR4. Further, we found that LMW-HA favors repair of vaginal epithelial injury, involving TLR2 and TLR4, and independently from its classical receptor CD44. This wound-healing activity of LMW-HA is dependent from an Akt/phosphatidylinositol 3 kinase pathway. Therefore, these findings suggest that the vaginal epithelium is more than a simple physical barrier to protect against invading pathogens: on the contrary, this surface acts as efficient player of innate host defense, which may modulate its antimicrobial properties and injury restitution activity, following LMW-HA stimulation; this activity may furnish an additional protective activity to this body compartment, highly and constantly exposed to microbiota, ameliorating the self-defense of the vaginal epithelium in both basal and pathological conditions.

Interaction of Low Molecular Weight Hyaluronan (LMW-HA) with CD44 and Toll-Like Receptors Promotes the Actin Filament-Associated Protein (AFAP-110)-Actin Binding and MyD88-NFκB Signaling Leading to Pro-inflammatory Cytokine/Chemokine Production and Breast Tumor Invasion

PubMed Central

Bourguignon, Lilly Y.W.; Wong, Gabriel; Earle, Christine A.; Xia, Weiliang

2011-01-01

Both high and low molecular weight hyaluronan (HMW-HA vs. LMW-HA) exist in various tissues and cells. In this study we investigated LMW-HA-mediated CD44 interaction with Toll-like receptors (TLRs), the actin filament-associated protein (AFAP-110) and a myeloid differentiation factor (MyD88) in breast tumor cells (MDA-MB-231 cells). Our data indicate that LMW-HA (but not HMW-HA) preferentially stimulates a physical association between CD44 and TLRs followed by a concomitant recruitment of AFAP-110 and MyD88 into receptor-containing complexes in breast tumor cells. LMW-HA-activated AFAP-110 then binds to F-actin resulting in MyD88/NF-κB nuclear translocation, NF-κB-specific transcription and target gene (IL-1β and IL-8) expression. These signaling events lead to pro-inflammatory cytokine/chemokine production in the breast tumor cells. AFAP-110-F-actin (activated by LMW-HA) also promotes tumor cell invasion. Downregulation of AFAP-110 or MyD88 by transfecting breast tumor cells with AFAP-110 siRNA or MyD88 siRNA, respectively not only blocks the ability of LMW-HA to stimulate AFAP-110-actin function, but also impairs MyD88-NF-κB nuclear translocation and NF-κB transcriptional activation. Consequently, both IL-1β/IL-8 production and tumor cell invasion are impaired. Taken together, these findings suggest that LMW-HA plays an important role in CD44-TLR-associated AFAP-110-actin interaction and MyD88-NF-κB signaling required for tumor cell behaviors which may contribute to the progression of breast cancer. PMID:22031535

Influences of animal mucins on lysozyme activity in solution and on hydroxyapatite surfaces.

PubMed

Park, Won-Kyu; Chung, Jin-Woo; Kim, Young-Ku; Chung, Sung-Chang; Kho, Hong-Seop

2006-10-01

The purpose of this study was to investigate the influence of animal mucins on lysozyme activity in solution and on the surface of hydroxyapatite (HA) beads. The effects of animal mucins on lysozyme activity in solution were examined by incubating porcine gastric mucin (PGM) or bovine submaxillary mucin (BSM) with hen egg-white lysozyme (HEWL) or salivary samples. HA-immobilised animal mucins or lysozyme were used to determine the influence of animal mucins on lysozyme activity on HA surfaces. Lysozyme activity was determined by turbidity measurement of a Micrococcus lysodeikticus substrate suspension. Protein concentration was determined by ninhydrin assay. PGM inhibited the activity of HEWL and salivary lysozyme in solution. The amount of inhibition was dependent on mucin concentration, incubation time and temperature, and the structural integrity of the mucin. The inhibition of salivary lysozyme activity by PGM was greater in submandibular/sublingual saliva than in parotid saliva. The inhibition of lysozyme activity by PGM was markedly dependent on pH. However, BSM did not inhibit the in-solution lysozyme activities of HEWL and clarified saliva. Both PGM and BSM bound to HA surfaces, and HA-adsorbed animal mucins increased the subsequent adsorption of lysozyme. When HA beads were exposed to a mixture of HEWL and PGM or BSM, lysozyme activity on the HA surfaces was significantly increased. The results suggest that animal mucins affect lysozyme activity, and the effects are different on HA surfaces compared with in solution. Further research is needed to determine the effect of animal mucins on lysozyme activity in vivo.

Somatodendritic surface expression of epitope-tagged and KChIP binding-deficient Kv4.2 channels in hippocampal neurons.

PubMed

Prechtel, Helena; Hartmann, Sven; Minge, Daniel; Bähring, Robert

2018-01-01

Kv4.2 channels mediate a subthreshold-activating somatodendritic A-type current (ISA) in hippocampal neurons. We examined the role of accessory Kv channel interacting protein (KChIP) binding in somatodendritic surface expression and activity-dependent decrease in the availability of Kv4.2 channels. For this purpose we transfected cultured hippocampal neurons with cDNA coding for Kv4.2 wild-type (wt) or KChIP binding-deficient Kv4.2 mutants. All channels were equipped with an externally accessible hemagglutinin (HA)-tag and an EGFP-tag, which was attached to the C-terminal end. Combined analyses of EGFP self-fluorescence, surface HA immunostaining and patch-clamp recordings demonstrated similar dendritic trafficking and functional surface expression for Kv4.2[wt]HA,EGFP and the KChIP binding-deficient Kv4.2[A14K]HA,EGFP. Coexpression of exogenous KChIP2 augmented the surface expression of Kv4.2[wt]HA,EGFP but not Kv4.2[A14K]HA,EGFP. Notably, activity-dependent decrease in availability was more pronounced in Kv4.2[wt]HA,EGFP + KChIP2 coexpressing than in Kv4.2[A14K]HA,EGFP + KChIP2 coexpressing neurons. Our results do not support the notion that accessory KChIP binding is a prerequisite for dendritic trafficking and functional surface expression of Kv4.2 channels, however, accessory KChIP binding may play a potential role in Kv4.2 modulation during intrinsic plasticity processes.

Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-RasV12 -induced cell transformation.

PubMed

Lin, Hsiu-Kuan; Lin, Hsi-Hui; Chiou, Yu-Wei; Wu, Ching-Lung; Chiu, Wen-Tai; Tang, Ming-Jer

2018-05-01

Caveolin-1 (Cav1) is down-regulated during MK4 (MDCK cells harbouring inducible Ha-Ras V12 gene) transformation by Ha-Ras V12 . Cav1 overexpression abrogates the Ha-Ras V12 -driven transformation of MK4 cells; however, the targeted down-regulation of Cav1 is not sufficient to mimic this transformation. Cav1-silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction-related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I-CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha-Ras V12 -inducing MK4 cells increased exosome-like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I-CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I-CM (MK4+I-EXs). Wnt5a, a downstream product of Ha-Ras V12 , was markedly secreted by MK4+I-CM and MK4+I-EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha-Ras V12 - and MK4+I-CM-induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down-regulation, either by Ha-Ras V12 or targeted shRNA, increased frizzled-2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I-EXs in MDCK cells. These data suggest that Cav1-dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha-Ras V12 -Wnt5a-Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha-Ras V12 -driven cell transformation. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Potentially harmful microalgal distribution in an area of the NW Adriatic coastline: Sampling procedure and correlations with environmental factors

NASA Astrophysics Data System (ADS)

Penna, Antonella; Ingarao, Cristina; Ercolessi, Manuela; Rocchi, Marco; Penna, Nunzio

2006-10-01

In this study, the trend of potentially Harmful Algal (HA) taxa (genera and species), was analysed along a coastal area of the NW Adriatic Sea on a monthly scale. The study included the use of a phytoplankton net for sample collection. The investigation was carried out in four sampling stations characterised by different ecological features. The composition of potentially HA phytoplankton taxa and their succession were related to the environmental factors. The potentially HA group abundance accounted for 8% of all the phytoplankton taxa considered. Multivariate analyses of environmental factors suggested that potentially HA taxa are sensitive to phosphate content: potential DSP-YTX (Diarrhetic Shellfish Poisoning-Yessotoxin) producers were positively correlated with P content ( p = 0.023), while potential ASP (Amnesic Shellfish Poisoning) producers were negatively correlated with P content ( p = 0.006). Phosphorus could be considered to be the limiting factor for phytoplankton taxa density in the NW Adriatic Sea. There was a highly positive correlation between the occurrences of potentially HA taxa and low values of salinity ( p = 0.001 for potential producers of ASP, p = 0.029 for potential DSP-YTX producers). The counting of potential HA dinoflagellates in net samples represented a more accurate estimation of potential HA abundances in the water column making it possible to concentrate a greater number of potential HA dinoflagellate cells by net sampling along the entire water column rather than by sampling only at the surface as in routine monitoring procedures.

Hydroxyapatite-coated magnesium implants with improved in vitro and in vivo biocorrosion, biocompatibility, and bone response.

PubMed

Kim, Sae-Mi; Jo, Ji-Hoon; Lee, Sung-Mi; Kang, Min-Ho; Kim, Hyoun-Ee; Estrin, Yuri; Lee, Jong-Ho; Lee, Jung-Woo; Koh, Young-Hag

2014-02-01

Magnesium and its alloys are candidate materials for biodegradable implants; however, excessively rapid corrosion behavior restricts their practical uses in biological systems. For such applications, surface modification is essential, and the use of anticorrosion coatings is considered as a promising avenue. In this study, we coated Mg with hydroxyapatite (HA) in an aqueous solution containing calcium and phosphate sources to improve its in vitro and in vivo biocorrosion resistance, biocompatibility and bone response. A layer of needle-shaped HA crystals was created uniformly on the Mg substrate even when the Mg sample had a complex shape of a screw. In addition, a dense HA-stratum between this layer and the Mg substrate was formed. This HA-coating layer remarkably reduced the corrosion rate of the Mg tested in a simulated body fluid. Moreover, the biological response, including cell attachment, proliferation and differentiation, of the HA-coated samples was enhanced considerably compared to samples without a coating layer. The preliminary in vivo experiments also showed that the biocorrosion of the Mg implant was significantly retarded by HA coating, which resulted in good mechanical stability. In addition, in the case of the HA-coated implants, biodegradation was mitigated, particularly over the first 6 weeks of implantation. This considerably promoted bone growth at the interface between the implant and bone. These results confirmed that HA-coated Mg is a promising material for biomedical implant applications. © 2013 Wiley Periodicals, Inc.

Synthesis, characterization and cell behavior of fluoridated hydroxyapatite

NASA Astrophysics Data System (ADS)

Qu, Haibo

Fluorine-containing hydroxyapatite (Ca5(PO4) 3(OH)1-xFx FHA), where F- partially replaces OH- in hydroxyapatite (HA), is recognized as a possible biomaterial for bone and tooth implants and gaining attention in the last several years as a possible alternative to HA. In this study, FHA powders were synthesized through a pH-cycling method. It was discovered that fluorine incorporation increased with the fluorine content in the initial solution and the number of pH cycles employed. A relatively low fluorine incorporation efficiency, ˜60%, was attained for most of the FHA samples. The short time of stay at each pH cycle and the limited number of cycles used are believed to be the main reasons of the low fluorine incorporation into the apatite structure. It was also revealed that the FHA particles produced by the pH-cycling method were inhomogeneous. They were a mixture of hydroxyapatite and F-rich apatite (or FA) particles. The mechanisms of incorporation of fluorine ions into hydroxyapatite by a pH cyclicing method were studied using TEM, XRD and fluorine measurement. Instead of forming laminated structures as reported by other research groups, a mixture of nano-sized F-rich apatite (FHA) and hydroxyapatite (HA) particles were obtained using the pH-cyclicing method. After calcination, these FHA particles were homogenized and became single phased FHA. The effect of fluorine content, preparing method, and sintering temperature on both the bulk density and biaxial flexural strength of sintered FHA was studied. Both uniaxially pressed un-milled (UPU) and cold isostatically pressed milled (IPM) FHA discs were sintered at temperatures between 1200˜400°C at an interval of 100°C. It was found that the fluorine content had a significant impact on the sintering behavior, densification, and mechanical properties of FHA discs. A close correlation between the sintered density and biaxial flexural strength of the specimens was revealed, where the biaxial flexural strength increased exponentially with the sintered density. FHA discs with various fluorine contents have been used to investigate the effect of fluorine content on osteoblastic cell behaviors. Rat osteosarcoma (ROS 17/28) cells were cultured on FHA discs for appropriate times. The osteoblastic cell behaviors were examined in terms of cell attachment, proliferation, morphology and differentiation. The fluorine content in FHA strongly affected the cell activities. More cell attachment and proliferation were observed on the fluorine-containing FHA than pure HA. Fluorine content also affected the differentiation behaviors of osteoblastic cells. Cells on fluorine-containing FHA had higher alkaline phosphatase (ALP) activity than pure HA in 2 weeks. The morphology of the cells showed that it took less time for cells to cover the surface of fluorine-containing samples than that of pure HA. These results suggested that fluorine ions had a significant impact on osteoblastic cell behaviors.

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition

PubMed Central

Nicholls, Sarah; Piper, Karen P.; Mohammed, Fiyaz; Dafforn, Timothy R.; Tenzer, Stefan; Salim, Mahboob; Mahendra, Premini; Craddock, Charles; van Endert, Peter; Schild, Hansjörg; Cobbold, Mark; Engelhard, Victor H.; Moss, Paul A. H.; Willcox, Benjamin E.

2009-01-01

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1H versus HA-1R) in the HMHA1 gene. The HA-1H peptide is restricted by HLA-A2 and is immunogenic in HA-1R/R into HA-1H transplants, while HA-1R has been suggested to be a “null allele” in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1R variant. To understand these findings, we determined the structure of an HLA-A2-HA-1H complex to 1.3Å resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1H-specific T-cells bound HA-1H peptide with moderate affinity but failed to bind HA-1R, indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition. PMID:19234124

Cell responses to two kinds of nanohydroxyapatite with different sizes and crystallinities.

PubMed

Liu, Xiaochen; Zhao, Minzhi; Lu, Jingxiong; Ma, Jian; Wei, Jie; Wei, Shicheng

2012-01-01

Hydroxyapatite (HA) is the principal inorganic constituent of human bone. Due to its good biocompatibility and osteoconductivity, all kinds of HA particles were prepared by different methods. Numerous reports demonstrated that the properties of HA affected its biological effects. Two kinds of nanohydroxyapatite with different sizes and crystallinities were obtained via a hydrothermal treatment method under different temperatures. It was found that at a temperature of 140°C, a rod-like crystal (n-HA1) with a diameter of 23 ± 5 nm, a length of 47 ± 14 nm, and crystallinity of 85% ± 5% was produced, while at a temperature of 80°C, a rod-like crystal (n-HA2) with a diameter of 16 ± 3 nm, a length of 40 ± 10 nm, and crystallinity of 65% ± 3% was produced. The influence of nanohydroxyapatite size and crystallinity on osteoblast viability was studied by MTT, scanning electron microscopy, and flow cytometry. n-HA1 gave a better biological response than n-HA2 in promoting cell growth and inhibiting cell apoptosis, and also exhibited much more active cell morphology. Alkaline phosphatase activity for both n-HA2 and n-HA1 was obviously higher than for the control, and no significant difference was found between n-HA1 and n-HA2. The same trend was observed on Western blotting for expression of type I collagen and osteopontin. In addition, it was found by transmission electron microscopy that large quantities of n-HA2 entered into the cell and damaged the cellular morphology. Release of tumor necrosis factor alpha from n-HA2 was markedly higher than from n-HA1, indicating that n-HA2 might trigger a severe inflammatory response. This work indicates that not all nanohydroxyapatite should be considered a good biomaterial in future clinical applications.

Establishment of a cell model of X-linked sideroblastic anemia using genome editing.

PubMed

Kaneko, Kiriko; Kubota, Yoshiko; Nomura, Kazumi; Hayashimoto, Haruka; Chida, Taisei; Yoshino, Naoto; Wayama, Marina; Ogasawara, Katsutoshi; Nakamura, Yukio; Tooyama, Ikuo; Furuyama, Kazumichi

2018-06-13

ALAS2 gene mutations cause X-linked sideroblastic anemia. The presence of ring sideroblasts in a patient's bone marrow is the hallmark of sideroblastic anemia, but the precise mechanisms underlying sideroblast formation are largely unknown. Using a genome editing system, a mutation was introduced in the erythroid-specific enhancer of the ALAS2 gene in HUDEP2 cells, which were derived from human umbilical stem cells and can produce erythrocytes. The established cell line, termed HA2low, expressed less ALAS2 mRNA than did wild-type cells, even after erythroid differentiation. Although the mRNA expression of α-globin, β-globin, and the mitochondrial iron importer mitoferrin-1 was induced similarly in wild-type and HA2low cells, hemoglobinization of differentiated cells was limited in HA2low cells compared to wild-type cells. Importantly, Prussian blue staining revealed that approximately one-third of differentiated HA2low cells exhibited intracellular iron deposition, and these cells looked like ring sideroblasts. Electron microscopy confirmed that the mitochondria in HA2low cells contained high-density deposits that might contain iron. Ring sideroblastic cells appeared among HA2low cells only after differentiation, while the induced expression of mitochondrial ferritin was observed in both cell types during differentiation. These results suggest that the induction of mitochondrial ferritin expression might be essential for, but not the primary cause of, ring sideroblast formation. Our results also suggest that the insufficient supply of protoporphyrin IX due to ALAS2 deficiency, in combination with increased iron import into mitochondria during erythroid differentiation, results in the formation of ring sideroblasts. Furthermore, HA2low cells are a useful tool for characterizing ring sideroblasts in vitro. Copyright © 2018. Published by Elsevier Inc.

Ameliorative Effect of Curcumin-Encapsulated Hyaluronic Acid-PLA Nanoparticles on Thioacetamide-Induced Murine Hepatic Fibrosis.

PubMed

Chen, Yu-Nong; Hsu, Shih-Lan; Liao, Ming-Yuan; Liu, Yi-Ting; Lai, Chien-Hung; Chen, Ji-Feng; Nguyen, Mai-Huong Thi; Su, Yung-Hsiang; Chen, Shang-Ting; Wu, Li-Chen

2016-12-24

In this study, we developed curcumin-encapsulated hyaluronic acid-polylactide nanoparticles (CEHPNPs) to be used for liver fibrosis amelioration. CD44, the hyaluronic acid (HA) receptor, is upregulated on the surface of cancer cells and on activated hepatic stellate cells (aHSCs) rather than normal cells. CEHPNPs could bind to CD44 and be internalized effectively through endocytosis to release curcumin, a poor water-soluble liver protective agent. Thus, CEHPNPs were potentially not only improving drug efficiency, but also targeting aHSCs. HA and polylactide (PLA) were crosslinked by adipic acid dihydrazide (ADH). The synthesis of HA-PLA was monitored by Fourier-transform infrared (FTIR) and Nuclear Magnetic Resonance (NMR). The average particle size was approximately 60-70 nm as determined by dynamic light scattering (DLS) and scanning electron microscope (SEM). Zeta potential was around -30 mV, which suggested a good stability of the particles. This drug delivery system induced significant aHSC cell death without affecting quiescent HSCs, hepatic epithelial, and parenchymal cells. This system reduced drug dosage without sacrificing therapeutic efficacy. The cytotoxicity IC 50 (inhibitory concentration at 50%) value of CEHPNPs was approximately 1/30 to that of the free drug treated group in vitro. Additionally, the therapeutic effects of CEHPNPs were as effective as the group treated with the same curcumin dose intensity in vivo. CEHPNPs significantly reduced serum aspartate transaminase/alanine transaminase (ALT/AST) significantly, and attenuated tissue collagen production and cell proliferation as revealed by liver biopsy. Conclusively, the advantages of superior biosafety and satisfactory therapeutic effect mean that CEHPNPs hold great potential for treating hepatic fibrosis.

Nanoparticles decorated with viral antigens are more immunogenic at low surface density.

PubMed

Brewer, Matthew G; DiPiazza, Anthony; Acklin, Joshua; Feng, Changyong; Sant, Andrea J; Dewhurst, Stephen

2017-02-01

There is an urgent need to develop protective vaccines for high priority viral pathogens. One approach known to enhance immune responses to viral proteins is to display them on a nanoparticle (NP) scaffold. However, little is known about the effect of protein density on the B cell response to antigens displayed on NPs. To address this question HIV-1 Envelope (Env) and influenza hemagglutinin (HA) were displayed on a polystyrene-based NP scaffold at various densities - corresponding to mean antigen distances that span the range encountered on naturally occurring virions. Our studies revealed that NPs displaying lower densities of Env or HA more efficiently stimulated antigen-specific B cells in vitro, as measured by calcium flux, than did NPs displaying higher antigen densities. Similarly, NPs displaying a low density of Env or HA also elicited higher titers of antigen-specific serum IgG in immunized BALB/c mice (including elevated titers of hemagglutination-inhibiting antibodies), as well as an increased frequency of antigen-specific antibody secreting cells in the lymph node, spleen and bone marrow. Importantly, our studies showed that the enhanced B cell response elicited by the lower density NPs is likely secondary to more efficient development of follicular helper CD4 T cells and germinal center B cells. These findings demonstrate that the density of antigen on a NP scaffold is a critical determinant of the humoral immune response elicited, and that high density display does not always result in an optimal response. Copyright © 2017 Elsevier Ltd. All rights reserved.

Anatomic Mesenchymal Stem Cell-Based Engineered Cartilage Constructs for Biologic Total Joint Replacement

PubMed Central

Saxena, Vishal; Kim, Minwook; Keah, Niobra M.; Neuwirth, Alexander L.; Stoeckl, Brendan D.; Bickard, Kevin; Restle, David J.; Salowe, Rebecca; Wang, Margaret Ye; Steinberg, David R.

2016-01-01

Cartilage has a poor healing response, and few viable options exist for repair of extensive damage. Hyaluronic acid (HA) hydrogels seeded with mesenchymal stem cells (MSCs) polymerized through UV crosslinking can generate functional tissue, but this crosslinking is not compatible with indirect rapid prototyping utilizing opaque anatomic molds. Methacrylate-modified polymers can also be chemically crosslinked in a cytocompatible manner using ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED). The objectives of this study were to (1) compare APS/TEMED crosslinking with UV crosslinking in terms of functional maturation of MSC-seeded HA hydrogels; (2) generate an anatomic mold of a complex joint surface through rapid prototyping; and (3) grow anatomic MSC-seeded HA hydrogel constructs using this alternative crosslinking method. Juvenile bovine MSCs were suspended in methacrylated HA (MeHA) and crosslinked either through UV polymerization or chemically with APS/TEMED to generate cylindrical constructs. Minipig porcine femoral heads were imaged using microCT, and anatomic negative molds were generated by three-dimensional printing using fused deposition modeling. Molded HA constructs were produced using the APS/TEMED method. All constructs were cultured for up to 12 weeks in a chemically defined medium supplemented with TGF-β3 and characterized by mechanical testing, biochemical assays, and histologic analysis. Both UV- and APS/TEMED-polymerized constructs showed increasing mechanical properties and robust proteoglycan and collagen deposition over time. At 12 weeks, APS/TEMED-polymerized constructs had higher equilibrium and dynamic moduli than UV-polymerized constructs, with no differences in proteoglycan or collagen content. Molded HA constructs retained their hemispherical shape in culture and demonstrated increasing mechanical properties and proteoglycan and collagen deposition, especially at the edges compared to the center of these larger constructs. Immunohistochemistry showed abundant collagen type II staining and little collagen type I staining. APS/TEMED crosslinking can be used to produce MSC-seeded HA-based neocartilage and can be used in combination with rapid prototyping techniques to generate anatomic MSC-seeded HA constructs for use in filling large and anatomically complex chondral defects or for biologic joint replacement. PMID:26871863

The use of acetone to enhance the infiltration of HA nanoparticles into a demineralized dentin collagen matrix.

PubMed

Besinis, Alexandros; van Noort, Richard; Martin, Nicolas

2016-03-01

This study investigates the role of acetone, as a carrier for nano-hydroxyapatite (nano-HA) in solution, to enhance the infiltration of fully demineralized dentin with HA nanoparticles (NPs). Dentin specimens were fully demineralized and subsequently infiltrated with two types of water-based nano-HA solutions (one containing acetone and one without). Characterization of the dentin surfaces and nano-HA particles was performed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The surface wettability and infiltration capacity of the nano-HA solutions were quantified by means of contact angle measurements and energy dispersive X-ray spectroscopy (EDS), respectively. Contact angle measurements were taken at baseline and repeated at regular intervals to assess the effect of acetone. The P and Ca levels of infiltrated dentin specimens were measured and compared to sound dentin and non-infiltrated controls. The presence of acetone resulted in an eight-fold decrease in the contact angles of the nano-HA solutions recorded on the surface of demineralized dentin compared to nano-HA solutions without acetone (one-way ANOVA, p<0.05). Perfect wetting of the demineralized dentin surface was achieved 5min after the application of the nano-HA solution containing acetone. Infiltration of demineralized dentin with the nano-HA solution containing acetone restored the lost mineral content by 50%, whereas the mean mineralization values for P and Ca in dentin treated with the acetone-free nano-HA solution were less than 6%. Acetone was shown to act as a vehicle to enhance the capacity to infiltrate demineralized dentin with HA NPs. The successful infiltration of dentin collagen with HA NPs provides a suitable scaffold, whereby the infiltrated HA NPs have the potential to act as seeds that may initiate heterogenous mineral growth when exposed to an appropriate mineral-rich environment. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Novel Hyaluronan Formulation Enhances the Efficacy of Boron Neutron Capture Therapy for Murine Mesothelioma.

PubMed

Sasai, Masao; Nakamura, Hiroyuki; Sougawa, Nagako; Sakurai, Yoshinori; Suzuki, Minoru; Lee, Chun Man

2016-03-01

Malignant pleural mesothelioma (MPM) is a refractory cancer of the pleura caused by asbestos exposure. MPM is difficult to treat because it easily disseminates. Boron neutron capture therapy (BNCT) is a radiotherapy in which cancer cells that selectively take up (10)Boron-containing compounds are destroyed, and normal cells are uninjured. Hyaluronan (HA) is a ligand of cluster of differentiation 44 (CD44), that is expressed on MPM cells. In order to enhance BNCT for MPM tumors, we developed a novel HA-containing (10)B (sodium borocaptate: BSH) formulation (HA-BND-S). We examined the efficacy of HA-BND-S using MPM cells and a mouse MPM model. HA-BND-S preferentially bound MPM cells dose-dependently, and increased the cytotoxicity of BNCT compared to BSH in vitro. HA-BND-S administration significantly increased the survival of MPM tumor-bearing mice compared to BSH at the same (10)B dosage in BNCT. Modifying BSH with HA is a promising strategy for enhancing the efficacy of BNCT for therapy of MPM. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Evaluation of the potentials of humic acid removal in water by gas phase surface discharge plasma.

PubMed

Wang, Tiecheng; Qu, Guangzhou; Ren, Jingyu; Yan, Qiuhe; Sun, Qiuhong; Liang, Dongli; Hu, Shibin

2016-02-01

Degradation of humic acid (HA), a predominant type of natural organic matter in ground water and surface waters, was conducted using a gas phase surface discharge plasma system. HA standard and two surface waters (Wetland, and Weihe River) were selected as the targets. The experimental results showed that about 90.9% of standard HA was smoothly removed within 40 min's discharge plasma treatment at discharge voltage 23.0 kV, and the removal process fitted the first-order kinetic model. Roles of some active species in HA removal were studied by evaluating the effects of solution pH and OH radical scavenger; and the results presented that O3 and OH radical played significant roles in HA removal. Scanning electron microscope (SEM) and FTIR analysis showed that HA surface topography and molecular structure were changed during discharge plasma process. The mineralization of HA was analyzed by UV-Vis spectrum, dissolved organic carbon (DOC), specific UV absorbance (SUVA), UV absorption ratios, and excitation-emission matrix (EEM) fluorescence. The formation of disinfection by-products during HA sample chlorination was also identified, and CHCl3 was detected as the main disinfection by-product, but discharge plasma treatment could suppress its formation to a certain extent. In addition, approximately 82.3% and 67.9% of UV254 were removed for the Weihe River water and the Wetland water after 40 min of discharge plasma treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Differential Expression and Enzymatic Activity of DPPIV/CD26 Affects Migration Ability of Cervical Carcinoma Cells

PubMed Central

Beckenkamp, Aline; Willig, Júlia Biz; Santana, Danielle Bertodo; Nascimento, Jéssica; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Bruno, Alessandra Nejar; Pilger, Diogo André; Wink, Márcia Rosângela; Buffon, Andréia

2015-01-01

Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane glycoprotein that inactivates or degrades some bioactive peptides and chemokines. For this reason, it regulates cell proliferation, migration and adhesion, showing its role in cancer processes. This enzyme is found mainly anchored onto the cell membrane, although it also has a soluble form, an enzymatically active isoform. In the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and non-tumorigenic HaCaT cells. The effect of the DPPIV/CD26 inhibitor (sitagliptin phosphate) on cell migration and adhesion was also evaluated. Cervical cancer cells and keratinocytes exhibited DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form. DPPIV/CD26 expression was observed in HaCaT, SiHa and C33A, while in HeLa cells it was almost undetectable. We observed higher migratory capacity of HeLa, when compared to SiHa. But in the presence of sitagliptin SiHa showed an increase in migration, indicating that, at least in part, cell migration is regulated by DPPIV/CD26 activity. Furthermore, in the presence of sitagliptin phosphate, SiHa and HeLa cells exhibited a significant reduction in adhesion. However this mechanism seems to be mediated independent of DPPIV/CD26. This study demonstrates, for the first time, the activity and expression of DPPIV/CD26 in cervical cancer cells and the effect of sitagliptin phosphate on cell migration and adhesion. PMID:26222679

Differential Expression and Enzymatic Activity of DPPIV/CD26 Affects Migration Ability of Cervical Carcinoma Cells.

PubMed

Beckenkamp, Aline; Willig, Júlia Biz; Santana, Danielle Bertodo; Nascimento, Jéssica; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Bruno, Alessandra Nejar; Pilger, Diogo André; Wink, Márcia Rosângela; Buffon, Andréia

2015-01-01

Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane glycoprotein that inactivates or degrades some bioactive peptides and chemokines. For this reason, it regulates cell proliferation, migration and adhesion, showing its role in cancer processes. This enzyme is found mainly anchored onto the cell membrane, although it also has a soluble form, an enzymatically active isoform. In the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and non-tumorigenic HaCaT cells. The effect of the DPPIV/CD26 inhibitor (sitagliptin phosphate) on cell migration and adhesion was also evaluated. Cervical cancer cells and keratinocytes exhibited DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form. DPPIV/CD26 expression was observed in HaCaT, SiHa and C33A, while in HeLa cells it was almost undetectable. We observed higher migratory capacity of HeLa, when compared to SiHa. But in the presence of sitagliptin SiHa showed an increase in migration, indicating that, at least in part, cell migration is regulated by DPPIV/CD26 activity. Furthermore, in the presence of sitagliptin phosphate, SiHa and HeLa cells exhibited a significant reduction in adhesion. However this mechanism seems to be mediated independent of DPPIV/CD26. This study demonstrates, for the first time, the activity and expression of DPPIV/CD26 in cervical cancer cells and the effect of sitagliptin phosphate on cell migration and adhesion.

Development of Novel Biocomposite Scaffold of Chitosan-Gelatin/Nanohydroxyapatite for Potential Bone Tissue Engineering Applications

NASA Astrophysics Data System (ADS)

Dan, Yang; Liu, Ouyang; Liu, Yong; Zhang, Yuan-Yuan; Li, Shuai; Feng, Xiao-bo; Shao, Zeng-wu; Yang, Cao; Yang, Shu-Hua; Hong, Ji-bo

2016-11-01

In this study, a three-dimensional chitosan-gelatin/nanohydroxyapatite (ChG/nHaP) scaffold was successfully fabricated and characterized in terms of swelling, degradation, cell proliferation, cell attachment, and mineralization characterizations. The ChG/nHaP scaffold was fabricated with a mean pore size of 100-180 μm. Our results showed that the physicochemical and biological properties of the scaffolds were affected by the presence of HaP. The swelling and degradation characteristics of the ChG scaffold were remarkably decreased by the addition of HaP. On the other hand, the presence of HaP remarkably improved the MC3T3-E1 cell attachment and cell growth in the scaffold membrane. The biocompatible nature of the ChG/nHaP scaffold leads to the development of finely scaled mineral deposits on the scaffold membrane. Thus, HaP played an important role in improving the biological performance of the scaffold. Therefore, the ChG/nHaP scaffold could be applied as a suitable material for bone tissue engineering applications.

Unique Directional Motility of Influenza C Virus Controlled by Its Filamentous Morphology and Short-Range Motions.

PubMed

Sakai, Tatsuya; Takagi, Hiroaki; Muraki, Yasushi; Saito, Mineki

2018-01-15

Influenza virus motility is based on cooperation between two viral spike proteins, hemagglutinin (HA) and neuraminidase (NA), and is a major determinant of virus infectivity. To translocate a virus particle on the cell surface, HA molecules exchange viral receptors and NA molecules accelerate the receptor exchange of HA. This type of virus motility was recently identified in influenza A virus (IAV). To determine if other influenza virus types have a similar receptor exchange mechanism-driven motility, we investigated influenza C virus (ICV) motility on a receptor-fixed glass surface. This system excludes receptor mobility, which makes it more desirable than a cell surface for demonstrating virus motility by receptor exchange. Like IAV, ICV was observed to move across the receptor-fixed surface. However, in contrast to the random movement of IAV, a filamentous ICV strain, Ann Arbor/1/50 (AA), moved in a straight line, in a directed manner, and at a constant rate, whereas a spherical ICV strain, Taylor/1233/47 (Taylor), moved randomly, similar to IAV. The AA and Taylor viruses each moved with a combination of gradual (crawling) and rapid (gliding) motions, but the distances of crawling and gliding for the AA virus were shorter than those of the Taylor virus. Our findings indicate that like IAV, ICV also has a motility that is driven by the receptor exchange mechanism. However, compared with IAV movement, filamentous ICV movement is highly regulated in both direction and speed. Control of ICV movement is based on its specific motility employing short crawling and gliding motions as well as its own filamentous morphology. IMPORTANCE Influenza virus enters into a host cell for infection via cellular endocytosis. Human influenza virus infects epithelial cells of the respiratory tract, the surfaces of which are hidden by abundant cilia that are inactive in endocytosis. An open question is the manner by which the virus migrates to endocytosis-active domains. In analyzing individual virus behaviors through single-virus tracking, we identified a novel function of the hemagglutinin and esterase of influenza C virus (ICV) as the motility machinery. Hemagglutinin iteratively exchanges a viral receptor, causing virus movement. Esterase degrades the receptors along the trajectory traveled by the virus and prevents the virus from moving backward, causing directional movement. We propose that ICV has a unique motile machinery directionally controlled via hemagglutinin sensing the receptor density manipulated by esterase. Copyright © 2018 Sakai et al.

Effect of humic acid on the sorption of perfluorooctane sulfonate (PFOS) and perfluorobutane sulfonate (PFBS) on boehmite.

PubMed

Wang, Fei; Shih, Kaimin; Leckie, James O

2015-01-01

The sorption of PFOS and PFBS on boehmite was significantly retarded by the competitive sorption of humic acid (HA), implying that PFOS and PFBS are likely more mobile in water and groundwater systems enriched with HA. The sorption behavior of PFOS and PFBS on the HA-modified boehmite surface were also found to differ due to their different chain lengths. For a partially HA-modified boehmite surface, the isotherm study showed that PFOS had a much higher maximum sorption capacity than PFBS and that PFOS might possess additional surface interactions besides electrostatic interaction. For a HA-saturated boehmite, a linear sorption isotherm was found for PFOS while nearly no PFBS sorption was observed. This indicates that sorption behavior between PFOS and the sorbed HA on boehmite was dominated by hydrophobic interactions, instead of electrostatic interaction. In addition, a conceptual model combining hydrophobic and electrostatic interaction was established to explain the sorption behavior of PFOS and PFBS on HA-modified boehmite. Finally, the results revealed that the sorption of PFOS and PFBS on HA-modified boehmite is pH-dependent. The neutralization of negative sites on HA-modified boehmite reduced the electrostatic repulsion and enhanced the partitioning of PFBS on the sorbed HA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Trophoblast expression of the minor histocompatibility antigen HA-1 is regulated by oxygen and is increased in placentas from preeclamptic women.

PubMed

Linscheid, C; Heitmann, E; Singh, P; Wickstrom, E; Qiu, L; Hodes, H; Nauser, T; Petroff, M G

2015-08-01

Maternal T-cells reactive towards paternally inherited fetal minor histocompatibility antigens are expanded during pregnancy. Placental trophoblast cells express at least four fetal antigens, including human minor histocompatibility antigen 1 (HA-1). We investigated oxygen as a potential regulator of HA-1 and whether HA-1 expression is altered in preeclamptic placentas. Expression and regulation of HA-1 mRNA and protein were examined by qRT-PCR and immunohistochemistry, using first, second, and third trimester placentas, first trimester placental explant cultures, and term purified cytotrophoblast cells. Low oxygen conditions were achieved by varying ambient oxygen, and were mimicked using cobalt chloride. HA-1 mRNA and protein expression levels were evaluated in preeclamptic and control placentas. HA-1 protein expression was higher in the syncytiotrophoblast of first trimester as compared to second trimester and term placentas (P<0.01). HA-1 mRNA was increased in cobalt chloride-treated placental explants and purified cytotrophoblast cells (P = 0.04 and P<0.01, respectively) and in purified cytotrophoblast cells cultured under 2% as compared to 8% and 21% oxygen (P<0.01). HA-1 mRNA expression in preeclamptic vs. control placentas was increased 3.3-fold (P = 0.015). HA-1 protein expression was increased in syncytial nuclear aggregates and the syncytiotrophoblast of preeclamptic vs. control placentas (P = 0.02 and 0.03, respectively). Placental HA-1 expression is regulated by oxygen and is increased in the syncytial nuclear aggregates and syncytiotrophoblast of preeclamptic as compared to control placentas. Increased HA-1 expression, combined with increased preeclamptic syncytiotrophoblast deportation, provides a novel potential mechanism for exposure of the maternal immune system to increased fetal antigenic load during preeclampsia. Published by Elsevier Ltd.

Cytotoxicity and genotoxicity property of hydroxyapatite-mullite eluates.

PubMed

Kalmodia, Sushma; Sharma, Vyom; Pandey, Alok K; Dhawan, Alok; Basu, Bikramjit

2011-02-01

Long-term biomedical applications of implant materials may cause osteolysis, aseptic losing and toxicity. Therefore, we investigated the cytotoxic and genotoxic potential of hydroxyapatite (HA) mullite eluates in L929 mouse fibroblast cells. The spark plasma sintered HA-20% mullite biocomposite (HA20M) were ground using mortar and pestle as well as ball milling. The cells were exposed for 6 h to varying concentrations (10, 25, 50, 75 and 100%) of the eluates of HA-20% mullite (87 nm), HA (171 nm) and mullite (154 nm). The scanning electron microscopy and MTT assay revealed the concentration dependent toxicity of H20M eluate at and above 50%. The analysis of the DNA damaging potential of HA, mullite and HA20M eluates using Comet assay demonstrated a significant DNA damage by HA20M which was largely related to the presence of mullite. The results collectively demonstrate the cytotoxic and genotoxic potential of HA20M eluate in L929 cells is dependent on particle size, concentration and composition.

Mechanical properties of natural chitosan/hydroxyapatite/magnetite nanocomposites for tissue engineering applications.

PubMed

Heidari, Fatemeh; Razavi, Mehdi; E Bahrololoom, Mohammad; Bazargan-Lari, Reza; Vashaee, Daryoosh; Kotturi, Hari; Tayebi, Lobat

2016-08-01

Chitosan (CS), hydroxyapatite (HA), and magnetite (Fe3O4) have been broadly employed for bone treatment applications. Having a hybrid biomaterial composed of the aforementioned constituents not only accumulates the useful characteristics of each component, but also provides outstanding composite properties. In the present research, mechanical properties of pure CS, CS/HA, CS/HA/magnetite, and CS/magnetite were evaluated by the measurements of bending strength, elastic modulus, compressive strength and hardness values. Moreover, the morphology of the bending fracture surfaces were characterized using a scanning electron microscope (SEM) and an image analyzer. Studies were also conducted to examine the biological response of the human Mesenchymal Stem Cells (hMSCs) on different composites. We conclude that, although all of these composites possess in-vitro biocompatibility, adding hydroxyapatite and magnetite to the chitosan matrix can noticeably enhance the mechanical properties of the pure chitosan. Copyright © 2016 Elsevier B.V. All rights reserved.

In Vitro Corrosion and Cytocompatibility of ZK60 Magnesium Alloy Coated with Hydroxyapatite by a Simple Chemical Conversion Process for Orthopedic Applications

PubMed Central

Wang, Bing; Huang, Ping; Ou, Caiwen; Li, Kaikai; Yan, Biao; Lu, Wei

2013-01-01

Magnesium and its alloys—a new class of degradable metallic biomaterials—are being increasingly investigated as a promising alternative for medical implant and device applications due to their advantageous mechanical and biological properties. However, the high corrosion rate in physiological environments prevents the clinical application of Mg-based materials. Therefore, the objective of this study was to develop a hydroxyapatite (HA) coating on ZK60 magnesium alloy substrates to mediate the rapid degradation of Mg while improving its cytocompatibility for orthopedic applications. A simple chemical conversion process was applied to prepare HA coating on ZK60 magnesium alloy. Surface morphology, elemental compositions, and crystal structures were characterized using scanning electron microscopy, energy dispersive spectroscopy, and X-ray diffraction, respectively. The corrosion properties of samples were investigated by immersion test and electrochemical test. Murine fibroblast L-929 cells were harvested and cultured with coated and non-coated ZK60 samples to determine cytocompatibility. The degradation results suggested that the HA coatings decreased the degradation of ZK60 alloy. No significant deterioration in compression strength was observed for all the uncoated and coated samples after 2 and 4 weeks’ immersion in simulated body fluid (SBF). Cytotoxicity test indicated that the coatings, especially HA coating, improved cytocompatibility of ZK60 alloy for L929 cells. PMID:24300096

Highly stretchable HA/SA hydrogels for tissue engineering.

PubMed

Zhu, Chengcheng; Yang, Rui; Hua, Xiaobin; Chen, Hong; Xu, Jumei; Wu, Rile; Cen, Lian

2018-04-01

A highly stretchable hyaluronic acid (HA)/sodium alginate (SA) hydrogel was developed in this study based on an interpenetrating polymer network. HA/SA hydrogels were prepared by mixing two polysaccharides followed by covalent crosslinking via epoxy groups on HA molecules and ionic crosslinking via divalent ions on SA chains sequentially. The effect of HA/SA ratio on the pore size and distribution, swelling ratio, elongation and rheological properties as well as protein loading and release properties of HA/SA hydrogels was explored. Moreover, a surface modification method, layer-by-layer (LBL) assembly technique, was applied to modify the hydrogel to evaluate the hydrogel's tenability in varying biological performance. It was then shown that the hydrogels had the pore sizes ranging from 100 to 50 μm. With the increase in SA content of the resulting hydrogels, the pore size, swelling ratio, and storage modulus (G') and loss modulus (G″) of the hydrogel all decreased, whereas the in vitro bulk weight loss was fastened. Moreover, elongation at break (EB) value increased first, reached a peak value and then decreased, that is HA8/SA1 (HA:SA = 8:1) had the highest EB value of 417%. This hydrogel could retain 33.2% of the pre-loaded protein even after 72 h, which could be further attenuated when LBL was used to shell the hydrogel. The growth of fibroblasts on HA8/SA1 hydrogel gave preliminary assessment on its suitability as a cellular carrier, while the LBL modified HA8/SA1 hydrogel also favored the anchoring of keratinocytes, further enhancing its cell carrier role for tissue regeneration, especially skin engineering.

Targeting Antigens to Dec-205 on Dendritic Cells Induces Immune Protection in Experimental Colitis in Mice

PubMed Central

Wadwa, Munisch; Klopfleisch, Robert; Buer, Jan; Westendorf, Astrid M.

2016-01-01

The endocytotic c-type lectin receptor DEC-205 is highly expressed on immature dendritic cells. In previous studies, it was shown that antigen-targeting to DEC-205 is a useful tool for the induction of antigen-specific Foxp3+ regulatory T cells and thereby can prevent inflammatory processes. However, whether this approach is sufficient to mediate tolerance in mucosal tissues like the gut is unknown. In this study, we established a new mouse model in which the adoptive transfer of naive hemagglutinin (HA)-specific CD4+Foxp3– T cells into VILLIN-HA transgenic mice leads to severe colitis. To analyze if antigen-targeting to DEC-205 could protect against inflammation of the gut, VILLIN-HA transgenic mice were injected with an antibody–antigen complex consisting of the immunogenic HA110–120 peptide coupled to an α-DEC-205 antibody (DEC-HA) before adoptive T cell transfer. DEC-HA-treated mice showed significantly less signs of intestinal inflammation as was demonstrated by reduced loss of body weight and histopathology in the gut. Strikingly, abrogated intestinal inflammation was mediated via the conversion of naive HA-specific CD4+Foxp3– T cells into HA-specific CD4+Foxp3+ regulatory T cells. In this study, we provide evidence that antigen-targeting to DEC-205 can be utilized for the induction of tolerance in mucosal organs that are confronted with large numbers of exogenous antigens. PMID:27141310

A comparison study on the behavior of human endometrial stem cell-derived osteoblast cells on PLGA/HA nanocomposite scaffolds fabricated by electrospinning and freeze-drying methods.

PubMed

Namini, Mojdeh Salehi; Bayat, Neda; Tajerian, Roxana; Ebrahimi-Barough, Somayeh; Azami, Mahmoud; Irani, Shiva; Jangjoo, Saranaz; Shirian, Sadegh; Ai, Jafar

2018-03-27

An engineered tissue structure is an artificial scaffold combined with cells and signaling factors. Among various polymers, the polylactide-co-glycolide/hydroxyapatite (PLGA/HA) has attracted much attention due to their optimal properties. The aim of this study was to study the behavior of human endometrial stem cell (hEnSC)-derived osteoblast cells cultured on PLGA/HA nanocomposite scaffolds. hEnSCs were isolated and exposed to osteogenic media for 21 days. Differentiated cells were cultured on PLGA/HA synthetic scaffolds. The PLGA/HA-based nanocomposite scaffolds were fabricated using either electrospinning or freeze-drying methods. Behavior of the cells was evaluated a week after seeding hEnSC-derived osteoblast-like cells on these scaffolds. Osteogenesis was investigated in terms of alkaline phosphatase activity, gene expression, immunocytochemistry (ICC), proliferation, and scanning electron microscopy (SEM). Moreover, scaffold properties, such as pore size and morphology of the cells, onto the scaffolds were evaluated using SEM. Furthermore, biocompatibility of these scaffolds was confirmed by 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The matrix mineralization was proved by alizarin red staining, and the osteogenic media-treated cultures positively expressed osteocalcin and osteopontin markers. Moreover, qRT-PCR results confirmed the positive gene expression of osteopontin and osteonectin in the differentiated osteoblast-like cells. The results of behavior assessment of the cultured cells on electrospinning and freeze-dried scaffolds showed that the behavior of the cultured cells on the freeze-dried PLGA/HA scaffolds was significantly better than the electrospinning PLGA/HA scaffolds. It has been shown that the freeze-dried PLGA/HA nanocomposite scaffolds can appropriately support the attachment and proliferation of the differentiated osteoblast cells and are a suitable candidate for bone tissue engineering.

Biochemical characterization of domain-specific glycoproteins of the rat hepatocyte plasma membrane.

PubMed

Bartles, J R; Braiterman, L T; Hubbard, A L

1985-10-15

Seven integral proteins (CE 9, HA 21, HA 116, HA 16, HA 4, HA 201, and HA 301) were isolated from rat hepatocyte plasma membranes by immunoaffinity chromatography on monoclonal antibody-Sepharose. Six of the proteins (all but HA 16) exhibit domain-specific localizations (either bile canalicular or sinusoidal/lateral) about the hepatocyte surface. We identified three of these protein antigens as leucine aminopeptidase (HA 201), dipeptidyl peptidase IV (HA 301), and the asialoglycoprotein receptor (HA 116). We also developed 125I-lectin blotting procedures that, when used in conjunction with chemical and glycosidase treatments, permitted a comparison of the types of oligosaccharides present on the seven proteins. All seven are sialoglycoproteins, based upon the effects of prior neuraminidase and periodate-aniline-cyanoborohydride treatments of blots on labeling by 125I-wheat germ agglutinin. 125I-labeled Ricinus communis agglutinin I and 125I-peanut agglutinin blotting of the desialylated proteins revealed few if any conventional O-linked oligosaccharides, suggesting that the sialyl residues represent termini of N-linked complex-type oligosaccharides. Depending upon the protein, we estimated the presence of 2-26 N-linked oligosaccharides/polypeptide chain from the Mr reductions accompanying chemical or enzymatic deglycosylation. Three of these mature plasma membrane proteins (HA 21, HA 116, and HA 4) have both high mannose-type and complex-type oligosaccharides on every copy of their polypeptide chains. The labeling of these three proteins by 125I-concanavalin A was sensitive to treatment with endoglycosidase H, and each exhibited a quantitative reduction in Mr after the treatment, as assessed independently by 125I-wheat germ agglutinin blotting. At this level of analysis, we were unable to discern differences in the types of oligosaccharides present on these seven glycoproteins that correlate with their patterns of expression within the plasma membrane domains of this polarized epithelial cell.

The Impact of HA Oligomer Content on Physical, Mechanical, and Biologic Properties of Divinyl Sulfone-Crosslinked HA Hydrogels

PubMed Central

Ibrahim, Samir; Kang, Qian K; Ramamurthi, Anand

2009-01-01

In recent studies, we showed that exogenous hyaluronic acid oligomers (HA-o) stimulate functional endothelialization, though native long-chain HA is more bioinert and possibly more biocompatible. Thus, in this study, hydrogels containing high molecular weight (HMW) HA (1×106 Da) and HA oligomer mixtures (HA-o: 0.75–10 kDa) were created by crosslinking with divinyl sulfone (DVS). The incorporation of HA oligomers was found to compromise the physical and mechanical properties of the gels (rheology, apparent crosslinking density, swelling ratio, degradation) and to very mildly enhance inflammatory cell recruitment in vivo; increasing the DVS crosslinker content within the gels in general, had the opposite effect, though the relatively high concentration of DVS within these gels (necessary to create a solid gel) also stimulated a mild sub-cutaneous inflammatory response in vivo and VCAM-1 expression by ECs cultured atop; ICAM-expression levels remained very low irrespective extent of DVS crosslinking or HA-o content. The greatest EC attachment and proliferation (MTT assay) was observed on gels that contained the highest amount of HA-o. The study shows that the beneficial EC response to HA oligomers and biocompatibility of HA is mostly unaltered by their chemical derivatization and crosslinking into a hydrogel. However, the study also demonstrates that the relatively high concentrations of DVS, necessary to create solid gels, compromises their biocompatibility. Moreover, the poor mechanics of even these heavily crosslinked gels, in the context of vascular implantation, necessitates the investigation of other, more appropriate crosslinking agents. Alternately, the outcomes of this study may be used to guide an approach based on chemical immobilization and controlled surface-presentation of both bioactive HA oligomers and more biocompatible HMW HAon synthetic or tissue engineered grafts already in use, without the use of a crosslinker, so that improved, predictable, and functional endothelialization can be achieved, and the need to create a mechanically compliant biomaterial for standalone use, circumvented. PMID:20186732

Micro-/Nano- sized hydroxyapatite directs differentiation of rat bone marrow derived mesenchymal stem cells towards an osteoblast lineage

NASA Astrophysics Data System (ADS)

Huang, Yan; Zhou, Gang; Zheng, Lisha; Liu, Haifeng; Niu, Xufeng; Fan, Yubo

2012-03-01

Regenerative medicine consisting of cells and materials provides a new way for the repair and regeneration of tissues and organs. Nano-biomaterials are highlighted due to their advantageous features compared with conventional micro-materials. The aim of this study is to investigate the effects of micro-/nano- sized hydroxyapatite (μ/n-HA) on the osteogenic differentiation of rat bone marrow derived mesenchymal stem cells (rBMSCs). μ/n-HA were prepared by a microwave synthesizer and precipitation method, respectively. Different sizes of μ/n-HA were characterized by IR, XRD, SEM, TEM and co-cultured with rBMSCs. It was shown that rBMSCs expressed higher levels of osteoblast-related markers by n-HA than μ-HA stimulation. The size of HA is an important factor for affecting the osteogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.

Effects of SiO2 substitution on wettability of laser deposited Ca-P biocoating on Ti-6Al-4V.

PubMed

Yang, Yuling; Paital, Sameer R; Dahotre, Narendra B

2010-09-01

Silicon (Si) substitution in the crystal structure of calcium phosphate (CaP) ceramics has proved to generate materials with improved bioactivity than their stoichiometric counterpart. In light of this, in the current work, 100 wt% hydroxyapatite (HA) precursor and 25 wt% SiO(2)-HA precursors were used to prepare bioactive coatings on Ti-6Al-4V substrates by a laser cladding technique. The effects of SiO(2) on phase constituents, crystallite size, surface roughness, and surface energy of the CaP coatings were studied. Furthermore, on the basis of these results, the effects and roles of SiO(2) substitution in HA were systematically discussed. X-ray diffraction analysis of the coated samples indicated the presence of various phases such as CaTiO(3), Ca(2)SiO(4), Ca(3)(PO(4))(2), TiO(2) (Anatase), and TiO(2) (Rutile). The addition of SiO(2) in the HA precursor resulted in the refinement of grain size. Confocal laser microscopy characterization of the surface morphology demonstrated an improved surface roughness for samples with 25 wt% SiO(2)-HA precursor compared to the samples with 100 wt% HA precursor processed at 125 cm/min laser speed. The addition of SiO(2) in the HA precursor resulted in the highest surface energy, increased hydrophilicity, and improved biomineralization as compared to the control (untreated Ti-6Al-4V) and the sample with 100 wt% HA as precursor. The microstructural evolution observed using a scanning electron microscopy indicated that the addition of SiO(2) in the HA precursor resulted in the presence of reduced cracking across the cross-section of the bioceramic coating.

Surface-enrichment with hydroxyapatite nanoparticles in stereolithography-fabricated composite polymer scaffolds promotes bone repair.

PubMed

Guillaume, O; Geven, M A; Sprecher, C M; Stadelmann, V A; Grijpma, D W; Tang, T T; Qin, L; Lai, Y; Alini, M; de Bruijn, J D; Yuan, H; Richards, R G; Eglin, D

2017-05-01

Fabrication of composite scaffolds using stereolithography (SLA) for bone tissue engineering has shown great promises. However, in order to trigger effective bone formation and implant integration, exogenous growth factors are commonly combined to scaffold materials. In this study, we fabricated biodegradable composite scaffolds using SLA and endowed them with osteopromotive properties in the absence of biologics. First we prepared photo-crosslinkable poly(trimethylene carbonate) (PTMC) resins containing 20 and 40wt% of hydroxyapatite (HA) nanoparticles and fabricated scaffolds with controlled macro-architecture. Then, we conducted experiments to investigate how the incorporation of HA in photo-crosslinked PTMC matrices improved human bone marrow stem cells osteogenic differentiation in vitro and kinetic of bone healing in vivo. We observed that bone regeneration was significantly improved using composite scaffolds containing as low as 20wt% of HA, along with difference in terms of osteogenesis and degree of implant osseointegration. Further investigations revealed that SLA process was responsible for the formation of a rich microscale layer of HA corralling scaffolds. To summarize, this work is of substantial importance as it shows how the fabrication of hierarchical biomaterials via surface-enrichment of functional HA nanoparticles in composite polymer stereolithographic structures could impact in vitro and in vivo osteogenesis. This study reports for the first time the enhance osteopromotion of composite biomaterials, with controlled macro-architecture and microscale distribution of hydroxyapatite particles, manufactured by stereolithography. In this process, the hydroxyapatite particles are not only embedded into an erodible polymer matrix, as reported so far in the literature, but concentrated at the surface of the structures. This leads to robust in vivo bone formation at low concentration of hydroxyapatite. The reported 3D self-corralling composite architecture provides significant opportunities to develop functional biomaterials for bone repair and tissue engineering. Copyright © 2017. Published by Elsevier Ltd.

Treatments to induce the nucleation and growth of apatite-like layers on polymeric surfaces and foams.

PubMed

Reis, R L; Cunha, A M; Fernandes, M H; Correia, R N

1997-12-01

In this work, a bioactive glass is used as a percusor of calcium-phosphate (Ca-P) film deposition onto several polymer-based materials. Both bioinert (high molecular weight polyethylene, HMWPE), and biodegradable (corn starch-based blends, SEVA-C) polymers, unreinforced or reinforced with hydroxylapatite (HA), were coated by the very simple proposed route. Also polyurethane (PU) foams, with an open-cell structure, were mineralized by the proposed method. In fact, it was possible to induce the growth of the Ca-P films not only at the surface, but also in the bulk of the PU foam. These cellular materials are intended for cancellous bone replacement applications. The morphology of the formed films was strongly dependent on the used substrate, its polar character, and on the presence of HA in its composition, as observed by SEM. Nevertheless, a well defined needly like structure was observed in all samples at high magnifications. The Ca:P ratios of the films were between 1.5 and 1.7, i.e. in the range of tricalcium phosphate-hydroxylapatite. Raman spectroscopy and thin-film x-ray diffraction (XRD) evidenced the formation of mostly amorphous calcium-phosphate films. After scraping the coating from the polymer surface and heat-treating the resulting powder at 1000 degrees C for 1 h, HA and beta-tricalcium phosphate (TCP) typical peaks were found on XRD patterns.

CD8+ T cell recognition of an endogenously processed epitope is regulated primarily by residues within the epitope

PubMed Central

1992-01-01

Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides associated with cell surface class I major histocompatibility complex (MHC) molecules. This association presumably occurs between newly synthesized class I MHC molecules and peptide fragments in a pre-Golgi compartment. Little is known about the factors that regulate the formation of these antigenic peptide fragments within the cell. To examine the role of residues within a core epitope and in the flanking sequences for the generation and presentation of the newly synthesized peptide fragment recognized by CD8+ CTL, we have mutagenized the coding sequence for the CTL epitope spanning residues 202-221 in the influenza A/Japan/57 hemagglutinin (HA). In this study over 60 substitution mutations in the epitope were tested for their effects on target cell sensitization using a cytoplasmic viral expression system. The HA202- 221 site contains two overlapping subsites defined by CTL clones 11-1 and 40-2. Mutations in HA residues 204-213 or residues 210-219 often abolished target cell lysis by CTL clones 11-1 and 40-2, respectively. Although residues outside the core epitope did not usually affect the ability to be lysed by CTL clones, substitution of a Gly residue for Val-214 abolished lysis by clone 11-1. These data suggest that residues within a site that affect MHC binding and T cell receptor recognition appear to play the predominant role in dictating the formation of the antigenic complex recognized by CD8+ CTL, and therefore the antigenicity of the protein antigen presented to CD8+ T cells. Most alterations in residues flanking the endogenously expressed epitope do not appreciably affect the generation and recognition of the site. PMID:1383384

Distribution of PLGA-modified nanoparticles in 3D cell culture models of hypo-vascularized tumor tissue.

PubMed

Sims, Lee B; Huss, Maya K; Frieboes, Hermann B; Steinbach-Rankins, Jill M

2017-10-05

Advanced stage cancer treatments are often invasive and painful-typically comprised of surgery, chemotherapy, and/or radiation treatment. Low transport efficiency during systemic chemotherapy may require high chemotherapeutic doses to effectively target cancerous tissue, resulting in systemic toxicity. Nanotherapeutic platforms have been proposed as an alternative to more safely and effectively deliver therapeutic agents directly to tumor sites. However, cellular internalization and tumor penetration are often diametrically opposed, with limited access to tumor regions distal from vasculature, due to irregular tissue morphologies. To address these transport challenges, nanoparticles (NPs) are often surface-modified with ligands to enhance transport and longevity after localized or systemic administration. Here, we evaluate stealth polyethylene-glycol (PEG), cell-penetrating (MPG), and CPP-stealth (MPG/PEG) poly(lactic-co-glycolic-acid) (PLGA) NP co-treatment strategies in 3D cell culture representing hypo-vascularized tissue. Smaller, more regularly-shaped avascular tissue was generated using the hanging drop (HD) method, while more irregularly-shaped masses were formed with the liquid overlay (LO) technique. To compare NP distribution differences within the same type of tissue as a function of different cancer types, we selected HeLa, cervical epithelial adenocarcinoma cells; CaSki, cervical epidermoid carcinoma cells; and SiHa, grade II cervical squamous cell carcinoma cells. In HD tumors, enhanced distribution relative to unmodified NPs was measured for MPG and PEG NPs in HeLa, and for all modified NPs in SiHa spheroids. In LO tumors, the greatest distribution was observed for MPG and MPG/PEG NPs in HeLa, and for PEG and MPG/PEG NPs in SiHa spheroids. Pre-clinical evaluation of PLGA-modified NP distribution into hypo-vascularized tumor tissue may benefit from considering tissue morphology in addition to cancer type.

A phase IIa study of HA-irinotecan, formulation of hyaluronic acid and irinotecan targeting CD44 in extensive-stage small cell lung cancer.

PubMed

Alamgeer, Muhammad; Neil Watkins, D; Banakh, Ilia; Kumar, Beena; Gough, Daniel J; Markman, Ben; Ganju, Vinod

2018-04-01

Preclinical studies in small cell lung cancer (SCLC) have shown that hyaluronic acid (HA) can be effectively used to deliver chemotherapy and selectively decrease CD44 expressing (stem cell-like) tumour cells. The current study aimed to replicate these findings and obtain data on safety and activity of HA-irinotecan (HA-IR). Eligible patients with extensive stage SCLC were consented. A safety cohort (n = 5) was treated with HA-IR and Carboplatin (C). Subsequently, the patients were randomised 1:1 to receive experimental (HA-IR + C) or standard (IR + C) treatment, to a maximum of 6 cycles. The second line patients were added to the study and treated with open label HA-IR + C. Tumour response was measured after every 2 cycles. Baseline tumour specimens were stained for CD44s and CD44v6 expression. Circulating tumour cells (CTCs) were enumerated before each treatment cycle. Out of 39 patients screened, 34 were evaluable for the study. The median age was 66 (range 39-83). The overall response rates were 69% and 75% for experimental and standard arms respectively. Median progression free survival was 42 and 28 weeks, respectively (p = 0.892). The treatments were well tolerated. The incidence of grade III/IV diarrhea was more common in the standard arm, while anaemia was more common in the experimental arm. IHC analysis suggested that the patients with CD44s positive tumours may gain survival benefit from HA-IR. HA-IR is well tolerated and active in ES-SCLC. The effect of HA-IR on CD44s + cancer stem-like cells provide an early hint towards a potential novel target.

Hormone and pathogen content in soil after litter applications

USDA-ARS?s Scientific Manuscript database

Poultry litter applications at agronomic rates established for a crop’s P or N requirements may contain as much as 385 mg estradiol ha-1, 605 mg testosterone ha-1, 4.4(10)12 Escherichia coli cells ha-1, and 4.4(10)13 fecal enterococci cells ha-1. Field experiments from small plot- to small watershed...

Biological evaluation of partially stabilized zirconia added HA/HDPE composites with osteoblast and fibroblast cell lines.

PubMed

Yari Sadi, Amir; Shokrgozar, Mohammad Ali; Homaeigohar, Seyed Shahin; Khavandi, Alireza

2008-06-01

In the present study, the biocompatibility of partially stabilized zirconia (PSZ) added hydroxyapatite (HA)--high density polyethylene (HDPE) composites was evaluated by proliferation and cell attachment assays on two osteoblast cell lines (G-292, Saos-2) and a type of fibroblast cell isolated from bone tissue namely HBF in different time intervals. Cell-material interactions on the surface of the composites were observed by scanning electron microscopy (SEM). The effect of composites on the behavior of osteoblast and fibroblast cells was compared with those of HDPE and Tissue Culture Poly Styrene (TPS) (as negative control) samples. Results showed that the composite samples supported a higher proliferation rate of osteoblast cells in the presence of composite samples as compared to the HDPE and TPS samples after 3, 7 and 14 days of incubation period. It was showed that an equal or in some cases an even higher proliferation rate of G-292 and Saos-2 osteoblast cells on composite samples in compare to negative controls in culture period (P < 0.05). The number of adhered cells on the composite samples was equal and in some cases higher than the number adhered on the HDPE and TPS samples after the above mentioned incubation periods (P < 0.05). Adhered cells presented a normal morphology by SEM and many of the cells were seen to be undergoing cell division.

Neovascularization Induced by the Hyaluronic Acid-Based Spongy-Like Hydrogels Degradation Products.

PubMed

Silva, Lucília P da; Pirraco, Rogério P; Santos, Tírcia C; Novoa-Carballal, Ramon; Cerqueira, Mariana T; Reis, Rui L; Correlo, Vitor M; Marques, Alexandra P

2016-12-14

Neovascularization has been a major challenge in many tissue regeneration strategies. Hyaluronic acid (HA) of 3-25 disaccharides is known to be angiogenic due to its interaction with endothelial cell receptors. This effect has been explored with HA-based structures but a transitory response is observed due to HA burst biodegradation. Herein we developed gellan gum (GG)-HA spongy-like hydrogels from semi-interpenetrating network hydrogels with different HA amounts. Enzymatic degradation was more evident in the GG-HA with high HA amount due to their lower mechanical stability, also resulting from the degradation itself, which facilitated the access of the enzyme to the HA in the bulk. GG-HA spongy-like hydrogels hyaluronidase-mediated degradation lead to the release of HA oligosaccharides of different amounts and sizes in a HA content-dependent manner which promoted in vitro proliferation of human umbilical cord vein endothelial cells (HUVECs) but not their migration. Although no effect was observed in human dermal microvascular endothelial cells (hDMECs) in vitro, the implantation of GG-HA spongy-like hydrogels in an ischemic hind limb mice model promoted neovascularization in a material-dependent manner, consistent with the in vitro degradation profile. Overall, GG-HA spongy-like hydrogels with a sustained release of HA oligomers are valuable options to improve tissue vascularization, a critical issue in several applications in the tissue engineering and regenerative medicine field.

Absence of differences among low, middle, and high molecular weight hyaluronan in activating murine immune cells in vitro.

PubMed

Šafránková, Barbora; Hermannová, Martina; Nešporová, Kristina; Velebný, Vladimír; Kubala, Lukáš

2018-02-01

Hyaluronan (HA) effects on immune response are suggested to be dependent on HA molecular weight (MW), as low MW HA should activate immune cells in contrast to high MW HA. However, some current studies do not support this conception and emphasize the importance of the form of preparation of HA, particularly with respect to its purity and origin. We compared the activation of mouse immune cells by HA samples (100kDa, 500kDa, and 997kDa) prepared from HA originating from rooster comb, and HA samples (71kDa, 500kDa, and 1000kDa) prepared from pharmacological grade HA originating from Streptococcus equi. Interestingly, in contrast to established theory, only middle and high MW HA originating from rooster comb induced the production of tumor necrosis factor-α by macrophages and in whole blood. Further, all tested preparations of HA failed to induce the expression of inducible nitric oxide synthase, the production of nitric oxide, or the expression of cyclooxygenase 2 in macrophages and splenocytes. Importantly, all HA samples originating from rooster comb were found to be contaminated by endotoxin (up to 1.23EU/ml). Hence, low MW HA did not reveal itself to have significantly higher immunostimulatory activity compared to HA of higher MW. Copyright © 2017 Elsevier B.V. All rights reserved.

Dendritic Cells Program Non-Immunogenic Prostate-Specific T Cell Responses Beginning at Early Stages of Prostate Tumorigenesis

PubMed Central

Mihalyo, Marianne A.; Hagymasi, Adam T.; Slaiby, Aaron M.; Nevius, Erin E.; Adler, Adam J.

2010-01-01

BACKGROUND Prostate cancer promotes the development of T cell tolerance towards prostatic antigens, potentially limiting the efficacy of prostate cancer vaccines targeting these antigens. Here, we sought to determine the stage of disease progression when T cell tolerance develops, as well as the role of steady state dendritic cells (DC) and CD4+CD25+ T regulatory cells (Tregs) in programming tolerance. METHODS The response of naïve HA-specific CD4+ T cells were analyzed following adoptive transfer into Pro-HA × TRAMP transgenic mice harboring variably-staged HA-expressing prostate tumors on two genetic backgrounds that display different patterns and kinetics of tumorigenesis. The role of DC and Tregs in programming HA-specific CD4 cell responses were assessed via depletion. RESULTS HA-specific CD4 cells underwent non-immunogenic responses at all stages of tumorigenesis in both genetic backgrounds. These responses were completely dependent on DC, but not appreciably influenced by Tregs. CONCLUSIONS These results suggest that tolerogenicity is an early and general property of prostate tumors. PMID:17221844

PCL-HA microscaffolds for in vitro modular bone tissue engineering.

PubMed

Totaro, Alessandra; Salerno, Aurelio; Imparato, Giorgia; Domingo, Concepción; Urciuolo, Francesco; Netti, Paolo Antonio

2017-06-01

The evolution of microscaffolds and bone-bioactive surfaces is a pivotal point in modular bone tissue engineering. In this study, the design and fabrication of porous polycaprolactone (PCL) microscaffolds functionalized with hydroxyapatite (HA) nanoparticles by means of a bio-safe and versatile thermally-induced phase separation process is reported. The ability of the as-prepared nanocomposite microscaffolds to support the adhesion, growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in standard and osteogenic media and using dynamic seeding/culture conditions was investigated. The obtained results demonstrated that the PCL-HA nanocomposite microparticles had an enhanced interaction with hMSCs and induced their osteogenic differentiation, even without the exogenous addition of osteogenic factors. In particular, calcium deposition, alizarin red assay, histological analysis, osteogenic gene expression and collagen I secretion were assessed. The results of these tests demonstrated the formation of bone microtissue precursors after 28 days of dynamic culture. These findings suggest that PCL-HA nanocomposite microparticles represent an excellent platform for in vitro modular bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Dual Targeting Biomimetic Liposomes for Paclitaxel/DNA Combination Cancer Treatment

PubMed Central

Liu, Guo-Xia; Fang, Gui-Qing; Xu, Wei

2014-01-01

Combinations of chemotherapeutic drugs with nucleic acid has shown great promise in cancer therapy. In the present study, paclitaxel (PTX) and DNA were co-loaded in the hyaluronic acid (HA) and folate (FA)-modified liposomes (HA/FA/PPD), to obtain the dual targeting biomimetic nanovector. The prepared HA/FA/PPD exhibited nanosized structure and narrow size distributions (247.4 ± 4.2 nm) with appropriate negative charge of −25.40 ± 2.7 mV. HA/FA/PD (PTX free HA/FA/PPD) showed almost no toxicity on murine malignant melanoma cell line (B16) and human hepatocellular carcinoma cell line (HepG2) (higher than 80% cell viability), demonstrating the safety of the blank nanovector. In comparison with the FA-modified PTX/DNA co-loaded liposomes (FA/PPD), HA/FA/PPD showed significant superiority in protecting the nanoparticles from aggregation in the presence of plasma and degradation by DNase I. Moreover, HA/FA/PPD could also significantly improve the transfection efficiency and cellular internalization rates on B16 cells comparing to that of FA/PPD (p < 0.05) and PPD (p < 0.01), demonstrating the great advantages of dual targeting properties. Furthermore, fluorescence microscope and flow cytometry results showed that PTX and DNA could be effectively co-delivered into the same tumor cell via HA/FA/PPD, contributing to PTX/DNA combination cancer treatment. In conclusion, the obtained HA/FA/PPD in the study could effectively target tumor cells, enhance transfection efficiency and subsequently achieve the co-delivery of PTX and DNA, displaying great potential for optimal combination therapy. PMID:25177862

Lopinavir up-regulates expression of the antiviral protein ribonuclease L in human papillomavirus-positive cervical carcinoma cells.

PubMed

Batman, Gavin; Oliver, Anthony W; Zehbe, Ingeborg; Richard, Christina; Hampson, Lynne; Hampson, Ian N

2011-01-01

We have previously shown that the HIV protease inhibitor lopinavir has selective toxicity against human papillomavirus (HPV)-positive cervical carcinoma cells via an unknown mechanism. SiHa cervical carcinoma cells were stably transfected with the proteasome sensor vector pZsProSensor-1 to confirm lopinavir inhibits the proteasome in these cells. The Panorama Xpress profiler 725 antibody array was then used to analyse specific changes in protein expression in lopinavir-treated versus control untreated SiHa cells followed by PCR and western blotting. Colorimetric growth assays of lopinavir-treated E6/E7 immortalised versus control human keratinocytes were performed. Targeted small interfering RNA gene silencing followed by growth assay comparison of lopinavir-treated/untreated SiHa cells was also used. Lopinavir induced an increase in the fluorescence of pZsProSensor-1 transfected SiHa cells, indicative of proteasomal inhibition. Ribonuclease L (RNASEL) protein was shown to be up-regulated in lopinavir-treated SiHa cells, which was confirmed by PCR and western blot. Targeted silencing of RNASEL reduced the sensitivity of SiHa cells to lopinavir. Selective toxicity against E6/E7 immortalised keratinocytes versus control cells was also seen with lopinavir and was associated with up-regulated RNASEL expression. These data are consistent with the toxicity of lopinavir against HPV-positive cervical carcinoma cells being related to its ability to block viral proteasome activation and induce an up-regulation of the antiviral protein RNASEL. This is supported by the drug's selective toxicity and up-regulation of RNASEL in E6/E7 immortalised keratinocytes combined with the increased resistance to lopinavir observed in SiHa cells following silencing of RNASEL gene expression.

[Neoretinal antigen expression: a comparison of anatomical and clinical features of a murine uveoretinitis model].

PubMed

Terrada, C; Pâques, M; Fisson, S; De Kozak, Y; Klatzmann, D; Salomon, B; LeHoang, P; Bodaghi, B

2008-02-01

Uveitis is an inflammation involving the retina. The antigens targeted by the experimental models are located in the pigmentary epithelium-photoreceptor complex. To gain insights into the variations in topographic expression of the antigen in the retina, we studied a new mouse model. and methods: Stable retinal expression of the influenza virus hemagglutinin (HA) was obtained after intravitreal or subretinal injection of recombinant adeno-associated virus carrying HA (AAV-HA). One month later, we transferred HA-specific T cells, followed by a subcutaneous immunization of the cognate antigen emulsified in CFA. The animals were clinically examined with a slit lamp biomicroscope. Infiltration of donor cells was detected by immunostaining on retina flatmounts with anti-Thy-1.1 antibody, and infiltrating cells were studied using FACS analysis. Whatever the location of the HA expression, intraocular inflammation was clinically and histologically detected in all animals, between 10 and 15 days after immunization with HA. Lesions were identified with histopathological analysis. The ocular infiltrate was mostly composed of macrophages and HA-specific T cells in different proportions. The topographic variations of targeted ocular antigens do not seem to modify the development of inflammatory reactions in our model. By targeting different antigen-presenting cells, ocular infiltrating cells are different.

Influence of pretreatment on the surface characteristics of PLLA fibers and subsequent hydroxyapatite coating.

PubMed

Peng, F; Olson, J R; Shaw, M T; Wei, M

2009-01-01

A fibrous precursor for bone repair composites was made by coating poly(L-lactide) (PLLA) fibers with hydroxyapatite (HA) using a biomimetic method. To enhance the bonding between the HA coating and the PLLA fiber, PLLA fibers were etched with either sodium hydroxide or sodium hypochlorite to generate carboxyl groups on fiber surfaces. The experiments were designed to determine the influence of etching on the fiber surface morphology and chemistry as well as the subsequent HA coating on the etched fiber surfaces. It was found that the etching pretreatment increased the roughness as well as the hydrophilicity of fibers, indicating that hydrolysis of PLLA chains had taken place on fiber surfaces. The etching pretreatment also promoted HA coating formation by introducing thicker coating on the surface of fibers with a longer etching time, a higher etching concentration, or with NaOCl as the etching agent. A mechanism of surface hydrolysis and oxidation of PLLA was proposed. (c) 2008 Wiley Periodicals, Inc.

CD44-targeted hyaluronic acid-curcumin prodrug protects renal tubular epithelial cell survival from oxidative stress damage.

PubMed

Hu, Jing-Bo; Li, Shu-Juan; Kang, Xu-Qi; Qi, Jing; Wu, Jia-Hui; Wang, Xiao-Juan; Xu, Xiao-Ling; Ying, Xiao-Ying; Jiang, Sai-Ping; You, Jian; Du, Yong-Zhong

2018-08-01

Based on the abnormally increased expression of CD44 receptors on renal tubule epithelial cells during ischemia/reperfusion-induced acute kidney injury (AKI), we developed a hyaluronic acid-curcumin (HA-CUR) polymeric prodrug targeting to epithelial cells and then relieving oxidative stress damages. The water solubility of HA-CUR was significantly enhanced and approximately 27-fold higher than that of CUR. Cellular uptake test showed HA-CUR was preferably internalized by H 2 O 2 -pretreated tubular epithelial (HK-2) cells compared with free CUR benefiting from the specific binding between HA and CD44 receptors. Biodistribution results further demonstrated the increased accumulation of HA-CUR in kidneys with 13.9-fold higher than that of free CUR. Pharmacodynamic studies indicated HA-CUR effectively ameliorated AKI, and the exact mechanism was that HA-CUR protected renal tubule epithelial cells from oxidative stress damage via inhibiting PtdIns3K-AKT-mTOR signaling pathway. Taken together, this study provides a new therapeutic strategy for the treatment of AKI based on the pathogenesis of the disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Behavior of Osteoblast-Like Cells on a β-Tricalcium Phosphate Synthetic Scaffold Coated With Calcium Phosphate and Magnesium.

PubMed

Park, Ki-Deog; Jung, Young-Suk; Lee, Kyung-Ku; Park, Hong-Ju

2016-06-01

Tricalcium phosphate (TCP) is one of the most useful synthetic scaffolds for bone grafts and has several advantages. However, the rapid degradation of TCP makes it less osteoconductive than the other candidates, and represents a major shortcoming. To overcome this problem, the authors investigated magnesium (Mg) and/or hydroxyapatite (HA) coating on a β-TCP substrate using a sputtering technique. Biocompatibility tests were carried out on β-TCP discs that were either uncoated (TCP), coated with HA by radio frequency magnetron sputtering (HA-TCP), coated with Mg by DC sputtering (Mg-TCP), or multicoated with Mg and HA by DC and radio frequency magnetron sputtering (MgHA-TCP). Cells showed similar morphology in all 4 groups, and were widely spread, had flattened elongated shapes, and were connected to adjacent cells by pseudopods. An MTT assay revealed higher cell proliferation on HA-TCP, Mg-TCP, and MgHA-TCP compared with TCP at 3 and 5 days. MgHA-TCP also showed significantly higher alkaline phosphatase activity levels compared with TCP, HA-TCP, and Mg-TCP (P < 0.05). Results suggest that Mg-coated β-TCP could have great potential as a bone graft material for future applications in hard tissue regeneration.

Hyaluronan Induces the Selective Accumulation of Matrix- and Cell-Associated Proteoglycans by Mesangial Cells

PubMed Central

Kastner, Sabine; Thomas, Gareth J.; Jenkins, Robert H.; Davies, Malcolm; Steadman, Robert

2007-01-01

Mesangial cells (MCs) are essential for normal renal function through the synthesis of their own extracellular matrix, which forms the structural support of the renal glomerulus. In many renal diseases this matrix is reorganized in response to a variety of cytokines and growth factors. This study examines proteoglycan and hyaluronan (HA) synthesis by MCs triggered by proinflammatory agents and investigates the effect of an exogenous HA matrix on matrix synthesis by MCs. Metabolic labeling, ion exchange and size exclusion chromatography, Western blotting, and immunocytochemistry were used to identify changes in matrix accumulation. When incubated with interleukin-1, platelet-derived growth factor, or fetal calf serum, MCs initiated rapid HA synthesis associated with the up-regulation of HA synthase-2 and increased the synthesis of versican, perlecan, and decorin/biglycan. HA was both released into the medium and incorporated into extensive pericellular coats. Adding exogenous HA to unstimulated cells that had undetectable pericellular coats of HA selectively reduced perlecan and versican turnover, whereas other proteoglycans were unaffected. These results suggest that high levels of HA in the mesangium in disease is a mechanism controlling the accumulation of specific mesangial matrix components. HA may thus be an attractive target for therapeutic intervention. PMID:17974600

Hyaluronan Production Regulates Metabolic and Cancer Stem-like Properties of Breast Cancer Cells via Hexosamine Biosynthetic Pathway-coupled HIF-1 Signaling*

PubMed Central

Chanmee, Theerawut; Ontong, Pawared; Izumikawa, Tomomi; Higashide, Miho; Mochizuki, Nobutoshi; Chokchaitaweesuk, Chatchadawalai; Khansai, Manatsanan; Nakajima, Kazuki; Kakizaki, Ikuko; Kongtawelert, Prachya; Taniguchi, Naoyuki; Itano, Naoki

2016-01-01

Cancer stem cells (CSCs) represent a small subpopulation of self-renewing oncogenic cells. As in many other stem cells, metabolic reprogramming has been implicated to be a key characteristic of CSCs. However, little is known about how the metabolic features of cancer cells are controlled to orchestrate their CSC-like properties. We recently demonstrated that hyaluronan (HA) overproduction allowed plastic cancer cells to revert to stem cell states. Here, we adopted stable isotope-assisted tracing and mass spectrometry profiling to elucidate the metabolic features of HA-overproducing breast cancer cells. These integrated approaches disclosed an acceleration of metabolic flux in the hexosamine biosynthetic pathway (HBP). A metabolic shift toward glycolysis was also evident by quantitative targeted metabolomics, which was validated by the expression profiles of key glycolytic enzymes. Forced expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), an HBP rate-limiting enzyme, resembled the results of HA overproduction with regard to HIF-1α accumulation and glycolytic program, whereas GFAT1 inhibition significantly decreased HIF-1α protein level in HA-overproducing cancer cells. Moreover, inhibition of the HBP-HIF-1 axis abrogated HA-driven glycolytic enhancement and reduced the CSC-like subpopulation. Taken together, our results provide compelling evidence that HA production regulates the metabolic and CSC-like properties of breast cancer cells via HBP-coupled HIF-1 signaling. PMID:27758869

Involvement of a Gardos-type potassium channel in head activator-induced mitosis of BON cells.

PubMed

Kayser, S T; Ulrich, H; Schaller, H C

1998-06-01

The human neuroendocrine cell line BON was used to study second messengers involved in signal transduction for entry into mitosis. BON cells produce the neuropeptide head activator (HA) and use it as autocrine growth factor. HA stimulates BON cell proliferation by triggering entry into mitosis. HA-induced mitosis is mediated by an inhibitory G protein, the action of which is blocked by pertussis toxin. HA signaling requires inhibition of the cAMP pathway, calcium influx, and hyperpolarization of cells. The latter is a very important and sensitive step involving a calcium-activated potassium channel. Cell cycle progression and proliferation of BON cells are most efficiently inhibited with specific inhibitors of this potassium channel. Pharmacology and RNA analysis suggest identity with the recently cloned Gardos-type potassium channel.

Hyaluronan (HA) interacting proteins RHAMM and hyaluronidase impact prostate cancer cell behavior and invadopodia formation in 3D HA-based hydrogels.

PubMed

Gurski, Lisa A; Xu, Xian; Labrada, Lyana N; Nguyen, Ngoc T; Xiao, Longxi; van Golen, Kenneth L; Jia, Xinqiao; Farach-Carson, Mary C

2012-01-01

To study the individual functions of hyaluronan interacting proteins in prostate cancer (PCa) motility through connective tissues, we developed a novel three-dimensional (3D) hyaluronic acid (HA) hydrogel assay that provides a flexible, quantifiable, and physiologically relevant alternative to current methods. Invasion in this system reflects the prevalence of HA in connective tissues and its role in the promotion of cancer cell motility and tissue invasion, making the system ideal to study invasion through bone marrow or other HA-rich connective tissues. The bio-compatible cross-linking process we used allows for direct encapsulation of cancer cells within the gel where they adopt a distinct, cluster-like morphology. Metastatic PCa cells in these hydrogels develop fingerlike structures, "invadopodia", consistent with their invasive properties. The number of invadopodia, as well as cluster size, shape, and convergence, can provide a quantifiable measure of invasive potential. Among candidate hyaluronan interacting proteins that could be responsible for the behavior we observed, we found that culture in the HA hydrogel triggers invasive PCa cells to differentially express and localize receptor for hyaluronan mediated motility (RHAMM)/CD168 which, in the absence of CD44, appears to contribute to PCa motility and invasion by interacting with the HA hydrogel components. PCa cell invasion through the HA hydrogel also was found to depend on the activity of hyaluronidases. Studies shown here reveal that while hyaluronidase activity is necessary for invadopodia and inter-connecting cluster formation, activity alone is not sufficient for acquisition of invasiveness to occur. We therefore suggest that development of invasive behavior in 3D HA-based systems requires development of additional cellular features, such as activation of motility associated pathways that regulate formation of invadopodia. Thus, we report development of a 3D system amenable to dissection of biological processes associated with cancer cell motility through HA-rich connective tissues.

Genoprotective effect of hyaluronic acid against benzalkonium chloride-induced DNA damage in human corneal epithelial cells

PubMed Central

Wu, Han; Zhang, Huina; Wang, Changjun; Wu, Yihua; Xie, Jiajun; Jin, Xiuming; Yang, Jun

2011-01-01

Purpose The aim of this study was to investigate hyaluronic acid (HA) protection on cultured human corneal epithelial cells (HCEs) against benzalkonium chloride (BAC)-induced DNA damage and intracellular reactive oxygen species (ROS) increase. Methods Cells were incubated with different concentrations of BAC with or without the presence of 0.2% HA for 30 min. DNA damage to HCEs was examined by alkaline comet assay and by immunofluorescence microscopic detection of the phosphorylated form of histone variant H2AX (γH2AX) foci. ROS production was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Cell apoptosis was determined with annexin V staining by flow cytometry. Results HA significantly reduced BAC-induced DNA damage as indicated by the tail length (TL) and tail moment (TM) of alkaline comet assay and by γH2AX foci formation, respectively. Moreover, HA significantly decreased BAC-induced ROS increase and cell apoptosis. However, exposure to HA alone did not produce any significant change in DNA damage, ROS generation, or cell apoptosis. Conclusions BAC could induce DNA damage and cell apoptosis in HCEs, probably through increasing oxidative stress. Furthermore, HA was an effective protective agent that had antioxidant properties and could decrease DNA damage and cell apoptosis induced by BAC. PMID:22219631

Nucleation and growth of hydroxyapatite on arc-deposited TiO2 surfaces studied by quartz crystal microbalance with dissipation

NASA Astrophysics Data System (ADS)

Lilja, Mirjam; Butt, Umer; Shen, Zhijian; Bjöörn, Dorota

2013-11-01

Understanding of nucleation and growth kinetics of biomimetically deposited hydroxyapatite (HA) on crystalline TiO2 surfaces is important with respect to the application and performance of HA as functional implant coatings. Arc-evaporation was used to deposit TiO2 coatings dominated by anatase phase, rutile phase or their mixtures. Subsequent formation of HA from phosphate buffered saline solution (PBS) was investigated in real-time using in situ quartz crystal microbalance with dissipation technique (QCM-D). Scanning electron microscopy (SEM) and X-ray diffraction (XRD) were employed to characterize the presence, morphology and crystal structure of TiO2 coatings and the formed HA. Increasing temperature of the PBS, increasing flow rate and applying a higher ion concentration in solution were found to accelerate HA nucleation process and hence affect growth kinetics. Lower PBS temperature resulted in the formation of HA coatings with flake-like morphology and increasing HA porosity. All TiO2 coatings under study enabled HA formation at body temperature, while in contrast Ti reference surfaces only supported HA nucleation and growth at elevated temperatures. QCM-D technique is a powerful tool for studying the impact of process parameters during biomimetic coating deposition on coating structure evolution in real time and provides valuable information for understanding, optimizing as well as tailoring the biomimetic HA growth processes.

Particle size, charge and colloidal stability of humic acids coprecipitated with Ferrihydrite.

PubMed

Angelico, Ruggero; Ceglie, Andrea; He, Ji-Zheng; Liu, Yu-Rong; Palumbo, Giuseppe; Colombo, Claudio

2014-03-01

Humic acids (HA) have a colloidal character whose size and negative charge are strictly dependent on surface functional groups. They are able to complex large amount of poorly ordered iron (hydr)oxides in soil as a function of pH and other environmental conditions. Accordingly, with the present study we intend to assess the colloidal properties of Fe(II) coprecipitated with humic acids (HA) and their effect on Fe hydroxide crystallinity under abiotic oxidation and order of addition of both Fe(II) and HA. TEM, XRD and DRS experiments showed that Fe-HA consisted of Ferrihydrite with important structural variations. DLS data of Fe-HA at acidic pH showed a bimodal size distribution, while at very low pH a slow aggregation process was observed. Electrophoretic zeta-potential measurements revealed a negative surface charge for Fe-HA macromolecules, providing a strong electrostatic barrier against aggregation. Under alkaline conditions HA chains swelled, which resulted in an enhanced stabilization of the colloid particles. The increasing of zeta potential and size of the Fe-HA macromolecules, reflects a linear dependence of both with pH. The increase in the size and negative charge of the Fe-HA precipitate seems to be more affected by the ionization of the phenolic acid groups, than by the carboxylic acid groups. The main cause of negative charge generation of Fe/HA is due to increased dissociation of phenolic groups in more expanded structure. The increased net negative surface potential induced by coprecipitation with Ferrihydrite and the correspondent changes in configuration of the HA could trigger the inter-particle aggregation with the formation of new negative surface. The Fe-HA coprecipitation can reduce electrosteric repulsive forces, which in turn may inhibit the aggregation process at different pH. Therefore, coprecipitation of Ferrihydrite would be expected to play an important role in the carbon stabilization and persistence not only in organic soils, but also in waters containing dissolved organic matter. Copyright © 2013 Elsevier Ltd. All rights reserved.

Hyaluronic acid and Arg-Gly-Asp peptide modified Graphene oxide with dual receptor-targeting function for cancer therapy.

PubMed

Guo, Yufeng; Xu, Haixing; Li, Yiping; Wu, Fengzheng; Li, Yixuan; Bao, Yun; Yan, Xiumei; Huang, Zhijun; Xu, Peihu

2017-07-01

Graphene oxide (GO) modified with hyaluronic acid (HA) and Arg-gly-asp peptide (RGD) was designed as a dual-receptor targeting drug delivery system to enhance the specificity and efficiency of anticancer drug delivery. Firstly, GO-HA-RGD conjugate was characterized to reveal its structure and morphology. Whereafter, doxorubicin (Dox) as a model drug was loaded on GO-HA-RGD carrier, which displayed a high loading rate (72.9%, GO:Dox (w/w) = 1:1), pH-response and sustained drug release behavior. Cytotoxicity experiments showed that GO-HA-RGD possessed excellent biocompatibility towards SKOV-3 and HOSEpiC cells. Additionally, the GO-HA-RGD/Dox had a stronger cytotoxicity for SKOV-3 cells than either GO-HA/Dox (single receptor) or GO/Dox (no receptor). Moreover, celluar uptake studies illustrated that GO-HA-RGD conjugate could be effectively taken up by SKOV-3 cells via a synergic effect of CD44-HA and integrin-RGD mediated endocytosis. Hence, GO-HA-RGD nanocarrier is able to be a promising platform for targeted cancer therapeutic.

Humic acids contribution to sedimentary organic matter on a shallow continental shelf (northern Adriatic Sea)

NASA Astrophysics Data System (ADS)

Giani, M.; Rampazzo, F.; Berto, D.

2010-12-01

The shallow northern Adriatic Sea receives large river runoff, predominantly from the Po River, which is the main allochthonous source of nutrients and organic matter. The origin and quality of organic matter deposited in the sediments can influence the degradation processes and oxygen consumption in the bottom waters as well as the fate of many pollutants. Therefore the humic acids (HA) were quantified in surface and sub-surface sediments collected in an area of the north-western Adriatic platform south of Po River. HA showed to have a relevant contribution to sedimentary organic matter. HA content in sediments were positively correlated with the organic carbon concentration and negatively with redox potential and pH, particularly in sub-surface reduced sediments, suggesting their important role in the diagenetic processes taking place in anoxic conditions. Elemental composition of HA extracted from surface and sub-surface sediments showed a wide range of variation of the C org/N ratios which could be due to a mixed (terrestrial and marine) origin and/or an elevated bacteria degradation of nitrogen during diagenesis processes in sediments. The spectroscopic ratios A 2/A 4 and A 4/A 6 of HA confirmed a mixed origin with a high degree of condensation of the HA extracted from sediments.

Oral vaccine of Lactococcus lactis harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice.

PubMed

Joan, Stella Siaw Xiu; Pui-Fong, Jee; Song, Adelene Ai-Lian; Chang, Li-Yen; Yusoff, Khatijah; AbuBakar, Sazaly; Rahim, Raha Abdul

2016-05-01

An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus. Recombinant L. lactis strains expressed HA1-nisP fusion proteins when induced with nisin, as confirmed through western blotting. However, immunofluorescense did not detect any surface-displayed proteins, suggesting that the protein was either unsuccessfully translocated or improperly displayed. Despite this, oral administration of recombinant L. lactis strains to BALB/c mice revealed that significant levels of anti-HA1 sIgA antibodies were detected in mice fecal suspension samples of mice group NZ9000 (pNZ:HN) when compared to the negative control NZ9000 (pNZ8048) group. Specific anti-HA1 sIgA antibodies were locally produced and live recombinant lactococcal vaccine was able to elicit humoral response of BALB/c mice despite unsuccessful surface display of the HA1 epitope.

[Establishment of MDCK-pHaMDR cell model and standard operation procedure for assessing blood-brain barrier permeability of chemical components of traditional Chinese medicine].

PubMed

Yang, Yan-Fang; Wu, Ni; Yang, Xiu-Wei

2016-07-01

To establish MDCK-pHaMDR cell model and standard operation procedure for assessing the blood-brain barrier permeability of chemical components of traditional Chinese medicine. MDCK-pHaMDR cell model was evaluated by determining the morphology features, transepithelial electrical resistance, bidirectional transport and intracellular accumulation of Rhodamine 123 and the apparent permeability of positive control drugs caffeine and atenolol. The MDCK-pHaMDR cell model had satisfactory integrity and tightness, and stable expression of P-gp. In addition, the transport results of the positive control drugs were consistent with the reported values in literature. All the parameters tested of the MDCK-pHaMDR cell model were consistent with the requirements, so the model can be used to study the blood-brain barrier permeability of chemical components of traditional Chinese medicine. Copyright© by the Chinese Pharmaceutical Association.

Cold-spray coating of hydroxyapatite on a three-dimensional polyetheretherketone implant and its biocompatibility evaluated by in vitro and in vivo minipig model.

PubMed

Lee, Jae Hyup; Jang, Hae Lin; Lee, Kyung Mee; Baek, Hae-Ri; Jin, Kyoungsuk; Noh, Jun Hong

2017-04-01

PEEK is a bioinert material that does not chemically bind to native bone tissue and thus formation of natural bone-like hydroxyapatite (HA) coating layer on PEEK has been an important challenge to improve biocompatibility and to preserve mechanical property of PEEK. Among various coating techniques, cold-spray coating method is suitable to form stable HA coating layer on PEEK while maintaining their chemical properties, because it can be conducted in relatively low-temperature range. Therefore, in this research, we used cold-spray coating method to form a thick layer of HA on the topographically complex PEEK substrates with periodic ridges on the surface and implanted in iliac bone defects of minipigs which is known to be similar with human body system. In addition, PEEK cage for clinical usage was coated with HA and inserted in the lumbar intervertebral disc space of minipig. We observed higher ALP activity, calcium production, and BSP production of human bone marrow mesenchymal stem cells on the HA-coated PEEK implants than the bare PEEK group in in vitro test. In addition, two-dimensional histological analysis and three-dimensional micro CT analysis demonstrated that implantation of complex shape of HA-PEEK hybrid implant in in vivo minipig model resulted sufficient biocompatibility and osseointegration for further clinical applications. Notably, due to the enhanced stability of PEEK cage induced from HA coating layer, osseointegration rate of the small HA blocks loaded inside the PEEK cage was also significantly improved which indicates overall increased fusion rate and adherence of the HA-coated PEEK cage. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 647-657, 2017. © 2015 Wiley Periodicals, Inc.

MiR-30e suppresses proliferation of hepatoma cells via targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Guoxing; Shi, Hui; Li, Jiong

Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3′-untranslated region (3′UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3′UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 atmore » the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • P4HA1 is a novel target gene of miR-30e. • P4HA1 is increased in clinical HCC tissues. • MiR-30e is negatively correlated with P4HA1 in clinical HCC tissues. • MiR-30e suppresses the proliferation of HCC cells through targeting P4HA1 mRNA.« less

Cervical hyaluronan biology in pregnancy, parturition and preterm birth.

PubMed

Mahendroo, Mala

2018-03-03

Cervical hyaluronan (HA) synthesis is robustly induced in late pregnancy in numerous species including women and mice. Recent evidence highlights the diverse and dynamic functions of HA in cervical biology that stem from its expression in the cervical stroma, epithelia and immune cells, changes in HA molecular weight and cell specific expression of HA binding partners. Mice deficient in HA in the lower reproductive tract confirm a structural role of HA to increase spacing and disorganization of fibrillar collagen, though this function is not critical for pregnancy and parturition. In addition, cervical HA depletion via targeted deletion of HA synthase genes, disrupts cell signaling required for the differentiation of epithelia and their mucosal and junctional barrier, resulting in increased susceptibility to ascending infection-mediated preterm birth. Finally the generation of HA disaccharides by bacterial hyaluronidases as made by Group B streptococcus can ligate toll like receptors TLR2/4 thus preventing appropriate inflammatory responses as needed to fight ascending infection and preterm birth. This review summarizes our current understanding of HA's novel and unique roles in cervical remodeling in the process of birth. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Differential effects of miR-34c-3p and miR-34c-5p on SiHa cells proliferation apoptosis, migration and invasion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez, Jesus Adrian; Alvarez-Salas, Luis Marat, E-mail: lalvarez@cinvestav.mx

Highlights: {yields} In this study we examine miR-34c-3p and miR-34c-5p functions in SiHa cells. {yields} We study miRNA effect on cell proliferation, anchorage independent growth, apoptosis, cell motility and invasion. {yields} We find that miR-34c-3p and miR-34c-5p inhibition of proliferation and anchorage independent growth are exclusive to SiHa cells. {yields} miR-34c-3p induces apoptosis and inhibits cell motility and invasion in SiHa cells. {yields} In this study we conclude that miR-34c-3p functions as a tumor suppressor differ from miR-34c-5p. -- Abstract: MicroRNAs (miRNA) regulate expression of several genes associated with human cancer. Here, we analyzed the function of miR-34c, an effectormore » of p53, in cervical carcinoma cells. Expression of either miR-34c-3p or miR-34c-5p mimics caused inhibition of cell proliferation in the HPV-containing SiHa cells but not in other cervical cells irrespective of tumorigenicity and HPV content. These results suggest that SiHa cells may lack of regulatory mechanisms for miR-34c. Monolayer proliferation results showed that miR-34c-3p produced a more pronounced inhibitory effect although both miRNAs caused inhibition of anchorage independent growth at similar extent. However, ectopic expression of pre-miR-34c-3p, but not pre-miR-34c-5p, caused S-phase arrest in SiHa cells triggering a strong dose-dependent apoptosis. A significant inhibition was observed only for miR-34c-3p on SiHa cells migration and invasion, therefore implying alternative regulatory pathways and targets. These results suggest differential tumor suppressor roles for miR-34c-3p and miR-34c-5p and provide new insights in the understanding of miRNA biology.« less

Abiotic stress of ambient cold temperature regulates the host receptivity to pathogens by cell surfaced sialic acids.

PubMed

Moon, Seong-Cheol; Joo, Su-Yeon; Chung, Tae-Wook; Choi, Hee-Jung; Park, Mi-Ju; Choi, Hee-Jin; Bae, Sung-Jin; Kim, Keuk-Jun; Kim, Cheorl-Ho; Joo, Myungsoo; Ha, Ki-Tae

2016-07-29

Ambient cold temperature, as an abiotic stress, regulates the survival, stability, transmission, and infection of pathogens. However, the effect of cold temperature on the host receptivity to the pathogens has not been fully studied. In this study, the expression of terminal α-2,3- and α-2,6-sialic acids were increased in murine lung tissues, especially bronchial epithelium, by exposure to cold condition. The expression of several sialyltransferases were also increased by exposure to cold temperature. Furthermore, in human bronchial epithelial BEAS-2B cells, the expressions of α-2,3- and α-2,6-sialic acids, and mRNA levels of sialyltransferases were increased in the low temperature condition at 33 °C. On the other hand, the treatment of Lith-Gly, a sialyltransferase inhibitor, blocked the cold-induced expression of sialic acids on surface of BEAS-2B cells. The binding of influenza H1N1 hemagglutinin (HA) toward BEAS-2B cells cultured at low temperature condition was increased, compared to 37 °C. In contrast, the cold-increased HA binding was blocked by treatment of lithocholicglycine and sialyl-N-acetyl-D-lactosamines harboring α-2,3- and α-2,6-sialyl motive. These results suggest that the host receptivity to virus at cold temperature results from the expressions of α-2,3- and α-2,6-sialic acids through the regulation of sialyltransferase expression. Copyright © 2016 Elsevier Inc. All rights reserved.

In Vitro and In Vivo Dentinogenic Efficacy of Human Dental Pulp-Derived Cells Induced by Demineralized Dentin Matrix and HA-TCP

PubMed Central

Kang, Kyung-Jung; Lee, Min Suk; Moon, Chan-Woong; Lee, Jae-Hoon

2017-01-01

Human dental pulp cells have been known to have the stem cell features such as self-renewal and multipotency. These cells are differentiated into hard tissue by addition of proper cytokines and biomaterials. Hydroxyapatite-tricalcium phosphates (HA-TCPs) are essential components of hard tissue and generally used as a biocompatible material in tissue engineering of bone. Demineralized dentin matrix (DDM) has been reported to increase efficiency of bone induction. We compared the efficiencies of osteogenic differentiation and in vivo bone formation of HA-TCP and DDM on human dental pulp stem cells (hDPSCs). DDM contains inorganic components as with HA-TCP, and organic components such as collagen type-1. Due to these components, osteoinduction potential of DDM on hDPSCs was remarkably higher than that of HA-TCP. However, the efficiencies of in vivo bone formation are similar in HA-TCP and DDM. Although osteogenic gene expression and bone formation in immunocompromised nude mice were similar levels in both cases, dentinogenic gene expression level was slightly higher in DDM transplantation than in HA-TCP. All these results suggested that in vivo osteogenic potentials in hDPSCs are induced with both HA-TCP and DDM by osteoconduction and osteoinduction, respectively. In addition, transplantation of hDPSCs/DDM might be more effective for differentiation into dentin. PMID:28761445

Hyaluronan and Hyaluronan-Binding Proteins Accumulate in Both Human Type 1 Diabetic Islets and Lymphoid Tissues and Associate With Inflammatory Cells in Insulitis

PubMed Central

Bogdani, Marika; Johnson, Pamela Y.; Potter-Perigo, Susan; Nagy, Nadine; Day, Anthony J.; Bollyky, Paul L.

2014-01-01

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor–stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes. PMID:24677718

In Vitro and In Vivo Dentinogenic Efficacy of Human Dental Pulp-Derived Cells Induced by Demineralized Dentin Matrix and HA-TCP.

PubMed

Kang, Kyung-Jung; Lee, Min Suk; Moon, Chan-Woong; Lee, Jae-Hoon; Yang, Hee Seok; Jang, Young-Joo

2017-01-01

Human dental pulp cells have been known to have the stem cell features such as self-renewal and multipotency. These cells are differentiated into hard tissue by addition of proper cytokines and biomaterials. Hydroxyapatite-tricalcium phosphates (HA-TCPs) are essential components of hard tissue and generally used as a biocompatible material in tissue engineering of bone. Demineralized dentin matrix (DDM) has been reported to increase efficiency of bone induction. We compared the efficiencies of osteogenic differentiation and in vivo bone formation of HA-TCP and DDM on human dental pulp stem cells (hDPSCs). DDM contains inorganic components as with HA-TCP, and organic components such as collagen type-1. Due to these components, osteoinduction potential of DDM on hDPSCs was remarkably higher than that of HA-TCP. However, the efficiencies of in vivo bone formation are similar in HA-TCP and DDM. Although osteogenic gene expression and bone formation in immunocompromised nude mice were similar levels in both cases, dentinogenic gene expression level was slightly higher in DDM transplantation than in HA-TCP. All these results suggested that in vivo osteogenic potentials in hDPSCs are induced with both HA-TCP and DDM by osteoconduction and osteoinduction, respectively. In addition, transplantation of hDPSCs/DDM might be more effective for differentiation into dentin.

Influence of Magnesium Ion Substitution on Structural and Thermal Behavior of Nanodimensional Hydroxyapatite

NASA Astrophysics Data System (ADS)

Batra, Uma; Kapoor, Seema; Sharma, Sonia

2013-06-01

Hydroxyapatite (HA), incorporating small amount of magnesium, shows attractive biological performance in terms of improved bone metabolism, osteoblast and osteoclast activity, and bone in-growth. This article reports a systematic investigation on the influence of magnesium (Mg) substitution on structural and thermal behavior of nanodimensional HA. HA and Mg-substituted HA nanopowders were synthesized through sol-gel route. The morphology and size of nanopowders were characterized by transmission electron microscopy. The BET surface area was evaluated from N2 adsorption isotherms. Structural analysis and thermal behavior were investigated by means of Fourier transform infrared spectroscopy, x-ray diffraction, thermogravimetry, and differential thermal analysis. As-synthesized powders consisted of flake-like agglomerates of HA and calcium-deficient HA. The incorporation of magnesium in HA resulted in decrease of crystallite size, crystallinity, and lattice parameters a and c and increase in BET surface area. β-tricalcium phosphate formation occured at lower calcination temperature in Mg-substituted HA than HA.

SN38 conjugated hyaluronic acid gold nanoparticles as a novel system against metastatic colon cancer cells.

PubMed

Hosseinzadeh, Hosniyeh; Atyabi, Fatemeh; Varnamkhasti, Behrang Shiri; Hosseinzadeh, Reza; Ostad, Seyed Nasser; Ghahremani, Mohammad Hossein; Dinarvand, Rassoul

2017-06-30

Combination of chemotherapy and photothermal therapy has been proposed for better treatment of metastatic colon cancer. In this study SN38, a highly potent cytotoxic agent, was conjugated to negatively charged hyaluronic acid (HA), which was deposited on the surface of the positively charged gold nanoparticles via electrostatic interaction. The drug conjugation and its interaction with gold nanoparticles were verified by 1 H NMR and UV-vis spectroscopies, respectively. The prepared SN38-HA gold NPs are negatively charged spherical nanoparticles with an average size of 75±10nm. In vitro release study revealed that drug release in acidic conditions (pH 5.2) was faster than that in physiological pH. Red light emitting diode (LED, 630nm, 30mW) was used as a light source for photothermal experiments. The drug release in acidic conditions was increased up to 30% using red LED illumination (6min) in comparison with experiment carried out indark. The cytotoxicity study on MUC1 positive HT29, SW480 colon cancer cells and MUC1 negative CHO cells, showed higher toxicity of the nanoparticles on HT29 and SW480 cell lines compared to CHO cells. Confocal microscopy images along with flow cytometry analysis confirm the cytotoxicity results. The incubation time for reaching IC50 decreases from 48h to 24h by LED illumination after nanoparticle treatment. Migratory potential of the HT29 and SW480 cell lines was reduced by co-application of SN38-HA gold NPs and LED radiation. Also anti-proliferative study indicates that LED radiation has increased the cytotoxicity of the nanoparticles and this effect is remained up to 8days. Copyright © 2017 Elsevier B.V. All rights reserved.

Fabrication of nano-structured HA/CNT coatings on Ti6Al4V by electrophoretic deposition for biomedical applications.

PubMed

Zhang, Bokai; Kwok, Chi Tat; Cheng, Fai Tsun; Man, Hau Chung

2011-12-01

In order to improve the bone bioactivity and osteointegration of metallic implants, hydroxyapatite (HA) is often coated on their surface so that a real bond with the surrounding bone tissue can be formed. In the present study, cathodic electrophoretic deposition (EPD) has been attempted for depositing nanostructured HA coatings on titanium alloy Ti6Al4V followed by sintering at 800 degrees C. Nano-sized HA powder was used in the EPD process to produce dense coatings. Moreover, multiwalled carbon nanotubes (CNTs) were also used to reinforce the HA coating for enhancing its mechanical strength. The surface morphology, compositions and microstructure of the monolithic coating of HA and nanocomposite coatings of HA with different CNT contents (4 to 25%) on Ti6Al4V were investigated by scanning-electron microscopy, energy-dispersive X-ray spectroscopy and Xray diffractometry, respectively. Electrochemical corrosion behavior of the various coatings in Hanks' solution at 37 degrees C was investigated by means of open-circuit potential measurement and cyclic potentiodynamic polarization tests. Surface hardness, adhesion strength and bone bioactivity of the coatings were also studied. The HA and HA/CNT coatings had a thickness of about 10 microm, with corrosion resistance higher than that of the substrate and adhesion strength higher than that of plasma sprayed HA coating. The properties of the composite coatings were optimized by varying the CNT contents. The enhanced properties could be attributed to the use of nano-sized HA particles and CNTs. Compared with the monolithic HA coating, the CNT-reinforced HA coating markedly increased the coating hardness without deteriorating the corrosion resistance or adhesion strength.

The Effects of Bio-Lubricating Molecules on Flexor Tendon Reconstruction in A Canine Allograft Model In Vivo

PubMed Central

Zhao, Chunfeng; Wei, Zhuang; Kirk, Ramona L.; Thoreson, Andrew R.; Jay, Gregory D.; Moran, Steven L.; An, Kai-Nan; Amadio, Peter C.

2014-01-01

Background Using allograft is an attractive alternative for flexor tendon reconstruction because of the lack of donor morbidity, and better matching to the intrasynovial environment. The purpose of this study was to use biolubricant molecules to modify the graft surface to decrease adhesions and improve digit function. Methods 28 flexor digitorum profundus (FDP) tendons from the 2nd and 5th digits of 14 dogs were first lacerated and repaired to create a model with repair failure and scar digit for tendon reconstruction. Six weeks after the initial surgery, the tendons were reconstructed with FDP allograft tendons obtained from canine cadavers. One graft tendon in each dog was treated with saline as a control and the other was treated with gelatin, carbodiimide derivatized, hyaluronic acid and lubricin (cd-HA-Lubricin). Six weeks postoperatively, digit function, graft mechanics, and biology were analyzed. Results Allograft tendons treated with cd-HA-Lubricin had decreased adhesions at the proximal tendon/graft repair and within flexor sheath, improved digit function, and increased graft gliding ability. The treatment also reduced the strength at the distal tendon to bone repair, but the distal attachment rupture rate was similar for both graft types. Histology showed that viable cells migrated to the allograft, but these were limited to the tendon surface. Conclusion cd-HA-Lubricin treatment of tendon allograft improves digit functional outcomes after flexor tendon reconstruction. However, delayed bone-tendon healing should be a caution. Furthermore, the cell infiltration into the allograft tendons substance should be a target for future studies, to shorten the allograft self-regeneration period. PMID:24445876

Recent advances in hyaluronic acid-decorated nanocarriers for targeted cancer therapy

PubMed Central

Wickens, Jennifer M.; Alsaab, Hashem O.; Kesharwani, Prashant; Bhise, Ketki; Amin, Mohd Cairul Iqbal Mohd; Tekade, Rakesh Kumar; Gupta, Umesh; Iyer, Arun K.

2016-01-01

The cluster-determinant 44 (CD44) receptor has a high affinity for hyaluronic acid (HA) binding and is a desirable receptor for active targeting based on its overexpression in cancer cells compared with normal body cells. The nanocarrier affinity can be increased by conjugating drug-loaded carriers with HA, allowing enhanced cancer cell uptake via the HA-CD44 receptor-mediated endocytosis pathway. In this review, we discuss recent advances in HA-based nanocarriers and micelles for cancer therapy. In vitro and in vivo experiments have repeatedly indicated HA-based nanocarriers to be a target-specific drug and gene delivery platform with great promise for future applications in clinical cancer therapy. PMID:28017836

The importance of serum serotonin levels in the measurement of radiation-induced bystander cell death in HaCaT cells.

PubMed

Lyng, Fiona M; Desplanques, Maxime; Jella, Kishore Kumar; Garcia, Amaya; McClean, Brendan

2012-10-01

The aim of this study was to investigate the importance of serum serotonin levels in the measurement of bystander cell death. The study was undertaken as part of an intercomparison exercise involving seven European laboratories funded under the European Union Sixth Framework Programme (FP6) Non-Targeted Effects (NOTE) integrated project. Three batches of foetal bovine serum were tested; serum with high and low serotonin content from the intercomparison exercise as well as serum from the home laboratory. Three sets of human keratinocytes (HaCaT cell line) were cultured in DMEM:F12 medium supplemented with serum with high or low serotonin content or serum from the home laboratory and both donor and recipient HaCaT cells were plated. The donor HaCaT cells were irradiated (0.5 Gy) using a cobalt 60 teletherapy unit, the medium was harvested 1 hour post irradiation and transferred to the recipient HaCaT cells. Bystander induced cell death was measured by the clonogenic survival assay and the Alamar blue viability assay. A significant reduction in cell survival, as measured by the clonogenic assay, and in cell viability, as measured by the Alamar blue assay, was observed in the recipient HaCaT cells treated with medium from irradiated cells compared to the cells treated with medium from unirradiated cells. No significant difference was found between the three batches of serum. The data suggest that in our cell system and with our endpoints (clonogenic assay and Alamar blue assay), serum serotonin levels do not play a role in bystander-induced cell death.

Preparation, in vitro degradability, cytotoxicity, and in vivo biocompatibility of porous hydroxyapatite whisker-reinforced poly(L-lactide) biocomposite scaffolds.

PubMed

Xie, Lu; Yu, Haiyang; Yang, Weizhong; Zhu, Zhuoli; Yue, Li

2016-01-01

Biodegradable and bioactive scaffolds with interconnected macroporous structures, suitable biodegradability, adequate mechanical property, and excellent biocompatibility have drawn increasing attention in bone tissue engineering. Hence, in this work, porous hydroxyapatite whisker-reinforced poly(L-lactide) (HA-w/PLLA) composite scaffolds with different ratios of HA and PLLA were successfully developed through compression molding and particle leaching. The microstructure, in vitro mineralization, cytocompatibility, hemocompatibility, and in vivo biocompatibility of the porous HA-w/PLLA were investigated for the first time. The SEM results revealed that these HA-w/PLLA scaffolds possessed interconnected pore structures. Compared with porous HA powder-reinforced PLLA (HA-p/PLLA) scaffolds, HA-w/PLLA scaffolds exhibited better mechanical property and in vitro bioactivity, as more formation of bone-like apatite layers were induced on these scaffolds after mineralization in SBF. Importantly, in vitro cytotoxicity displayed that porous HA-w/PLLA scaffold with HA/PLLA ratio of 1:1 (HA-w1/PLLA1) produced no deleterious effect on human mesenchymal stem cells (hMSCs), and cells performed elevated cell proliferation, indicating a good cytocompatibility. Simultaneously, well-behaved hemocompatibility and favorable in vivo biocompatibility determined from acute toxicity test and histological evaluation were also found in the porous HA-w1/PLLA1 scaffold. These findings may provide new prospects for utilizing the porous HA whisker-based biodegradable scaffolds in bone repair, replacement, and augmentation applications.

Engineered Metal-Phenolic Capsules Show Tunable Targeted Delivery to Cancer Cells.

PubMed

Ju, Yi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Dai, Yunlu; Guo, Junling; Bertleff-Zieschang, Nadja; Suma, Tomoya; Richardson, Joseph J; Caruso, Frank

2016-06-13

We engineered metal-phenolic capsules with both high targeting and low nonspecific cell binding properties. The capsules were prepared by coating phenolic-functionalized hyaluronic acid (HA) and poly(ethylene glycol) (PEG) on calcium carbonate templates, followed by cross-linking the phenolic groups with metal ions and removing the templates. The incorporation of HA significantly enhanced binding and association with a CD44 overexpressing (CD44+) cancer cell line, while the incorporation of PEG reduced nonspecific interactions with a CD44 minimal-expressing (CD44-) cell line. Moreover, high specific targeting to CD44+ cells can be balanced with low nonspecific binding to CD44- cells simply by using an optimized feed-ratio of HA and PEG to vary the content of HA and PEG incorporated into the capsules. Loading an anticancer drug (i.e., doxorubicin) into the obtained capsules resulted in significantly higher cytotoxicity to CD44+ cells but lower cytotoxicity to CD44- cells.

Correlations between the in vitro and in vivo bioactivity of the Ti/HA composites fabricated by a powder metallurgy method.

PubMed

Ning, Congqin; Zhou, Yu

2008-11-01

Ti/HA composites were successfully prepared by a powder metallurgy method and the effect of phase composition on the in vitro and in vivo bioactivity of the Ti/HA composites was investigated in the present study. The correlations between the in vitro and in vivo biological behaviors were highlighted. The results showed that the in vitro and in vivo bioactivity of the Ti/HA composites was dependent on their phase composition. The in vitro bioactivity of the Ti/HA composites was evaluated in simulated body fluid with ion concentrations similar to those of human plasma. After immersion in the simulated body fluid for a certain time, apatite precipitations formed on the surface of the composites with an initial titanium content of 50 and 70 wt.%, and no apatite was found on the surface of the composite with 30% titanium. Ti(2)O was responsible for the apatite formation on the surfaces of the composites. For in vivo analysis, Ti/HA cylinders were implanted in the metaphases of the rabbit femur. At the early stage of implantation, the new bone formed on the surface of the composite with 30% titanium was much less than that on the surfaces of the composites with 50% and 70% titanium. All the Ti/HA composites formed a chemical bone-bonding interface with the host bone by 6 months after implantation. The Ti/HA composites formed the bone-bonding interface with the surrounding bone through an apatite layer. The results in the present study suggested that the in vivo results agreed well with the in vitro results.

Hyaluronan and hyaluronectin in the extracellular matrix of human brain tumour stroma.

PubMed

Delpech, B; Maingonnat, C; Girard, N; Chauzy, C; Maunoury, R; Olivier, A; Tayot, J; Creissard, P

1993-01-01

Hyaluronan (HA) and the hyaluronan-binding glycoprotein hyaluronectin (HN) were measured in 23 gliomas and 8 meningiomas and their location was revisited in 35 tumours. A clear-cut difference was found in the HN/HA ratio values of glioblastomas (below 0.5) and that of astrocytomas (above 0.5 P < 0.001). Besides their location in the intercellular part of gliomas, HA and HN displayed a perivascular location in 1/3 astrocytomas, 17/24 glioblastomas, and 3/7 meningiomas, suggesting they could be produced also by the vascular stroma of tumours and that they would characterise the neoangiogenesis. All cultivated glioma cells tested produced HA in vitro, whereas only 1/11 cell lines produced HN, at a low level. The results obtained suggest that glioma HA and HN are produced by both cancer cells and vascular stroma cells, which contribute to the edification of the extracellular matrix. In meningiomas only the stroma would be responsible for HA and HN production.

Substantial Humic Acid Adsorption to Activated Carbon Air Cathodes Produces a Small Reduction in Catalytic Activity.

PubMed

Yang, Wulin; Watson, Valerie J; Logan, Bruce E

2016-08-16

Long-term operation of microbial fuel cells (MFCs) can result in substantial degradation of activated carbon (AC) air-cathode performance. To examine a possible role in fouling from organic matter in water, cathodes were exposed to high concentrations of humic acids (HA). Cathodes treated with 100 mg L(-1) HA exhibited no significant change in performance. Exposure to 1000 mg L(-1) HA decreased the maximum power density by 14% (from 1310 ± 30 mW m(-2) to 1130 ± 30 mW m(-2)). Pore blocking was the main mechanism as the total surface area of the AC decreased by 12%. Minimization of external mass transfer resistances using a rotating disk electrode exhibited only a 5% reduction in current, indicating about half the impact of HA adsorption was associated with external mass transfer resistance and the remainder was due to internal resistances. Rinsing the cathodes with deionized water did not restore cathode performance. These results demonstrated that HA could contribute to cathode fouling, but the extent of power reduction was relatively small in comparison to large mass of humics adsorbed. Other factors, such as biopolymer attachment, or salt precipitation, are therefore likely more important contributors to long-term fouling of MFC cathodes.

High molecular weight hyaluronic acid limits astrocyte activation and scar formation after spinal cord injury

NASA Astrophysics Data System (ADS)

Khaing, Zin Z.; Milman, Brian D.; Vanscoy, Jennifer E.; Seidlits, Stephanie K.; Grill, Raymond J.; Schmidt, Christine E.

2011-08-01

A major hurdle for regeneration after spinal cord injury (SCI) is the ability of axons to penetrate and grow through the scar tissue. After SCI, inflammatory cells, astrocytes and meningeal cells all play a role in developing the glial scar. In addition, degradation of native high molecular weight (MW) hyaluronic acid (HA), a component of the extracellular matrix, has been shown to induce activation and proliferation of astrocytes. However, it is not known if the degradation of native HA actually enhances glial scar formation. We hypothesize that the presence of high MW HA (HA with limited degradation) after SCI will decrease glial scarring. Here, we demonstrate that high MW HA decreases cell proliferation and reduces chondroitin sulfate proteoglycan (CSPG) production in cultured neonatal and adult astrocytes. In addition, stiffness-matched high MW HA hydrogels crosslinked to resist degradation were implanted in a rat model of spinal dorsal hemisection injury. The numbers of immune cells (macrophages and microglia) detected at the lesion site in animals with HA hydrogel implants were significantly reduced at acute time points (one, three and ten days post-injury). Lesioned animals with HA implants also exhibited significantly lower CSPG expression at ten days post-injury. At nine weeks post-injury, animals with HA hydrogel implants exhibited a significantly decreased astrocytic response, but did not have significantly altered CSPG expression. Combined, these data suggest that high MW HA, when stabilized against degradation, mitigates astrocyte activation in vitro and in vivo. The presence of HA implants was also associated with a significant decrease in CSPG deposition at ten days after SCI. Therefore, HA-based hydrogel systems hold great potential for minimizing undesired scarring as part of future repair strategies after SCI.

High molecular weight hyaluronic acid limits astrocyte activation and scar formation after spinal cord injury.

PubMed

Khaing, Zin Z; Milman, Brian D; Vanscoy, Jennifer E; Seidlits, Stephanie K; Grill, Raymond J; Schmidt, Christine E

2011-08-01

A major hurdle for regeneration after spinal cord injury (SCI) is the ability of axons to penetrate and grow through the scar tissue. After SCI, inflammatory cells, astrocytes and meningeal cells all play a role in developing the glial scar. In addition, degradation of native high molecular weight (MW) hyaluronic acid (HA), a component of the extracellular matrix, has been shown to induce activation and proliferation of astrocytes. However, it is not known if the degradation of native HA actually enhances glial scar formation. We hypothesize that the presence of high MW HA (HA with limited degradation) after SCI will decrease glial scarring. Here, we demonstrate that high MW HA decreases cell proliferation and reduces chondroitin sulfate proteoglycan (CSPG) production in cultured neonatal and adult astrocytes. In addition, stiffness-matched high MW HA hydrogels crosslinked to resist degradation were implanted in a rat model of spinal dorsal hemisection injury. The numbers of immune cells (macrophages and microglia) detected at the lesion site in animals with HA hydrogel implants were significantly reduced at acute time points (one, three and ten days post-injury). Lesioned animals with HA implants also exhibited significantly lower CSPG expression at ten days post-injury. At nine weeks post-injury, animals with HA hydrogel implants exhibited a significantly decreased astrocytic response, but did not have significantly altered CSPG expression. Combined, these data suggest that high MW HA, when stabilized against degradation, mitigates astrocyte activation in vitro and in vivo. The presence of HA implants was also associated with a significant decrease in CSPG deposition at ten days after SCI. Therefore, HA-based hydrogel systems hold great potential for minimizing undesired scarring as part of future repair strategies after SCI.

Comparative Efficacy of Autologous Stromal Vascular Fraction and Autologous Adipose-Derived Mesenchymal Stem Cells Combined With Hyaluronic Acid for the Treatment of Sheep Osteoarthritis.

PubMed

Lv, Xiaoteng; He, Jiyin; Zhang, Xue; Luo, Xuan; He, Na; Sun, Zhongwei; Xia, Huitang; Liu, Victor; Zhang, Li; Lin, Xiangming; Lin, Liping; Yin, Huabin; Jiang, Dong; Cao, Wei; Wang, Richard; Zhou, Guangdong; Wang, Wen

2018-01-01

The current study explored whether intra-articular (IA) injection of autologous adipose mesenchymal stem cells (ASCs) combined with hyaluronic acid (HA) achieved better therapeutic efficacy than autologous stromal vascular fraction (SVF) combined with HA to prevent osteoarthritis (OA) progression and determined how long autologous ASCs combined with HA must remain in the joint to observe efficacy. OA models were established by performing anterior cruciate ligament transection (ACLT) and medial meniscectomy (MM). Autologous SVF (1×10 7 mononuclear cells), autologous low-dose ASCs (1×10 7 ), and autologous high-dose ASCs (5×10 7 ) combined with HA, and HA alone, or saline alone were injected into the OA model animals at 12 and 15 weeks after surgery, respectively. Compared with SVF+HA treatment, low-dose ASC+HA treatment yielded better magnetic resonance imaging (MRI) scores and macroscopic results, while the cartilage thickness of the tibial plateau did not differ between low, high ASC+HA and SVF+HA treatments detected by micro-computed tomography (µCT). Immunohistochemistry revealed that high-dose ASC+HA treatment rescued hypertrophic chondrocytes expressing collagen X in the deep area of articular cartilage. Western blotting analysis indicated the high- and low-dose ASC+HA groups expressed more collagen X than did the SVF+HA group. Enzyme-linked immunosorbent assay showed treatment with both ASC+HA and SVF+HA resulted in differing anti-inflammatory and trophic effects. Moreover, superparamagnetic iron oxide particle (SPIO)-labeled autologous ASC signals were detected by MRI at 2 and 18 weeks post-injection and were found in the lateral meniscus at 2 weeks and in the marrow cavity of the femoral condyle at 18 weeks post-injection. Thus, IA injection of autologous ASC+HA may demonstrate better efficacy than autologous SVF+HA in blocking OA progression and promoting cartilage regeneration, and autologous ASCs (5×10 7 cells) combined with HA potentially survive for at least 18 weeks after IA injection.

Structural characteristics of alkaline phosphatase from the moderately halophilic bacterium Halomonas sp. 593

PubMed Central

Arai, Shigeki; Yonezawa, Yasushi; Ishibashi, Matsujiro; Matsumoto, Fumiko; Adachi, Motoyasu; Tamada, Taro; Tokunaga, Hiroko; Blaber, Michael; Tokunaga, Masao; Kuroki, Ryota

2014-01-01

Alkaline phosphatase (AP) from the moderate halophilic bacterium Halomonas sp. 593 (HaAP) catalyzes the hydrolysis of phosphomonoesters over a wide salt-concentration range (1–4 M NaCl). In order to clarify the structural basis of its halophilic characteristics and its wide-range adaptation to salt concentration, the tertiary structure of HaAP was determined by X-ray crystallography to 2.1 Å resolution. The unit cell of HaAP contained one dimer unit corresponding to the biological unit. The monomer structure of HaAP contains a domain comprised of an 11-stranded β-sheet core with 19 surrounding α-helices similar to those of APs from other species, and a unique ‘crown’ domain containing an extended ‘arm’ structure that participates in formation of a hydrophobic cluster at the entrance to the substrate-binding site. The HaAP structure also displays a unique distribution of negatively charged residues and hydrophobic residues in comparison to other known AP structures. AP from Vibrio sp. G15-21 (VAP; a slight halophile) has the highest similarity in sequence (70.0% identity) and structure (Cα r.m.s.d. of 0.82 Å for the monomer) to HaAP. The surface of the HaAP dimer is substantially more acidic than that of the VAP dimer (144 exposed Asp/Glu residues versus 114, respectively), and thus may enable the solubility of HaAP under high-salt conditions. Conversely, the monomer unit of HaAP formed a substantially larger hydrophobic interior comprising 329 C atoms from completely buried residues, whereas that of VAP comprised 264 C atoms, which may maintain the stability of HaAP under low-salt conditions. These characteristics of HaAP may be responsible for its unique functional adaptation permitting activity over a wide range of salt concentrations. PMID:24598750

Hyaluronan synthesis in cultured tobacco cells (BY-2) expressing a chlorovirus enzyme: cytological studies.

PubMed

Rakkhumkaew, Numfon; Shibatani, Shigeo; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

2013-04-01

Extraction of hyaluronan from animals or microbial fermentation has risks including contamination with pathogens and microbial toxins. In this work, tobacco cultured-cells (BY-2) were successfully transformed with a chloroviral hyaluronan synthase (cvHAS) gene to produce hyaluronan. Cytological studies revealed accumulation of HA on the cells, and also in subcellular fractions (protoplasts, miniplasts, vacuoplasts, and vacuoles). Transgenic BY-2 cells harboring a vSPO-cvHAS construct containing the vacuolar targeting signal of sporamin connected to the N-terminus of cvHAS accumulated significant amounts of HA in vacuoles. These results suggested that cvHAS successfully functions on the vacuolar membrane and synthesizes/transports HA into vacuoles. Efficient synthesis of HA using this system provides a new method for practical production of HA. Copyright © 2012 Wiley Periodicals, Inc.

Mutational Analysis of Lassa Virus Glycoprotein Highlights Regions Required for Alpha-Dystroglycan Utilization.

PubMed

Acciani, Marissa; Alston, Jacob T; Zhao, Guohui; Reynolds, Hayley; Ali, Afroze M; Xu, Brian; Brindley, Melinda A

2017-09-15

Lassa virus (LASV) is an enveloped RNA virus endemic to West Africa and responsible for severe cases of hemorrhagic fever. Virus entry is mediated by the glycoprotein complex consisting of a stable-signal peptide, a receptor-binding subunit, GP1, and a viral-host membrane fusion subunit, GP2. Several cellular receptors can interact with the GP1 subunit and mediate viral entry, including alpha-dystroglycan (αDG) and lysosome-associated membrane protein 1 (LAMP1). In order to define the regions within GP1 that interact with the cellular receptors, we implemented insertional mutagenesis, carbohydrate shielding, and alanine scanning mutagenesis. Eighty GP constructs were engineered and evaluated for GP1-GP2 processing, surface expression, and the ability to mediate cell-to-cell fusion after low-pH exposure. To examine virus-to-cell entry, 49 constructs were incorporated onto vesicular stomatitis virus (VSV) pseudoparticles and transduction efficiencies were monitored in HAP1 and HAP1-ΔDAG1 cells that differentially produce the αDG cell surface receptor. Seven constructs retained efficient transduction in HAP1-ΔDAG1 cells yet poorly transduced HAP1 cells, suggesting that they are involved in αDG utilization. Residues H141, N146, F147, and Y150 cluster at the predicted central core of the trimeric interface and are important for GP-αDG interaction. Additionally, H92A-H93A, 150HA, 172HA, and 230HA displayed reduced transduction in both HAP1 and HAP1-ΔDAG1 cells, despite efficient cell-to-cell fusion activity. These mutations may interfere with interactions with the endosomal receptor LAMP1 or interfere at another stage in entry that is common to both cell lines. Insight gained from these data can aid in the development of more-effective entry inhibitors by blocking receptor interactions. IMPORTANCE Countries in which Lassa virus is endemic, such as Nigeria, Sierra Leone, Guinea, and Liberia, usually experience a seasonal outbreak of the virus from December to March. Currently, there is neither a preventative vaccine nor a therapeutic available to effectively treat severe Lassa fever. One way to thwart virus infection is to inhibit interaction with cellular receptors. It is known that the GP1 subunit of the Lassa glycoprotein complex plays a critical role in receptor recognition. Our results highlight a region within the Lassa virus GP1 protein that interacts with the cellular receptor alpha-dystroglycan. This information may be used for future development of new Lassa virus antivirals. Copyright © 2017 American Society for Microbiology.

Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

PubMed

Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

2013-10-01

Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

Divergent Mitochondrial Antioxidant Activities and Lung Alveolar Architecture in the Lungs of Rats and Mice at High Altitude.

PubMed

Jochmans-Lemoine, Alexandra; Revollo, Susana; Villalpando, Gabriella; Valverde, Ibana; Gonzales, Marcelino; Laouafa, Sofien; Soliz, Jorge; Joseph, Vincent

2018-01-01

Compared with mice, adult rats living at 3,600 m above sea level (SL-La Paz, Bolivia) have high hematocrit, signs of pulmonary hypertension, and low lung volume with reduced alveolar surface area. This phenotype is associated with chronic mountain sickness in humans living at high altitude (HA). We tested the hypothesis that this phenotype is associated with impaired gas exchange and oxidative stress in the lungs. We used rats and mice (3 months old) living at HA (La Paz) and SL (Quebec City, Canada) to measure arterial oxygen saturation under graded levels of hypoxia (by pulse oximetry), the alveolar surface area in lung slices and the activity of pro- (NADPH and xanthine oxidases-NOX and XO) and anti- (superoxide dismutase, and glutathione peroxidase-SOD and GPx) oxidant enzymes in cytosolic and mitochondrial lung protein extracts. HA rats have a lower arterial oxygen saturation and reduced alveolar surface area compared to HA mice and SL rats. Enzymatic activities (NOX, XO, SOD, and GPx) in the cytosol were similar between HA and SL animals, but SOD and GPx activities in the mitochondria were 2-3 times higher in HA vs. SL rats, and only marginally higher in HA mice vs. SL mice. Furthermore, the maximum activity of cytochrome oxidase-c (COX) measured in mitochondrial lung extracts was also 2 times higher in HA rats compared with SL rats, while there was only a small increase in HA mice vs. SL mice. Interestingly, compared with SL controls, alterations in lung morphology are not observed for young rats at HA (15 days after birth), and enzymatic activities are only slightly altered. These results suggest that rats living at HA have a gradual reduction of their alveolar surface area beyond the postnatal period. We can speculate that the elevation of SOD, GPx, and COX activities in the lung mitochondria are not sufficient to compensate for oxidative stress, leading to damage of the lung tissue in rats.

Divergent Mitochondrial Antioxidant Activities and Lung Alveolar Architecture in the Lungs of Rats and Mice at High Altitude

PubMed Central

Jochmans-Lemoine, Alexandra; Revollo, Susana; Villalpando, Gabriella; Valverde, Ibana; Gonzales, Marcelino; Laouafa, Sofien; Soliz, Jorge; Joseph, Vincent

2018-01-01

Compared with mice, adult rats living at 3,600 m above sea level (SL—La Paz, Bolivia) have high hematocrit, signs of pulmonary hypertension, and low lung volume with reduced alveolar surface area. This phenotype is associated with chronic mountain sickness in humans living at high altitude (HA). We tested the hypothesis that this phenotype is associated with impaired gas exchange and oxidative stress in the lungs. We used rats and mice (3 months old) living at HA (La Paz) and SL (Quebec City, Canada) to measure arterial oxygen saturation under graded levels of hypoxia (by pulse oximetry), the alveolar surface area in lung slices and the activity of pro- (NADPH and xanthine oxidases—NOX and XO) and anti- (superoxide dismutase, and glutathione peroxidase—SOD and GPx) oxidant enzymes in cytosolic and mitochondrial lung protein extracts. HA rats have a lower arterial oxygen saturation and reduced alveolar surface area compared to HA mice and SL rats. Enzymatic activities (NOX, XO, SOD, and GPx) in the cytosol were similar between HA and SL animals, but SOD and GPx activities in the mitochondria were 2–3 times higher in HA vs. SL rats, and only marginally higher in HA mice vs. SL mice. Furthermore, the maximum activity of cytochrome oxidase-c (COX) measured in mitochondrial lung extracts was also 2 times higher in HA rats compared with SL rats, while there was only a small increase in HA mice vs. SL mice. Interestingly, compared with SL controls, alterations in lung morphology are not observed for young rats at HA (15 days after birth), and enzymatic activities are only slightly altered. These results suggest that rats living at HA have a gradual reduction of their alveolar surface area beyond the postnatal period. We can speculate that the elevation of SOD, GPx, and COX activities in the lung mitochondria are not sufficient to compensate for oxidative stress, leading to damage of the lung tissue in rats. PMID:29670534

Low-concentration vemurafenib induces the proliferation and invasion of human HaCaT keratinocytes through mitogen-activated protein kinase pathway activation.

PubMed

Roh, Mi Ryung; Kim, Jung Min; Lee, Sang Hee; Jang, Hong Sun; Park, Kyu Hyun; Chung, Kee Yang; Rha, Sun Young

2015-09-01

Cutaneous squamous cell carcinomas and keratoacanthomas commonly occur in patients treated with BRAF inhibitors. We investigated the effect of the BRAF inhibitor vemurafenib on normal immortalized human HaCaT keratinocytes to explore the mechanism of hyperproliferative cutaneous neoplasia associated with the use of BRAF inhibitors. Vemurafenib induced an increase in viable cell number in BRAF wild-type cell lines (SK-MEL-2 and HaCaT) but not in BRAF mutant cell lines (SK-MEL-24 and G361). In HaCaT keratinocytes, a low concentration (2 μmol/L) of vemurafenib increased cell proliferation and activated mitogen-activated protein kinase kinase/extracellular signal-regulated kinase in a CRAF-dependent manner. Invasiveness of HaCaT cells in a Matrigel assay significantly increased upon cultivation of cells with 2 μmol/L vemurafenib for 24 h. Gelatin zymography, reverse transcription polymerase chain reaction and western blot results revealed that 2 μmol/L vemurafenib treatment increased matrix metalloproteinase (MMP)-2 and MMP-9 expressions and activities in HaCaT cells. These results offer additional insight into the complex mechanism of paradoxical mitogen-activated protein kinase signaling involved in hyperproliferative cutaneous neoplasias that arise after BRAF inhibition and suggest a possible role for MMP in tumor progression and invasion. © 2015 Japanese Dermatological Association.

Preliminary investigation of phosphorus adsorption onto two types of iron oxide-organic matter complexes.

PubMed

Yan, Jinlong; Jiang, Tao; Yao, Ying; Lu, Song; Wang, Qilei; Wei, Shiqiang

2016-04-01

Iron oxide (FeO) coated by natural organic matter (NOM) is ubiquitous. The associations of minerals with organic matter (OM) significantly changes their surface properties and reactivity, and thus affect the environmental fate of pollutants, including nutrients (e.g., phosphorus (P)). In this study, ferrihydrite/goethite-humic acid (FH/GE-HA) complexes were prepared and their adsorption characteristics on P at various pH and ionic strength were investigated. The results indicated that the FeO-OM complexes showed a decreased P adsorption capacity in comparison with bare FeO. The maximum adsorption capacity (Qmax) decreased in the order of FH (22.17 mg/g)>FH-HA (5.43 mg/g)>GE (4.67 mg/g)>GE-HA (3.27 mg/g). After coating with HA, the amorphous FH-HA complex still showed higher P adsorption than the crystalline GE-HA complex. The decreased P adsorption observed might be attributed to changes of the FeO surface charges caused by OM association. The dependence of P adsorption on the specific surface area of adsorbents suggests that the FeO component in the complexes is still the main contributor for the adsorption surfaces. The P adsorptions on FeO-HA complexes decreased with increasing initial pH or decreasing initial ionic strength. A strong dependence of P adsorption on ionic strength and pH may demonstrate that outer-sphere complexes between the OM component on the surface and P possibly coexist with inner-sphere surface complexes between the FeO component and P. Therefore, previous over-emphasis on the contributions of original minerals to P immobilization possibly over-estimates the P loading capacity of soils, especially in humic-rich areas. Copyright © 2015. Published by Elsevier B.V.

The macroscopic, microscopic and spectrometric effects of various chemotherapeutic agents on the plasma-sprayed hydroxyapatite-coated implant surface.

PubMed

Zablotsky, M; Meffert, R; Mills, O; Burgess, A; Lancaster, D

1992-12-01

The purpose of this research was to determine the nature of the residual hydroxyapatite (HA)-coated implant surface after treatment with various chemotherapeutic modalities, including: citric acid, chlorhexidine gluconate, hydrogen peroxide, tetracycline HCl, stannous fluoride, polymyxin B and a prototype plastic Cavitron tip. Implant surfaces were evaluated macroscopically, microscopically (scanning electron microscopy (SEM)) and spectrometrically (energy-dispersive spectrometry and X-ray diffraction). HA-substrate bond strength and dissolution testing was also performed for surfaces treated with a supersaturated citric acid solution. All treatments left either microscopic residues or a loss of surface roughness when viewed on SEM. A 30- to 60-s application of citric acid left a significantly greater coating thickness than all other treatments, whereas a 3-min application of citric acid removed significantly more HA than untreated controls. Significant changes in Ca/P ratios were seen with most treatments. The clinical significance of this phenomenon is not known. No treatments altered the crystallinity of the residual HA coating. A 1-min application of citric acid did not significantly alter the tensile bond strength of the coating to the substrate. The clinical significance of these findings is not known at present. However, when taken with results from previous studies, it appears that in treating the infected HA-coated implant surface, a 30- to 60-s application of citric acid (pH 1) may be beneficial in detoxifying the HA coating prior to regenerative procedures. Further in vitro and in vivo studies are necessary to evaluate the biological response to citric acid when used to detoxify the infected implant surface.

Hyaluronic acid-fabricated nanogold delivery of the inhibitor of apoptosis protein-2 siRNAs inhibits benzo[a]pyrene-induced oncogenic properties of lung cancer A549 cells

NASA Astrophysics Data System (ADS)

Lin, Chung-Ming; Kao, Wei-Chien; Yeh, Chun-An; Chen, Hui-Jye; Lin, Shinn-Zong; Hsieh, Hsien-Hsu; Sun, Wei-Shen; Chang, Chih-Hsuan; Hung, Huey-Shan

2015-03-01

Benzo[a]pyrene (BaP), a component of cooking oil fumes (COF), promotes lung cancer cell proliferation and survival via the induction of inhibitor of apoptosis protein-2 (IAP-2) proteins. Thus knockdown of IAP-2 would be a promising way to battle against lung cancer caused by COF. Functionalized gold nanoparticle (AuNP) is an effective delivery system for bio-active materials. Here, biocompatible hyaluronic acid (HA) was fabricated into nanoparticles to increase the target specificity by binding to CD44-over-expressed cancer cells. IAP-2-specific small-interfering RNA (siRNAs) or fluorescein isothiocyanate (FITC) were then incorporated into AuNP-HA. Conjugation of IAP-2 siRNA into AuNPs-HA was verified by the UV-vis spectrometer and Fourier transform infrared spectrometer. Further studies showed that AuNP-HA/FITC were effectively taken up by A549 cells through CD44-mediated endocytosis. Incubation of BaP-challenged cells with AuNP-HA-IAP-2 siRNAs silenced the expression of IAP-2, decreased cell proliferation and triggered pronounced cell apoptosis by the decrease in Bcl-2 protein and the increase in Bax protein as well as the active form of caspases-3. The BaP-elicited cell migration and enzymatic activity of the secreted matrix metalloproteinase-2 were also substantially suppressed by treatment with AuNP-HA-IAP-2 siRNAs. These results indicated that IAP-2 siRNAs can be efficiently delivered into A549 cells by functionalized AuNP-HA to repress the IAP-2 expression and BaP-induced oncogenic events, suggesting the potential therapeutic application of IAP-2 siRNA or other siRNA-conjugated AuNP-HA composites to COF-induced lung cancer and other gene-caused diseases in the future.

Surface modification of hydroxyapatite nanoparticles by poly( L-phenylalanine) via ROP of L-phenylalanine N-carboxyanhydride (Pha-NCA)

NASA Astrophysics Data System (ADS)

Dai, Yanfeng; Xu, Min; Wei, Junchao; Zhang, Haobin; Chen, Yiwang

2012-01-01

The surface of hydroxyapatite nanoparticles was modified by poly(L-phenylalanine) via the ring opening polymerization (ROP) of L-phenylalanine N-carboxyanhydride. The preparation procedure was monitored by Fourier transform infrared spectroscopy (FTIR), and the modified hydroxyapatite was characterized by thermal gravimetric analysis (TGA) and X-ray diffraction (XRD). The results showed that the surface grafting amounts of poly(L-phenylalanine) on HA ranging from 20.26% to 38.92% can be achieved by tuning the reaction condition. The XRD patterns demonstrated that the crystalline structure of the modified hydroxyapatite was nearly the same with that of HA, implying that the ROP was an efficient surface modification method. The MTT assay proved that the biocompatibility of modified HA was very good, which showed the potential application of modified HA in bone tissue engineering.

Potential negative effects of anti-histamines on male reproductive function.

PubMed

Mondillo, Carolina; Varela, María Luisa; Abiuso, Adriana María Belén; Vázquez, Ramiro

2018-05-01

Histamine (HA) is a pleiotropic biogenic amine synthesized exclusively by histidine decarboxylase (HDC) in most mammalian tissues. The literature on the role of HA within the male gonad has expanded over the last years, attracting attention to potential unexpected side-effects of anti-histamines on testicular function. In this regard, HA receptors (HRH1, HRH2 and HRH4) have been described in Leydig cells of different species, including human. Via these receptors, HA has been reported to trigger positive or negative interactions with the LH/hCG signaling pathway depending upon its concentration, thereby contributing to the local control of testicular androgen levels. It should then be considered that anti-histamines may affect testicular homeostasis by increasing or decreasing steroid production. Additionally, HRH1 and HRH2 receptors are present in peritubular and germ cells, and HRH2 antagonists have been found to negatively affect peritubular cells and reduce sperm viability. The potential negative impact of anti-histamines on male reproduction becomes even more dramatic if we consider that HA has also been associated with human sexual behavior and penile erection. What is more, although testicular mast cells are the major source of locally produced HA, recent studies have described HDC expression in macrophages, Leydig cells and germ cells, revealing the existence of multiple sources of HA within the testis. Undoubtedly, the more we learn about the testicular histaminergic system, the more opportunities there will be for rational design of drugs aimed at treating HA-related pathologies, with minimum or nule negative impact on fertility. © 2018 Society for Reproduction and Fertility.

Lubricin Surface Modification Improves Tendon Gliding After Tendon Repair in a Canine Model in Vitro

PubMed Central

Taguchi, Manabu; Sun, Yu-Long; Zhao, Chunfeng; Zobitz, Mark E.; Cha, Chung-Ja; Jay, Gregory D.; An, Kai-Nan; Amadio, Peter C.

2011-01-01

This study investigated the effects of lubricin on the gliding of repaired flexor digitorum profundus (FDP) tendons in vitro. Canine FDP tendons were completely lacerated, repaired with a modified Pennington technique, and treated with one of the following solutions: saline, carbodiimide derivatized gelatin/hyaluronic acid (cd-HA-gelatin), carbodiimide derivatized gelatin to which lubricin was added in a second step (cd-gelatin + lubricin), or carbodiimide derivatized gelatin/HA + lubricin (cd-HA-gelatin + lubricin). After treatment, gliding resistance was measured up to 1,000 cycles of simulated flexion/extension motion. The increase in average and peak gliding resistance in cd-HA-gelatin, cd-gelatin + lubricin, and cd-HA-gelatin + lubricin tendons was less than the control tendons after 1,000 cycles (p < 0.05). The increase in average gliding resistance of cd-HA-gelatin + lubricin treated tendons was also less than that of the cd-HA-gelatin treated tendons (p < 0.05). The surfaces of the repaired tendons and associated pulleys were assessed qualitatively with scanning electron microscopy and appeared smooth after 1,000 cycles of tendon motion for the cd-HA-gelatin, cd-gelatin + lubricin, and cd-HA-gelatin + lubricin treated tendons, while that of the saline control appeared roughened. These results suggest that tendon surface modification can improve tendon gliding ability, with a trend suggesting that lubricin fixed on the repaired tendon may provide additional improvement over that provided by HA and gelatin alone. PMID:18683890

Evaluation of nanoparticles of hydroxyapatite and MWCNT’s in scaffolds of poly lactic acid

NASA Astrophysics Data System (ADS)

Román-Doval, R.; Morales-Corona, J.; Olayo, R.; Escamilla-Rivera, V.; Uribe-Ramírez, M.; Ortega-López, M.

2016-12-01

In the tissue engineering, the cytotoxicity test is an important part of the biomaterials performance. This research reports the production and characterization of polylactic acid (PLA)-supported hydroxyapatite (HA) and multiwalled carbon nanotubes (MWCNT) scaffolds as a bone graft material. Samples containing different HA/MWCNT wt% ratios were prepared by electrospinning. The obtained samples displayed valuable characteristics for the cell adhesion because of their porous-spongy bone-like morphology. The Fourier transforms infrared and Raman analyses indicated no chemical interaction of HA and MWCNT with PLA molecules, but they appear to be only embedded into the PLA fibers. As indicated by x-ray diffraction, crystalline HA and MWCNT’s are supported in the amorphous PLA fibers. Under tensile stress, scaffolds display a Young’s Modulus about 86 MPa, whilst the scaffolds resistance increases with the HA-MWCNT’s ratio. However, the MTS in-vitro assays using the hFOB 1.19 (ATCC CRL-11372) cells, for cell exposure time of 24 and 48 h, revealed that viability reduces for HA-MWCNT’s ratio values over 25 wt%. Our results suggest that a maximum HA/MWCNT’s ratio of 19:1 could be acceptable for cell proliferation while maintaining HA at 200 mg.

Phytosome-hyaluronic acid systems for ocular delivery of L-carnosine

PubMed Central

Abdelkader, Hamdy; Longman, Michael R; Alany, Raid G; Pierscionek, Barbara

2016-01-01

This study reports on L-carnosine phytosomes as an alternative for the prodrug N-acetyl-L-carnosine as a novel delivery system to the lens. L-carnosine was loaded into lipid-based phytosomes and hyaluronic acid (HA)-dispersed phytosomes. L-carnosine-phospholipid complexes (PC) of different molar ratios, 1:1 and 1:2, were prepared by the solvent evaporation method. These complexes were characterized with thermal and spectral analyses. PC were dispersed in either phosphate buffered saline pH 7.4 or HA (0.1% w/v) in phosphate buffered saline to form phytosomes PC1:1, PC1:2, and PC1:2 HA, respectively. These phytosomal formulations were studied for size, zeta potential, morphology, contact angle, spreading coefficient, viscosity, ex vivo transcorneal permeation, and cytotoxicity using primary human corneal cells. L-carnosine-phospholipid formed a complex at a 1:2 molar ratio and phytosomes were in the size range of 380–450 nm, polydispersity index of 0.12–0.2. The viscosity of PC1:2 HA increased by 2.4 to 5-fold compared with HA solution and PC 1:2, respectively; significantly lower surface tension, contact angle, and greater spreading ability for phytosomes were also recorded. Ex vivo transcorneal permeation parameters showed significantly controlled corneal permeation of L-carnosine with the novel carrier systems without any significant impact on primary human corneal cell viability. Ex vivo porcine lenses incubated in high sugar media without and with L-carnosine showed concentration-dependent marked inhibition of lens brunescence indicative of the potential for delaying changes that underlie cataractogenesis that may be linked to diabetic processes. PMID:27366062

Gradient coatings of strontium hydroxyapatite/zinc β-tricalcium phosphate as a tool to modulate osteoblast/osteoclast response.

PubMed

Boanini, Elisa; Torricelli, Paola; Sima, Felix; Axente, Emanuel; Fini, Milena; Mihailescu, Ion N; Bigi, Adriana

2018-06-01

The chemistry, structure and morphology of the implant surface have a great influence on the integration of an implant material with bone tissue. In this work, we applied Combinatorial Matrix-Assisted Pulsed Laser Evaporation (C-MAPLE) to deposit gradient thin films with variable compositions of Sr-substituted hydroxyapatite (SrHA) and Zn-substituted β-tricalcium phosphate (ZnTCP) on Titanium substrates. Five samples with different SrHA/ZnTCP composition ratios were fabricated by a single step laser procedure. SrHA was synthesized in aqueous medium, whereas ZnTCP was obtained by reaction at high temperature. Both powders were separately suspended in deionized water, frozen at liquid nitrogen temperature and used as targets for C-MAPLE experiments, which proceed via simultaneous laser vaporization of two distinct material targets. X-ray diffraction, scanning electron microscopy and energy dispersive X-ray spectroscopy analyses confirmed that the coatings contain the same crystalline phases as the as-prepared powder samples, with a homogeneous distribution of the two phosphates along deposited thin films. Human osteoclast precursor 2T-110 and human osteoblast-like cells MG63 were co-cultured on the coatings. The results indicate that osteoblast viability and production of osteocalcin were promoted by the presence of ZnTCP. On the other hand, SrHA inhibited osteoclastogenesis and osteoclast differentiation, as demonstrated by the observed increase of the osteoprotegerin/RANKL ratio and decrease of the number of TRAP-positive multinucleated cells when increasing SrHA amount in the coatings. The results indicate that the possibility to tailor the composition of the coatings provides materials able to modulate bone growth and bone resorption. Copyright © 2018 Elsevier Inc. All rights reserved.

Endothelial Glycocalyx as a Shield Against Diabetic Vascular Complications: Involvement of Hyaluronan and Hyaluronidases.

PubMed

Dogné, Sophie; Flamion, Bruno; Caron, Nathalie

2018-07-01

The endothelial glycocalyx (EG), which covers the apical surface of the endothelial cells and floats into the lumen of the vessels, is a key player in vascular integrity and cardiovascular homeostasis. The EG is composed of PGs (proteoglycans), glycoproteins, glycolipids, and glycosaminoglycans, in particular hyaluronan (HA). HA seems to be implicated in most of the functions described for EG such as creating a space between blood and the endothelium, controlling vessel permeability, restricting leukocyte and platelet adhesion, and allowing an appropriate endothelial response to flow variation through mechanosensing. The amount of HA in the EG may be regulated by HYAL (hyaluronidase) 1, the most active somatic hyaluronidase. HYAL1 seems enriched in endothelial cells through endocytosis from the bloodstream. The role of the other main somatic hyaluronidase, HYAL2, in the EG is uncertain. Damage to the EG, accompanied by shedding of one or more of its components, is an early sign of various pathologies including diabetes mellitus. Shedding increases the blood or plasma concentration of several EG components, such as HA, heparan sulfate, and syndecan. The plasma levels of these molecules can then be used as sensitive markers of EG degradation. This has been shown in type 1 and type 2 diabetic patients. Recent experimental studies suggest that preserving the size and amount of EG HA in the face of diabetic insults could be a useful novel therapeutic strategy to slow diabetic complications. One way to achieve this goal, as suggested by a murine model of HYAL1 deficiency, may be to inhibit the function of HYAL1. The same approach may succeed in other pathological situations involving endothelial dysfunction and EG damage. © 2018 American Heart Association, Inc.

Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model

PubMed Central

Thomas, Reju George; Moon, Myeong Ju; Kim, Jo Heon; Lee, Jae Hyuk; Jeong, Yong Yeon

2015-01-01

Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis. PMID:26714035

Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model.

PubMed

Thomas, Reju George; Moon, Myeong Ju; Kim, Jo Heon; Lee, Jae Hyuk; Jeong, Yong Yeon

2015-01-01

Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis.

Independent effects of the chemical and microstructural surface properties of polymer/ceramic composites on proliferation and osteogenic differentiation of human MSCs.

PubMed

Sun, Lanying; Danoux, Charlène B; Wang, Qibao; Pereira, Daniel; Barata, David; Zhang, Jingwei; LaPointe, Vanessa; Truckenmüller, Roman; Bao, Chongyun; Xu, Xin; Habibovic, Pamela

2016-09-15

Within the general aim of finding affordable and sustainable regenerative solutions for damaged and diseased tissues and organs, significant efforts have been invested in developing synthetic alternatives to natural bone grafts, such as autografts. Calcium phosphate (CaP) ceramics are among widely used synthetic bone graft substitutes, but their mechanical properties and bone regenerative capacity are still outperformed by their natural counterparts. In order to improve the existing synthetic bone graft substitutes, it is imperative to understand the effects of their individual properties on a biological response, and to find a way to combine the desired properties into new, improved functional biomaterials. To this end, we studied the independent effects of the chemical composition and surface microstructure of a poly(lactic acid)/hydroxyapatite (PLA/HA) composite material on the proliferation and osteogenic differentiation of clinically relevant bone marrow-derived human mesenchymal stromal cells (hMSCs). While the molecular weight of the polymer and presence/absence of the ceramic phase were used as the chemical variables, a soft embossing technique was used to pattern the surfaces of all materials with either pits or pillars with identical microscale dimensions. The results indicated that, while cell morphology was affected by both the presence and availability of HA and by the surface microstructure, the effect of the latter parameter on cell proliferation was negligible. The osteogenic differentiation of hMSCs, and in particular the expression of bone morphogenetic protein 2 (BMP-2) and osteopontin (OP) were significantly enhanced when cells were cultured on the composite based on low-molecular-weight PLA, as compared to the high-molecular-weight PLA-based composite and the two pure polymers. The OP expression on the low-molecular-weight PLA-based composite was further enhanced when the surface was patterned with pits. Taken together, within this experimental set up, the individual effect of the chemistry, and in particular of the presence of CaP, was more pronounced than the individual effect of the surface microstructure, although their combined effects were, in some cases, synergistic. The approach presented here opens new routes to study the interactions of biomaterials with the biological environment in greater depths, which can serve as a starting point for developing biomaterials with improved bioactivity. The aim of the this study was to obtain insight into independent effects of the chemical composition and surface microstructure of a poly(lactic acid)/hydroxyapatite (PLA/HA) composite material on the morphology, proliferation and osteogenic differentiation of clinically relevant bone marrow-derived human mesenchymal stromal cells (hMSCs). While the need for synthetic alternatives for natural bone in bone regenerative strategies is rapidly increasing, the clinical performance of synthetic biomaterials needs to be further improved. To do this successfully, we believe that a better understanding of the relationship between a property of a material and a biological response is imperative. This study is a step forward in this direction, and we are therefore convinced that it will be of interest to the readers of Acta Biomaterialia. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Comparison of a low-tech vs. a high-tech method to evaluate surface fire temperatures

Treesearch

Daniel Yaussy; Joanne Rebbeck; Louis Iverson; Todd Hutchinson; Robert Long

2003-01-01

Prescribed surface fires were conducted in late March-early April 2001, at the Raccoon Ecological Management Area (two ~20 ha areas), and the Tar Hollow (~40 ha) and Zaleski (~40 ha) State Forests in thinned and unthinned mixed-oak forests of southeastern Ohio. Fires are being investigated as a silvicultural tool to aid in regenerating oaks, by removing understory...