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Sample records for 3-dimensional scaffold-embedded chondrocytes

  1. Evaluating Osteoarthritic Chondrocytes through a Novel 3-Dimensional In Vitro System for Cartilage Tissue Engineering and Regeneration.

    PubMed

    Li, Hanwei; Davison, Noel; Moroni, Lorenzo; Feng, Felicia; Crist, Joshua; Salter, Erin; Bingham, Clifton O; Elisseeff, Jennifer

    2012-04-01

    To characterize and evaluate osteoarthritic (OA) chondrocytes, in comparison to normal chondrocytes, through a novel 3-dimensional (3-D) culture system, poly(ethylene-glycol) diacrylate (PEGDA). The cytokine interleukin 1β (IL-1β) was also used to simulate an in vitro OA model. Normal and OA chondrocytes were cultured in monolayer and analyzed for changes in cartilage-specific gene expressions due to passage number. Then, cells were encapsulated in PEGDA to evaluate phenotype and matrix production capabilities through the in vitro culture system. Characterization was conducted with polymerase chain reaction (PCR), biochemical analyses, and histological staining. 3-D encapsulated chondrocytes (human and bovine) were also treated with IL-1β to characterize how the cytokine affects gene transcription and extracellular matrix (ECM) content. In 2-dimensional monolayer, anabolic genes were down-regulated significantly in both normal and OA chondrocytes. In 3-D culture, OA chondrocytes demonstrated significantly higher expressions of catabolic genes when compared to normal cells. Differentiation medium resulted in significantly more matrix production than growth medium from OA chondrocytes, indicated through histological staining. In addition, normal chondrocytes responded more significantly to exogenous administration of IL-1β than OA chondrocytes. Temporary initial stimulation of IL-1β to OA chondrocytes resulted in comparable gene expressions to untreated cells after 3 weeks of in vitro culture. Our findings demonstrate the use of OA chondrocytes in tissue engineering and their significance for potential future cartilage regeneration research through their matrix production capabilities and the use of a hydrogel culture system.

  2. Reconstruction of Hyaline Cartilage Deep Layer Properties in 3-Dimensional Cultures of Human Articular Chondrocytes.

    PubMed

    Nanduri, Vibudha; Tattikota, Surendra Mohan; T, Avinash Raj; Sriramagiri, Vijaya Rama Rao; Kantipudi, Suma; Pande, Gopal

    2014-06-01

    Articular cartilage (AC) injuries and malformations are commonly noticed because of trauma or age-related degeneration. Many methods have been adopted for replacing or repairing the damaged tissue. Currently available AC repair methods, in several cases, fail to yield good-quality long-lasting results, perhaps because the reconstructed tissue lacks the cellular and matrix properties seen in hyaline cartilage (HC). To reconstruct HC tissue from 2-dimensional (2D) and 3-dimensional (3D) cultures of AC-derived human chondrocytes that would specifically exhibit the cellular and biochemical properties of the deep layer of HC. Descriptive laboratory study. Two-dimensional cultures of human AC-derived chondrocytes were established in classical medium (CM) and newly defined medium (NDM) and maintained for a period of 6 weeks. These cells were suspended in 2 mm-thick collagen I gels, placed in 24-well culture inserts, and further cultured up to 30 days. Properties of chondrocytes, grown in 2D cultures and the reconstructed 3D cartilage tissue, were studied by optical and scanning electron microscopic techniques, immunohistochemistry, and cartilage-specific gene expression profiling by reverse transcription polymerase chain reaction and were compared with those of the deep layer of native human AC. Two-dimensional chondrocyte cultures grown in NDM, in comparison with those grown in CM, showed more chondrocyte-specific gene activity and matrix properties. The NDM-grown chondrocytes in 3D cultures also showed better reproduction of deep layer properties of HC, as confirmed by microscopic and gene expression analysis. The method used in this study can yield cartilage tissue up to approximately 1.6 cm in diameter and 2 mm in thickness that satisfies the very low cell density and matrix composition properties present in the deep layer of normal HC. This study presents a novel and reproducible method for long-term culture of AC-derived chondrocytes and reconstruction of cartilage

  3. One-Year Clinical and Radiological Results of a Prospective, Investigator-Initiated Trial Examining a Novel, Purely Autologous 3-Dimensional Autologous Chondrocyte Transplantation Product in the Knee

    PubMed Central

    Gerwien, Philip; Helmert, Benjamin; Schattenberg, Torsten; Weckbach, Sabine; Kaszkin-Bettag, Marietta; Lehmann, Lars

    2012-01-01

    Background: The 3-dimensional autologous chondrocyte transplantation (ACT3D) comprises isolation of chondrocytes from cartilage biopsies, cultivation to spheroids, and transplantation into the cartilage defect. Objectives: To evaluate the patients’ general health and functionality and to assess the defect repair after ACT3D with spheroids by MRI and MOCART scoring. Methods: Thirty-seven patients with isolated chondral lesions of the knee underwent ACT3D with spheroids through medial arthrotomy. Patient-administered scores were assessed at baseline (day before transplantation), at 6 weeks, and at 3, 6, and 12 months. MRI and MOCART scoring were performed at 3 and 12 months after ACT3D. Results: Patients were diagnosed with full-thickness patellofemoral (n = 16), femoral condylar (n = 18), or both defect types (n = 3), International Cartilage Repair Society (ICRS) grade 3 or 4, with defect sizes between 1.0 and 12.0 cm2. On average, 59.5 spheroids/cm2 in defect size were transplanted. An overall statistically significant improvement from baseline to 12 months was observed for all assessment scores (Lysholm, International Knee Documentation Committee [IKDC], SF-36, Tegner) combined with a significant reduction in the visual analog scale (VAS) for pain and an advanced defect filling. Subgroup analyses revealed a positive clinical outcome independent on defect size, defect locations, spheroid dosage, age, duration of symptoms, and severity of complaints at baseline. Seven patients experienced in total 8 adverse events, of which knee joint effusion and blocking were assessed as possibly or probably related to ACT3D. Conclusions: The patient-administered assessment scores along with the fast defect filling with ACT3D using spheroids demonstrated an increase in activity level and quality of life after a 1-year follow-up. PMID:26069617

  4. Fabrication and evaluation of a sustained-release chitosan-based scaffold embedded with PLGA microspheres.

    PubMed

    Song, Kedong; Liu, Yingchao; Macedo, Hugo M; Jiang, Lili; Li, Chao; Mei, Guanyu; Liu, Tianqing

    2013-04-01

    Nutrient depletion within three-dimensional (3D) scaffolds is one of the major hurdles in the use of this technology to grow cells for applications in tissue engineering. In order to help in addressing it, we herein propose to use the controlled release of encapsulated nutrients within polymer microspheres into chitosan-based 3D scaffolds, wherein the microspheres are embedded. This method has allowed maintaining a stable concentration of nutrients within the scaffolds over the long term. The polymer microspheres were prepared using multiple emulsions (w/o/w), in which bovine serum albumin (BSA) and poly (lactic-co-glycolic) acid (PLGA) were regarded as the protein pattern and the exoperidium material, respectively. These were then mixed with a chitosan solution in order to form the scaffolds by cryo-desiccation. The release of BSA, entrapped within the embedded microspheres, was monitored with time using a BCA kit. The morphology and structure of the PLGA microspheres containing BSA before and after embedding within the scaffold were observed under a scanning electron microscope (SEM). These had a round shape with diameters in the range of 27-55 μm, whereas the chitosan-based scaffolds had a uniform porous structure with the microspheres uniformly dispersed within their 3D structure and without any morphological change. In addition, the porosity, water absorption and degradation rate at 37 °C in an aqueous environment of 1% chitosan-based scaffolds were (92.99±2.51) %, (89.66±0.66) % and (73.77±3.21) %, respectively. The studies of BSA release from the embedded microspheres have shown a sustained and cumulative tendency with little initial burst, with (20.24±0.83) % of the initial amount released after 168 h (an average rate of 0.12%/h). The protein concentration within the chitosan-based scaffolds after 168 h was found to be (11.44±1.81)×10(-2) mg/mL. This novel chitosan-based scaffold embedded with PLGA microspheres has proven to be a promising technique

  5. Dependence of light attenuation and backscattering on collagen concentration and chondrocyte density in agarose scaffolds.

    PubMed

    Puhakka, P H; Ylärinne, J H; Lammi, M J; Saarakkala, S; Tiitu, V; Kröger, H; Virén, T; Jurvelin, J S; Töyräs, J

    2014-11-07

    Optical coherence tomography (OCT) has been applied for high resolution imaging of articular cartilage. However, the contribution of individual structural elements of cartilage on OCT signal has not been thoroughly studied. We hypothesize that both collagen and chondrocytes, essential structural components of cartilage, act as important light scatterers and that variation in their concentrations can be detected by OCT through changes in backscattering and attenuation. To evaluate this hypothesis, we established a controlled model system using agarose scaffolds embedded with variable collagen concentrations and chondrocyte densities. Using OCT, we measured the backscattering coefficient (µb) and total attenuation coefficient (µt) in these scaffolds. Along our hypothesis, light backscattering and attenuation in agarose were dependent on collagen concentration and chondrocyte density. Significant correlations were found between µt and chondrocyte density (ρ = 0.853, p < 0.001) and between µt and collagen concentration (ρ = 0.694, p < 0.001). µb correlated significantly with chondrocyte density (ρ = 0.504, p < 0.001) but not with collagen concentration (ρ = 0.103, p = 0.422) of the scaffold. Thus, quantitation of light backscattering and, especially, attenuation could be valuable when evaluating the integrity of soft tissues, such as articular cartilage with OCT.

  6. Induction of CD44 Cleavage in Articular Chondrocytes

    PubMed Central

    Takahashi, Nobunori; Knudson, Cheryl B.; Thankamony, Sai; Ariyoshi, Wataru; Mellor, Liliana; Im, Hee-Jeong; Knudson, Warren

    2010-01-01

    Objective The hyaluronan receptor CD44 provides chondrocytes with a mechanism for sensing and responding to changes in the extracellular matrix. The purpose of this study was to document the fragmentation and loss of CD44 and to determine the likely mechanisms involved. Methods A polyclonal anti-CD44 cytotail antibody was generated to detect CD44 fragmentation by Western blot analysis. Chondrocytes were isolated from human or bovine articular cartilage. Primary articular chondrocytes were treated with interleukin-1β (IL-1β), hyaluronan oligosaccharides, or phorbol myristate acetate or were passaged and subcultured in monolayer to induce dedifferentiation. Conditions that altered the capacity of CD44 to transit into lipid rafts, or pharmacologic inhibitors of metalloproteinase or γ-secretase activity were used to define the mechanism of fragmentation of CD44. Results Chondrocytes from osteoarthritic cartilage exhibited CD44 fragmentation as low molecular mass bands, corresponding to the CD44-EXT and CD44-ICD bands. Following dedifferentiation of chondrocytes or treatment of primary chondrocytes with hyaluronan oligosaccharides, IL-1β, or phorbol myristate acetate, CD44 fragmentation was enhanced. Subsequent culture of the dedifferentiated chondrocytes in 3-dimensional alginate beads rescued the chondrocyte phenotype and diminished the fragmentation of CD44. Fragmentation of CD44 in chondrocytes was blocked in the presence of the metalloproteinase inhibitor GM6001 and the γ-secretase inhibitor DAPT. Conclusion CD44 fragmentation, consistent with a signature pattern reported for sequential metalloproteinase/γ-secretase cleavage of CD44, is a common metabolic feature of chondrocytes that have undergone dedifferentiation in vitro and osteoarthritic chondrocytes. Transit of CD44 into lipid rafts may be required for its fragmentation. PMID:20178130

  7. Chondrocyte channel transcriptomics

    PubMed Central

    Lewis, Rebecca; May, Hannah; Mobasheri, Ali; Barrett-Jolley, Richard

    2013-01-01

    To date, a range of ion channels have been identified in chondrocytes using a number of different techniques, predominantly electrophysiological and/or biomolecular; each of these has its advantages and disadvantages. Here we aim to compare and contrast the data available from biophysical and microarray experiments. This letter analyses recent transcriptomics datasets from chondrocytes, accessible from the European Bioinformatics Institute (EBI). We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes. The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions. We discuss whether functional or protein data exist which support the microarray data. A microarray experiment comparing gene expression in osteoarthritis and healthy cartilage is also discussed and we verify the differential expression of 2 of these genes, namely the genes encoding large calcium-activated potassium (BK) and aquaporin channels. PMID:23995703

  8. The Interplay between Chondrocyte Redifferentiation Pellet Size and Oxygen Concentration

    PubMed Central

    Babur, Betul Kul; Ghanavi, Parisa; Levett, Peter; Lott, William B.; Klein, Travis; Cooper-White, Justin J.; Crawford, Ross; Doran, Michael R.

    2013-01-01

    Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×105–5×105 chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×105 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet

  9. [Chondrocytes application in regenerative medicine].

    PubMed

    Dziedzic, Katarzyna; Zalewski, Mateusz; Gadek, Artur; Drukała, Justyna

    2014-01-01

    Cartilage reconstruction is a crucial issue for tissue engineering because of high damage frequency in connection with low regenerative capacity. Microfractures and shaving are the oldest and most commonly used practices. The newest techniques are: Autologous Chondrocyte Implantation, Matrix Associated Chondrocytes Implantation and their derivatives. Dedifferentiation of chondrocytes due to low proliferation rate and phenotype loss makes isolation and in vitro culture of normal human chondrocytes very complex. Therefore, obtaining mesenchymal stem cells from various sources and differentiating them into chondrocytes is another interesting approach.

  10. Teleportation of a 3-dimensional GHZ State

    NASA Astrophysics Data System (ADS)

    Cao, Hai-Jing; Wang, Huai-Sheng; Li, Peng-Fei; Song, He-Shan

    2012-05-01

    The process of teleportation of a completely unknown 3-dimensional GHZ state is considered. Three maximally entangled 3-dimensional Bell states function as quantum channel in the scheme. This teleportation scheme can be directly generalized to teleport an unknown d-dimensional GHZ state.

  11. Chondroregulatory action of prolactin on proliferation and differentiation of mouse chondrogenic ATDC5 cells in 3-dimensional micromass cultures

    SciTech Connect

    Seriwatanachai, Dutmanee; Krishnamra, Nateetip; Charoenphandhu, Narattaphol

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Mouse chondrogenic ATDC5 cells expressed PRL receptor mRNAs and proteins. Black-Right-Pointing-Pointer Low PRL concentration (10 ng/mL) increased chondrocyte viability and differentiation. Black-Right-Pointing-Pointer Higher PRL concentrations ( Greater-Than-Or-Slanted-Equal-To 100 ng/mL) decreased viability and increased apoptosis. -- Abstract: A recent investigation in lactating rats has provided evidence that the lactogenic hormone prolactin (PRL) increases endochondral bone growth and bone elongation, presumably by accelerating apoptosis of hypertrophic chondrocytes in the growth plate and/or subsequent chondrogenic matrix mineralization. Herein, we demonstrated the direct chondroregulatory action of PRL on proliferation, differentiation and apoptosis of chondrocytes in 3-dimensional micromass culture of mouse chondrogenic ATDC5 cell line. The results showed that ATDC5 cells expressed PRL receptor (PRLR) transcripts, and responded typically to PRL by downregulating PRLR expression. Exposure to a low PRL concentration of 10 ng/mL, comparable to the normal levels in male and non-pregnant female rats, increased chondrocyte viability, differentiation, proteoglycan accumulation, and mRNA expression of several chondrogenic differentiation markers, such as Sox9, ALP and Hspg2. In contrast, high PRL concentrations of Greater-Than-Or-Slanted-Equal-To 100 ng/mL, comparable to the levels in pregnancy or lactation, decreased chondrocyte viability by inducing apoptosis, with no effect on chondrogenic marker expression. It could be concluded that chondrocytes directly but differentially responded to non-pregnant and pregnant/lactating levels of PRL, thus suggesting the stimulatory effect of PRL on chondrogenesis in young growing individuals, and supporting the hypothesis of hypertrophic chondrocyte apoptosis in the growth plate of lactating rats.

  12. Human telomerase reverse transcriptase and glucose-regulated protein 78 increase the life span of articular chondrocytes and their repair potential

    PubMed Central

    2012-01-01

    Background Like all mammalian cells, normal adult chondrocytes have a limited replicative life span, which decreases with age. To facilitate the therapeutic use of chondrocytes from older donors, a method is needed to prolong their life span. Methods We transfected chondrocytes with hTERT or GRP78 and cultured them in a 3-dimensional atelocollagen honeycomb-shaped scaffold with a membrane seal. Then, we measured the amount of nuclear DNA and glycosaminoglycans (GAGs) and the expression level of type II collagen as markers of cell proliferation and extracellular matrix formation, respectively, in these cultures. In addition, we allografted this tissue-engineered cartilage into osteochondral defects in old rabbits to assess their repair activity in vivo. Results Our results showed different degrees of differentiation in terms of GAG content between chondrocytes from old and young rabbits. Chondrocytes that were cotransfected with hTERT and GRP78 showed higher cellular proliferation and expression of type II collagen than those of nontransfected chondrocytes, regardless of the age of the cartilage donor. In addition, the in vitro growth rates of hTERT- or GRP78-transfected chondrocytes were higher than those of nontransfected chondrocytes, regardless of donor age. In vivo, the tissue-engineered cartilage implants exhibited strong repairing activity, maintained a chondrocyte-specific phenotype, and produced extracellular matrix components. Conclusions Focal gene delivery to aged articular chondrocytes exhibited strong repairing activity and may be therapeutically useful for articular cartilage regeneration. PMID:22472071

  13. 3-Dimensional Topographic Models for the Classroom

    NASA Technical Reports Server (NTRS)

    Keller, J. W.; Roark, J. H.; Sakimoto, S. E. H.; Stockman, S.; Frey, H. V.

    2003-01-01

    We have recently undertaken a program to develop educational tools using 3-dimensional solid models of digital elevation data acquired by the Mars Orbital Laser Altimeter (MOLA) for Mars as well as a variety of sources for elevation data of the Earth. This work is made possible by the use of rapid prototyping technology to construct solid 3-Dimensional models of science data. We recently acquired rapid prototyping machine that builds 3-dimensional models in extruded plastic. While the machine was acquired to assist in the design and development of scientific instruments and hardware, it is also fully capable of producing models of spacecraft remote sensing data. We have demonstrated this by using Mars Orbiter Laser Altimeter (MOLA) topographic data and Earth based topographic data to produce extruded plastic topographic models which are visually appealing and instantly engage those who handle them.

  14. 3-Dimensional Topographic Models for the Classroom

    NASA Technical Reports Server (NTRS)

    Keller, J. W.; Roark, J. H.; Sakimoto, S. E. H.; Stockman, S.; Frey, H. V.

    2003-01-01

    We have recently undertaken a program to develop educational tools using 3-dimensional solid models of digital elevation data acquired by the Mars Orbital Laser Altimeter (MOLA) for Mars as well as a variety of sources for elevation data of the Earth. This work is made possible by the use of rapid prototyping technology to construct solid 3-Dimensional models of science data. We recently acquired rapid prototyping machine that builds 3-dimensional models in extruded plastic. While the machine was acquired to assist in the design and development of scientific instruments and hardware, it is also fully capable of producing models of spacecraft remote sensing data. We have demonstrated this by using Mars Orbiter Laser Altimeter (MOLA) topographic data and Earth based topographic data to produce extruded plastic topographic models which are visually appealing and instantly engage those who handle them.

  15. 3-dimensional imaging at nanometer resolutions

    DOEpatents

    Werner, James H.; Goodwin, Peter M.; Shreve, Andrew P.

    2010-03-09

    An apparatus and method for enabling precise, 3-dimensional, photoactivation localization microscopy (PALM) using selective, two-photon activation of fluorophores in a single z-slice of a sample in cooperation with time-gated imaging for reducing the background radiation from other image planes to levels suitable for single-molecule detection and spatial location, are described.

  16. 3-dimensional fabrication of soft energy harvesters

    NASA Astrophysics Data System (ADS)

    McKay, Thomas; Walters, Peter; Rossiter, Jonathan; O'Brien, Benjamin; Anderson, Iain

    2013-04-01

    Dielectric elastomer generators (DEG) provide an opportunity to harvest energy from low frequency and aperiodic sources. Because DEG are soft, deformable, high energy density generators, they can be coupled to complex structures such as the human body to harvest excess mechanical energy. However, DEG are typically constrained by a rigid frame and manufactured in a simple planar structure. This planar arrangement is unlikely to be optimal for harvesting from compliant and/or complex structures. In this paper we present a soft generator which is fabricated into a 3 Dimensional geometry. This capability will enable the 3-dimensional structure of a dielectric elastomer to be customised to the energy source, allowing efficient and/or non-invasive coupling. This paper demonstrates our first 3 dimensional generator which includes a diaphragm with a soft elastomer frame. When the generator was connected to a self-priming circuit and cyclically inflated, energy was accumulated in the system, demonstrated by an increased voltage. Our 3D generator promises a bright future for dielectric elastomers that will be customised for integration with complex and soft structures. In addition to customisable geometries, the 3D printing process may lend itself to fabricating large arrays of small generator units and for fabricating truly soft generators with excellent impedance matching to biological tissue. Thus comfortable, wearable energy harvesters are one step closer to reality.

  17. Hydroelectric structures studies using 3-dimensional methods

    SciTech Connect

    Harrell, T.R.; Jones, G.V.; Toner, C.K. )

    1989-01-01

    Deterioration and degradation of aged, hydroelectric project structures can significantly affect the operation and safety of a project. In many cases, hydroelectric headworks (in particular) have complicated geometrical configurations, loading patterns and hence, stress conditions. An accurate study of such structures can be performed using 3-dimensional computer models. 3-D computer models can be used for both stability evaluation and for finite element stress analysis. Computer aided engineering processes facilitate the use of 3-D methods in both pre-processing and post-processing of data. Two actual project examples are used to emphasize the authors' points.

  18. 3-dimensional bioprinting for tissue engineering applications.

    PubMed

    Gu, Bon Kang; Choi, Dong Jin; Park, Sang Jun; Kim, Min Sup; Kang, Chang Mo; Kim, Chun-Ho

    2016-01-01

    The 3-dimensional (3D) printing technologies, referred to as additive manufacturing (AM) or rapid prototyping (RP), have acquired reputation over the past few years for art, architectural modeling, lightweight machines, and tissue engineering applications. Among these applications, tissue engineering field using 3D printing has attracted the attention from many researchers. 3D bioprinting has an advantage in the manufacture of a scaffold for tissue engineering applications, because of rapid-fabrication, high-precision, and customized-production, etc. In this review, we will introduce the principles and the current state of the 3D bioprinting methods. Focusing on some of studies that are being current application for biomedical and tissue engineering fields using printed 3D scaffolds.

  19. PGA-associated heterotopic chondrocyte cocultures: implications of nasoseptal and auricular chondrocytes in articular cartilage repair.

    PubMed

    El Sayed, K; Marzahn, U; John, T; Hoyer, M; Zreiqat, H; Witthuhn, A; Kohl, B; Haisch, A; Schulze-Tanzil, G

    2013-01-01

    The availability of autologous articular chondrocytes remains a limiting issue in matrix assisted autologous chondrocyte transplantation. Non-articular heterotopic chondrocytes could be an alternative autologous cell source. The aims of this study were to establish heterotopic chondrocyte cocultures to analyze cell-cell compatibilities and to characterize the chondrogenic potential of nasoseptal chondrocytes compared to articular chondrocytes. Primary porcine and human nasoseptal and articular chondrocytes were investigated for extracellular cartilage matrix (ECM) expression in a monolayer culture. 3D polyglycolic acid- (PGA) associated porcine heterotopic mono- and cocultures were assessed for cell vitality, types II, I, and total collagen-, and proteoglycan content. The type II collagen, lubricin, and Sox9 gene expressions were significantly higher in articular compared with nasoseptal monolayer chondrocytes, while type IX collagen expression was lower in articular chondrocytes. Only β1-integrin gene expression was significantly inferior in humans but not in porcine nasoseptal compared with articular chondrocytes, indicating species-dependent differences. Heterotopic chondrocytes in PGA cultures revealed high vitality with proteoglycan-rich hyaline-like ECM production. Similar amounts of type II collagen deposition and type II/I collagen ratios were found in heterotopic chondrocytes cultured on PGA compared to articular chondrocytes. Quantitative analyses revealed a time-dependent increase in total collagen and proteoglycan content, whereby the differences between heterotopic and articular chondrocyte cultures were not significant. Nasoseptal and auricular chondrocytes monocultured in PGA or cocultured with articular chondrocytes revealed a comparable high chondrogenic potential in a tissue engineering setting, which created the opportunity to test them in vivo for articular cartilage repair. Copyright © 2011 John Wiley & Sons, Ltd.

  20. Labial morphology: a 3-dimensional anthropometric study.

    PubMed

    Ferrario, Virgilio F; Rosati, Riccardo; Peretta, Redento; Dellavia, Claudia; Sforza, Chiarella

    2009-09-01

    To develop a noninvasive 3-dimensional method to evaluate labial morphology and to assess gender-related differences in healthy young adults. Dental and lip impressions of 11 men and 10 women aged 21 to 34 years, with sound, full, permanent dentition were obtained. The models were digitized and 3-dimensional virtual reproductions obtained. The labial thickness, vermilion area, and volume of the upper and lower lips were measured from the digital reconstructions. The male and female data were compared using Student's t test. The mean lip thickness was significantly larger (P = .02) in men (14.3 mm) than in women (12.3 mm). The lower lip was thicker than the upper lip. The vermilion width was larger in men (75 mm) than in women (70 mm), and no differences were found for vermilion height (10 mm). In the upper lip, the height/width ratio was significantly larger in women (14.1%) than in men (12.3%). The vermilion surface area was slightly larger in men than in women (upper lip area: women, 467 mm(2); men, 501 mm(2); lower lip area: women, 491 mm(2); men, 569 mm(2)). The labial volume was significantly larger in men (upper lip, 2,390 mm(3); lower lip, 2,902 mm(3)) than in women (upper lip, 1,743 mm(3); lower lip, 1,764 mm(3); P = .021). The upper/lower lip area and volume ratios were similar in the 2 genders. Overall, men had larger lips than women. The inferior lip height/width ratio was similar in both genders, and men had a relatively thinner upper lip than women.

  1. Doublecortin is expressed in articular chondrocytes.

    PubMed

    Zhang, Yi; Ryan, James A; Di Cesare, Paul E; Liu, Judy; Walsh, Christopher A; You, Zongbing

    2007-11-23

    Articular cartilage and cartilage in the embryonic cartilaginous anlagen and growth plates are both hyaline cartilages. In this study, we found that doublecortin (DCX) was expressed in articular chondrocytes but not in chondrocytes from the cartilaginous anlagen or growth plates. DCX was expressed by the cells in the chondrogenous layers but not intermediate layer of joint interzone. Furthermore, the synovium and cruciate ligaments were DCX-negative. DCX-positive chondrocytes were very rare in tissue engineered cartilage derived from in vitro pellet culture of rat chondrosarcoma, ATDC5, and C3H10T1/2 cells. However, the new hyaline cartilage formed in rabbit knee defect contained mostly DCX-positive chondrocytes. Our results demonstrate that DCX can be used as a marker to distinguish articular chondrocytes from other chondrocytes and to evaluate the quality of tissue engineered or regenerated cartilage in terms of their "articular" or "non-articular" nature.

  2. Classification of (n+3)-dimensional metric n-Lie algebras

    SciTech Connect

    Geng Qiaozhi; Ren Mingming; Chen Zhiqi

    2010-10-15

    In this paper, we focus on (n+3)-dimensional metric n-Lie algebras. To begin with, we give some properties on (n+3)-dimensional n-Lie algebras. Then based on the properties, we obtain the classification of (n+3)-dimensional metric n-Lie algebras.

  3. Induction of SHP2 deficiency in chondrocytes causes severe scoliosis and kyphosis in mice.

    PubMed

    Kim, Harry K W; Aruwajoye, Olumide; Sucato, Daniel; Richards, B Stephens; Feng, Gen-Sheng; Chen, Di; King, Philip D; Kamiya, Nobuhiro

    2013-10-01

    Genetic engineering techniques were used to develop an animal model of juvenile scoliosis during a postnatal skeletal-growth stage. To investigate the effect of targeted SHP2 (Src homology-2) deficiency in chondrocytes on the development of scoliosis during a juvenile growth stage in mice. Juvenile idiopathic scoliosis can lead to progressive severe spinal deformity. The pathophysiology and molecular mechanisms responsible for the deformity are unknown. Here, we investigated the role of SHP2 deficiency in chondrocytes as a potential cause of juvenile scoliosis. Genetically engineered mice with inducible deletion of SHP2 in chondrocytes were generated. The SHP2 function in chondrocytes was inactivated during a juvenile growth stage from the mouse age of 4 weeks. Radiographical, micro-computed tomographic, and histological assessments were used to analyze spinal changes. When SHP2 deficiency was induced during the juvenile stage, a progressive kyphoscoliotic deformity (thoracic lordosis and thoracolumbar kyphoscoliosis) developed within 2 weeks of the initiation of SHP2 deficiency. The 3-dimensional micro-computed tomography analysis confirmed the kyphoscoliotic deformity with a rotational deformity of the spine and osteophyte formation. The histological analysis revealed disorganization of the vertebral growth plate cartilage. Interestingly, when SHP2 was disrupted during the adolescent to adult stages, no spinal deformity developed. SHP2 plays an important role in normal spine development during skeletal maturation. Chondrocyte-specific deletion of SHP2 at a juvenile stage produced a kyphoscoliotic deformity. This new mouse model will be useful for future investigations of the role of SHP2 deficiency in chondrocytes as a mechanism leading to the development of juvenile scoliosis. N/A.

  4. Simvastatin inhibits CD44 fragmentation in chondrocytes.

    PubMed

    Terabe, Kenya; Takahashi, Nobunori; Takemoto, Toki; Knudson, Warren; Ishiguro, Naoki; Kojima, Toshihisa

    2016-08-15

    In human osteoarthritic chondrocytes, the hyaluronan receptor CD44 undergoes proteolytic cleavage at the cell surface. CD44 cleavage is thought to require transit of CD44 into cholesterol-rich lipid rafts. The purpose of this study was to investigate whether statins exert a protective effect on articular chondrocytes due to diminution of cholesterol. Three model systems of chondrocytes were examined including human HCS-2/8 chondrosarcoma cells, human osteoarthritic chondrocytes and normal bovine articular chondrocytes. Treatment with IL-1β + Oncostatin M resulted in a substantial increase in CD44 fragmentation in each of the three chondrocyte models. Pre-incubation with simvastatin prior to treatment with IL-1β + Oncostatin M decreased the level of CD44 fragmentation, decreased the proportion of CD44 that transits into the lipid raft fractions, decreased ADAM10 activity and diminished the interaction between CD44 and ADAM10. In HCS-2/8 cells and bovine articular chondrocytes, fragmentation of CD44 was blocked by the knockdown of ADAM10. Inhibition of CD44 fragmentation by simvastatin also resulted in improved retention of pericellular matrix. Addition of cholesterol and farnesyl-pyrophosphate reversed the protective effects of simvastatin. Thus, the addition of simvastatin exerts positive effects on chondrocytes including reduced CD44 fragmentation and enhanced the retention of pericellular matrix. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Infants’ Representations of 3-Dimensional Occluded Objects

    PubMed Central

    Woods, Rebecca J.; Wilcox, Teresa; Armstrong, Jennifer; Alexander, Gerianne

    2012-01-01

    Infants’ ability to represent objects has received significant attention from the developmental research community. With the advent of eye-tracking technology, detailed analysis of infants’ looking patterns during object occlusion have revealed much about the nature of infants’ representations. The current study continues this research by analyzing infants’ looking patterns in a novel manner and by comparing infants’ looking at a simple display in which a single 3-dimensional (3-D) object moves along a continuous trajectory to a more complex display in which two 3-D objects undergo trajectories that are interrupted behind an occluder. Six-month-old infants saw an occlusion sequence in which a ball moved along a linear path, disappeared behind a rectangular screen, and then a ball (ball-ball event) or a box (ball-box event) emerged at the other edge. An eye-tracking system recorded infants’ eye-movements during the event sequence. Results from examination of infants’ attention to the occluder indicate that during the occlusion interval infants looked longer to the side of the occluder behind which the moving occluded object was located, shifting gaze from one side of the occluder to the other as the object(s) moved behind the screen. Furthermore, when events included two objects, infants attended to the spatiotemporal coordinates of the objects longer than when a single object was involved. These results provide clear evidence that infants’ visual tracking is different in response to a one-object display than to a two-object display. Furthermore, this finding suggests that infants may require more focused attention to the hidden position of objects in more complex multiple-object displays and provides additional evidence that infants represent the spatial location of moving occluded objects. PMID:20926138

  6. Cardiothoracic Applications of 3-dimensional Printing.

    PubMed

    Giannopoulos, Andreas A; Steigner, Michael L; George, Elizabeth; Barile, Maria; Hunsaker, Andetta R; Rybicki, Frank J; Mitsouras, Dimitris

    2016-09-01

    Medical 3-dimensional (3D) printing is emerging as a clinically relevant imaging tool in directing preoperative and intraoperative planning in many surgical specialties and will therefore likely lead to interdisciplinary collaboration between engineers, radiologists, and surgeons. Data from standard imaging modalities such as computed tomography, magnetic resonance imaging, echocardiography, and rotational angiography can be used to fabricate life-sized models of human anatomy and pathology, as well as patient-specific implants and surgical guides. Cardiovascular 3D-printed models can improve diagnosis and allow for advanced preoperative planning. The majority of applications reported involve congenital heart diseases and valvular and great vessels pathologies. Printed models are suitable for planning both surgical and minimally invasive procedures. Added value has been reported toward improving outcomes, minimizing perioperative risk, and developing new procedures such as transcatheter mitral valve replacements. Similarly, thoracic surgeons are using 3D printing to assess invasion of vital structures by tumors and to assist in diagnosis and treatment of upper and lower airway diseases. Anatomic models enable surgeons to assimilate information more quickly than image review, choose the optimal surgical approach, and achieve surgery in a shorter time. Patient-specific 3D-printed implants are beginning to appear and may have significant impact on cosmetic and life-saving procedures in the future. In summary, cardiothoracic 3D printing is rapidly evolving and may be a potential game-changer for surgeons. The imager who is equipped with the tools to apply this new imaging science to cardiothoracic care is thus ideally positioned to innovate in this new emerging imaging modality.

  7. Organisation of the chondrocyte cytoskeleton and its response to changing mechanical conditions in organ culture

    PubMed Central

    DURRANT, L. A.; ARCHER, C. W.; BENJAMIN, M.; RALPHS, J. R.

    1999-01-01

    Articular cartilage undergoes cycles of compressive loading during joint movement, leading to its cyclical deformation and recovery. This loading is essential for chondrocytes to perform their normal function of maintenance of the extracellular matrix. Various lines of evidence suggest the involvement of the cytoskeleton in load sensing and response. The purpose of the present study is to describe the 3-dimensional (3D) architecture of the cytoskeleton of chondrocytes within their extracellular matrix, and to examine cytoskeletal responses to experimentally varied mechanical conditions. Uniformly sized explants of articular cartilage were dissected from adult rat femoral heads. Some were immediately frozen, cryosectioned and labelled for filamentous actin using phalloidin, and for the focal contact component vinculin or for vimentin by indirect immunofluorescence. Sections were examined by confocal microscopy and 3D modelling. Actin occurred in all chondrocytes, appearing as bright foci at the cell surface linked to an irregular network beneath the surface. Cell surface foci colocalised with vinculin, suggesting the presence of focal contacts between the chondrocyte and its pericellular matrix. Vimentin label occurred mainly in cells of the deep zone. It had a complex intracellular distribution, with linked networks of fibres surrounding the nucleus and beneath the plasma membrane. When cartilage explants were placed into organ culture, where in the absence of further treatments cartilage imbibes fluid from the culture medium and swells, cytoskeletal changes were observed. After 1 h in culture the vimentin cytoskeleton was disassembled, leading to diffuse labelling of cells. After a further hour in culture filamentous vimentin label reappeared in deep zone chondrocytes, and then over the next 48 h became more widespread in cells of the explants. Actin distribution was unaffected by culture. Further experiments were performed to test the effects of load on the

  8. Articular chondrocyte metabolism and osteoarthritis

    SciTech Connect

    Leipold, H.R.

    1989-01-01

    The three main objectives of this study were: (1) to determine if depletion of proteoglycans from the cartilage matrix that occurs during osteoarthritis causes a measurable increase of cartilage proteoglycan components in the synovial fluid and sera, (2) to observe what effect intracellular cAMP has on the expression of matrix components by chondrocytes, and (3) to determine if freshly isolated chondrocytes contain detectable levels of mRNA for fibronectin. Canine serum keratan sulfate and hyaluronate were measured to determine if there was an elevation of these serum glycosaminoglycans in a canine model of osteoarthritis. A single intra-articular injection of chymopapain into a shoulder joint increased serum keratan sulfate 10 fold and hyaluronate less than 2 fold in 24 hours. Keratan sulfate concentrations in synovial fluids of dogs about one year old were unrelated to the presence of spontaneous cartilage degeneration in the joints. High keratan sulfate in synovial fluids correlated with higher keratan sulfate in serum. The mean keratan sulfate concentration in sera of older dogs with osteoarthritis was 37% higher than disease-free controls, but the difference between the groups was not statistically significant. Treatment of chondrocytes with 0.5 millimolar (mM) dibutyryl cAMP (DBcAMP) caused the cells to adopt a more rounded morphology. There was no difference between the amount of proteins synthesized by cultures treated with DBcAMP and controls. The amount of fibronectin (FN) in the media of DBcAMP treated cultures detected by an ELISA was specifically reduced, and the amount of {sup 35}S-FN purified by gelatin affinity chromatography decreased. Moreover, the percentage of FN containing the extra domain. A sequence was reduced. Concomitant with the decrease in FN there was an increase in the concentration of keratan sulfate.

  9. Comparison of marker gene expression in chondrocytes from patients receiving autologous chondrocyte transplantation versus osteoarthritis patients

    PubMed Central

    Stoop, Reinout; Albrecht, Dirk; Gaissmaier, Christoph; Fritz, Jürgen; Felka, Tino; Rudert, Maximilian; Aicher, Wilhelm K

    2007-01-01

    Currently, autologous chondrocyte transplantation (ACT) is used to treat traumatic cartilage damage or osteochondrosis dissecans, but not degenerative arthritis. Since substantial refinements in the isolation, expansion and transplantation of chondrocytes have been made in recent years, the treatment of early stage osteoarthritic lesions using ACT might now be feasible. In this study, we determined the gene expression patterns of osteoarthritic (OA) chondrocytes ex vivo after primary culture and subculture and compared these with healthy chondrocytes ex vivo and with articular chondrocytes expanded for treatment of patients by ACT. Gene expression profiles were determined using quantitative RT-PCR for type I, II and X collagen, aggrecan, IL-1β and activin-like kinase-1. Furthermore, we tested the capability of osteoarthritic chondrocytes to generate hyaline-like cartilage by implanting chondrocyte-seeded collagen scaffolds into immunodeficient (SCID) mice. OA chondrocytes ex vivo showed highly elevated levels of IL-1β mRNA, but type I and II collagen levels were comparable to those of healthy chondrocytes. After primary culture, IL-1β levels decreased to baseline levels, while the type II and type I collagen mRNA levels matched those found in chondrocytes used for ACT. OA chondrocytes generated type II collagen and proteoglycan-rich cartilage transplants in SCID mice. We conclude that after expansion under suitable conditions, the cartilage of OA patients contains cells that are not significantly different from those from healthy donors prepared for ACT. OA chondrocytes are also capable of producing a cartilage-like tissue in the in vivo SCID mouse model. Thus, such chondrocytes seem to fulfil the prerequisites for use in ACT treatment. PMID:17596264

  10. Comparison of marker gene expression in chondrocytes from patients receiving autologous chondrocyte transplantation versus osteoarthritis patients.

    PubMed

    Stoop, Reinout; Albrecht, Dirk; Gaissmaier, Christoph; Fritz, Jürgen; Felka, Tino; Rudert, Maximilian; Aicher, Wilhelm K

    2007-01-01

    Currently, autologous chondrocyte transplantation (ACT) is used to treat traumatic cartilage damage or osteochondrosis dissecans, but not degenerative arthritis. Since substantial refinements in the isolation, expansion and transplantation of chondrocytes have been made in recent years, the treatment of early stage osteoarthritic lesions using ACT might now be feasible. In this study, we determined the gene expression patterns of osteoarthritic (OA) chondrocytes ex vivo after primary culture and subculture and compared these with healthy chondrocytes ex vivo and with articular chondrocytes expanded for treatment of patients by ACT. Gene expression profiles were determined using quantitative RT-PCR for type I, II and X collagen, aggrecan, IL-1beta and activin-like kinase-1. Furthermore, we tested the capability of osteoarthritic chondrocytes to generate hyaline-like cartilage by implanting chondrocyte-seeded collagen scaffolds into immunodeficient (SCID) mice. OA chondrocytes ex vivo showed highly elevated levels of IL-1beta mRNA, but type I and II collagen levels were comparable to those of healthy chondrocytes. After primary culture, IL-1beta levels decreased to baseline levels, while the type II and type I collagen mRNA levels matched those found in chondrocytes used for ACT. OA chondrocytes generated type II collagen and proteoglycan-rich cartilage transplants in SCID mice. We conclude that after expansion under suitable conditions, the cartilage of OA patients contains cells that are not significantly different from those from healthy donors prepared for ACT. OA chondrocytes are also capable of producing a cartilage-like tissue in the in vivo SCID mouse model. Thus, such chondrocytes seem to fulfil the prerequisites for use in ACT treatment.

  11. Morphological, genetic and phenotypic comparison between human articular chondrocytes and cultured chondrocytes.

    PubMed

    Mata-Miranda, Mónica Maribel; Martinez-Martinez, Claudia María; Noriega-Gonzalez, Jesús Emmanuel; Paredes-Gonzalez, Luis Enrique; Vázquez-Zapién, Gustavo Jesús

    2016-08-01

    Articular cartilage is an avascular and aneural tissue with limited capacity for regeneration. On large articular lesions, it is recommended to use regenerative medicine strategies, like autologous chondrocyte implantation. There is a concern about morphological changes that chondrocytes suffer once they have been isolated and cultured. Due to the fact that there is little evidence that compares articular cartilage chondrocytes with cultured chondrocytes, in this research we proposed to obtain chondrocytes from human articular cartilage, compare them with themselves once they have been cultured and characterize them through genetic, phenotypic and morphological analysis. Knee articular cartilage samples of 10 mm were obtained, and each sample was divided into two fragments; a portion was used to determine gene expression, and from the other portion, chondrocytes were obtained by enzymatic disaggregation, in order to be cultured and expanded in vitro. Subsequently, morphological, genetic and phenotypic characteristics were compared between in situ (articular cartilage) and cultured chondrocytes. Obtained cultured chondrocytes were rounded in shape, possessing a large nucleus with condensed chromatin and a clear cytoplasm; histological appearance was quite similar to typical chondrocyte. The expression levels of COL2A1 and COL10A1 genes were higher in cultured chondrocytes than in situ chondrocytes; moreover, the expression of COL1A1 was almost undetectable on cultured chondrocytes; likewise, COL2 and SOX9 proteins were detected by immunofluorescence. We concluded that chondrocytes derived from adult human cartilage cultured for 21 days do not tend to dedifferentiate, maintaining their capacity to produce matrix and also retaining their synthesis capacity and morphology.

  12. Incorporating 3-dimensional models in online articles.

    PubMed

    Cevidanes, Lucia H S; Ruellas, Antonio C O; Jomier, Julien; Nguyen, Tung; Pieper, Steve; Budin, Francois; Styner, Martin; Paniagua, Beatriz

    2015-05-01

    The aims of this article are to introduce the capability to view and interact with 3-dimensional (3D) surface models in online publications, and to describe how to prepare surface models for such online 3D visualizations. Three-dimensional image analysis methods include image acquisition, construction of surface models, registration in a common coordinate system, visualization of overlays, and quantification of changes. Cone-beam computed tomography scans were acquired as volumetric images that can be visualized as 3D projected images or used to construct polygonal meshes or surfaces of specific anatomic structures of interest. The anatomic structures of interest in the scans can be labeled with color (3D volumetric label maps), and then the scans are registered in a common coordinate system using a target region as the reference. The registered 3D volumetric label maps can be saved in .obj, .ply, .stl, or .vtk file formats and used for overlays, quantification of differences in each of the 3 planes of space, or color-coded graphic displays of 3D surface distances. All registered 3D surface models in this study were saved in .vtk file format and loaded in the Elsevier 3D viewer. In this study, we describe possible ways to visualize the surface models constructed from cone-beam computed tomography images using 2D and 3D figures. The 3D surface models are available in the article's online version for viewing and downloading using the reader's software of choice. These 3D graphic displays are represented in the print version as 2D snapshots. Overlays and color-coded distance maps can be displayed using the reader's software of choice, allowing graphic assessment of the location and direction of changes or morphologic differences relative to the structure of reference. The interpretation of 3D overlays and quantitative color-coded maps requires basic knowledge of 3D image analysis. When submitting manuscripts, authors can now upload 3D models that will allow readers to

  13. Incorporating 3-dimensional models in online articles

    PubMed Central

    Cevidanes, Lucia H. S.; Ruellasa, Antonio C. O.; Jomier, Julien; Nguyen, Tung; Pieper, Steve; Budin, Francois; Styner, Martin; Paniagua, Beatriz

    2015-01-01

    Introduction The aims of this article were to introduce the capability to view and interact with 3-dimensional (3D) surface models in online publications, and to describe how to prepare surface models for such online 3D visualizations. Methods Three-dimensional image analysis methods include image acquisition, construction of surface models, registration in a common coordinate system, visualization of overlays, and quantification of changes. Cone-beam computed tomography scans were acquired as volumetric images that can be visualized as 3D projected images or used to construct polygonal meshes or surfaces of specific anatomic structures of interest. The anatomic structures of interest in the scans can be labeled with color (3D volumetric label maps), and then the scans are registered in a common coordinate system using a target region as the reference. The registered 3D volumetric label maps can be saved in .obj, .ply, .stl, or .vtk file formats and used for overlays, quantification of differences in each of the 3 planes of space, or color-coded graphic displays of 3D surface distances. Results All registered 3D surface models in this study were saved in .vtk file format and loaded in the Elsevier 3D viewer. In this study, we describe possible ways to visualize the surface models constructed from cone-beam computed tomography images using 2D and 3D figures. The 3D surface models are available in the article’s online version for viewing and downloading using the reader’s software of choice. These 3D graphic displays are represented in the print version as 2D snapshots. Overlays and color-coded distance maps can be displayed using the reader’s software of choice, allowing graphic assessment of the location and direction of changes or morphologic differences relative to the structure of reference. The interpretation of 3D overlays and quantitative color-coded maps requires basic knowledge of 3D image analysis. Conclusions When submitting manuscripts, authors can

  14. Design of biphasic polymeric 3-dimensional fiber deposited scaffolds for cartilage tissue engineering applications.

    PubMed

    Moroni, L; Hendriks, J A A; Schotel, R; de Wijn, J R; van Blitterswijk, C A

    2007-02-01

    This report describes a novel system to create rapid prototyped 3-dimensional (3D) fibrous scaffolds with a shell-core fiber architecture in which the core polymer supplies the mechanical properties and the shell polymer acts as a coating providing the desired physicochemical surface properties. Poly[(ethylene oxide) terephthalate-co-poly(butylene) terephthalate] (PEOT/PBT) 3D fiber deposited (3DF) scaffolds were fabricated and examined for articular cartilage tissue regeneration. The shell polymer contained a higher molecular weight of the initial poly(ethylene glycol) (PEG) segments used in the copolymerization and a higher weight percentage of the PEOT domains compared with the core polymer. The 3DF scaffolds entirely produced with the shell or with the core polymers were also considered. After 3 weeks of culture, scaffolds were homogeneously filled with cartilage tissue, as assessed by scanning electron microscopy. Although comparable amounts of entrapped chondrocytes and of extracellular matrix formation were found for all analyzed scaffolds, chondrocytes maintained their rounded shape and aggregated during the culture period on shell-core 3DF scaffolds, suggesting a proper cell differentiation into articular cartilage. This finding was also observed in the 3DF scaffolds fabricated with the shell composition only. In contrast, cells spread and attached on scaffolds made simply with the core polymer, implying a lower degree of differentiation into articular cartilaginous tissue. Furthermore, the shell-core scaffolds displayed an improved dynamic stiffness as a result of a "prestress" action of the shell polymer on the core one. In addition, the dynamic stiffness of the constructs increased compared with the stiffness of the bare scaffolds before culture. These findings suggest that shell-core 3DF PEOT/PBT scaffolds with desired mechanical and surface properties are a promising solution for improved cartilage tissue engineering.

  15. Posttraumatic Chondrocyte Apoptosis in the Murine Xiphoid.

    PubMed

    Davis, Christopher G; Eisner, Eric; McGlynn, Margaret; Shelton, John M; Richardson, James; Borrelli, Joseph; Chen, Christopher C T

    2013-10-01

    To demonstrate posttraumatic chondrocyte apoptosis in the murine xiphoid after a crush-type injury and to ultimately determine the pathway (i.e., intrinsic or extrinsic) by which chondrocytes undergo apoptosis in response to mechanical injury. The xiphoids of adult female wild-type mice were injured with the use of a modified Kelly clamp. Postinjury xiphoid cartilage was analyzed via 3 well-described independent means of assessing apoptosis in chondrocytes: hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and activated caspase-3 staining. Injured specimens contained many chondrocytes with evidence of apoptosis, which is characterized by cell shrinkage, chromatin condensation, nuclear fragmentation, and the liberation of apoptotic bodies. There was a statistically significant increase in the number of chondrocytes undergoing apoptosis in the injured specimens as compared with the uninjured specimens. Chondrocytes can be stimulated to undergo apoptosis as a result of mechanical injury. These experiments involving predominantly cartilaginous murine xiphoid in vivo establish a baseline for future investigations that employ the genetic and therapeutic modulation of chondrocyte apoptosis in response to mechanical injury.

  16. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis

    PubMed Central

    Hwang, Hyun Sook; Kim, Hyun Ah

    2015-01-01

    Apoptosis is a highly-regulated, active process of cell death involved in development, homeostasis and aging. Dysregulation of apoptosis leads to pathological states, such as cancer, developmental anomalies and degenerative diseases. Osteoarthritis (OA), the most common chronic joint disease in the elderly population, is characterized by progressive destruction of articular cartilage, resulting in significant disability. Because articular cartilage depends solely on its resident cells, the chondrocytes, for the maintenance of extracellular matrix, the compromising of chondrocyte function and survival would lead to the failure of the articular cartilage. The role of subchondral bone in the maintenance of proper cartilage matrix has been suggested as well, and it has been proposed that both articular cartilage and subchondral bone interact with each other in the maintenance of articular integrity and physiology. Some investigators include both articular cartilage and subchondral bone as targets for repairing joint degeneration. In late-stage OA, the cartilage becomes hypocellular, often accompanied by lacunar emptying, which has been considered as evidence that chondrocyte death is a central feature in OA progression. Apoptosis clearly occurs in osteoarthritic cartilage; however, the relative contribution of chondrocyte apoptosis in the pathogenesis of OA is difficult to evaluate, and contradictory reports exist on the rate of apoptotic chondrocytes in osteoarthritic cartilage. It is not clear whether chondrocyte apoptosis is the inducer of cartilage degeneration or a byproduct of cartilage destruction. Chondrocyte death and matrix loss may form a vicious cycle, with the progression of one aggravating the other, and the literature reveals that there is a definite correlation between the degree of cartilage damage and chondrocyte apoptosis. Because current treatments for OA act only on symptoms and do not prevent or cure OA, chondrocyte apoptosis would be a valid

  17. [Chondrocyte mecanobiology. Application in cartilage tissue engineering].

    PubMed

    Stoltz, Jean François; Netter, Patrick; Huselstein, Céline; de Isla, Natalia; Wei Yang, Jing; Muller, Sylvaine

    2005-11-01

    Cartilage is a hydrated connective tissue that withstands and distributes mechanical forces within joints. Chondrocytes utilize mechanical signals to maintain cartilaginous tissue homeostasis. They regulate their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). Some mechanotransduction mechanisms are known, while many others no doubt remain to be discovered. Various aspects of chondrocyte mechanobiology have been applied to tissue engineering, with the creation of replacement tissue in vitro from bioresorbable or non-bioresorbable scaffolds and harvested cells. The tissues are maintained in a near-physiologic mechanical and biochemical environment. This paper is an overview of both chondrocyte mechanobiology and cartilage tissue engineering

  18. Autologous Chondrocytes and Next-Generation Matrix-Based Autologous Chondrocyte Implantation.

    PubMed

    Hinckel, Betina B; Gomoll, Andreas H

    2017-07-01

    Focal chondral defects of the knee are common and can significantly impair quality of life. The autologous chondrocyte implantation technique has evolved over the past 20 years; the newest third-generation technique is matrix-induced autologous chondrocyte implantation. Physical examination is important to characterize location and source of pain and identify associated injuries. Imaging studies allow characterization of the lesions, identification of associated lesions, and alignment. Conservative measures should be exhausted before proceeding with surgical treatment. Steps of surgical treatment are diagnostic arthroscopy and biopsy, chondrocyte culture, and chondrocyte implantation. The techniques and their outcomes are discussed in this article. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Chaotic Advection in a Bounded 3-Dimensional Potential Flow

    NASA Astrophysics Data System (ADS)

    Metcalfe, Guy; Smith, Lachlan; Lester, Daniel

    2012-11-01

    3-dimensional potential, or Darcy flows, are central to understanding and designing laminar transport in porous media; however, chaotic advection in 3-dimensional, volume-preserving flows is still not well understood. We show results of advecting passive scalars in a transient 3-dimensional potential flow that consists of a steady dipole flow and periodic reorientation. Even for the most symmetric reorientation protocol, neither of the two invarients of the motion are conserved; however, one invarient is closely shadowed by a surface of revolution constructed from particle paths of the steady flow, creating in practice an adiabatic surface. A consequence is that chaotic regions cover 3-dimensional space, though tubular regular regions are still transport barriers. This appears to be a new mechanism generating 3-dimensional chaotic orbits. These results contast with the experimental and theoretical results for chaotic scalar transport in 2-dimensional Darcy flows. Wiggins, J. Fluid Mech. 654 (2010).

  20. ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes

    SciTech Connect

    Matsumoto, Emi; Furumatsu, Takayuki; Kanazawa, Tomoko; Tamura, Masanori; Ozaki, Toshifumi

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer ROCK inhibitor stimulates chondrogenic gene expression of articular chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor prevents the dedifferentiation of monolayer-cultured chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor enhances the redifferentiation of cultured chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor is useful for preparation of un-dedifferentiated chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor may be a useful reagent for chondrocyte-based regeneration therapy. -- Abstract: Chondrocytes lose their chondrocytic phenotypes in vitro. The Rho family GTPase ROCK, involved in organizing the actin cytoskeleton, modulates the differentiation status of chondrocytic cells. However, the optimum method to prepare a large number of un-dedifferentiated chondrocytes is still unclear. In this study, we investigated the effect of ROCK inhibitor (ROCKi) on the chondrogenic property of monolayer-cultured articular chondrocytes. Human articular chondrocytes were subcultured in the presence or absence of ROCKi (Y-27632). The expression of chondrocytic marker genes such as SOX9 and COL2A1 was assessed by quantitative real-time PCR analysis. Cellular morphology and viability were evaluated. Chondrogenic redifferentiation potential was examined by a pellet culture procedure. The expression level of SOX9 and COL2A1 was higher in ROCKi-treated chondrocytes than in untreated cells. Chondrocyte morphology varied from a spreading form to a round shape in a ROCKi-dependent manner. In addition, ROCKi treatment stimulated the proliferation of chondrocytes. The deposition of safranin O-stained proteoglycans and type II collagen was highly detected in chondrogenic pellets derived from ROCKi-pretreated chondrocytes. Our results suggest that ROCKi prevents the dedifferentiation of monolayer-cultured chondrocytes, and may be a useful reagent to maintain chondrocytic phenotypes in vitro for chondrocyte

  1. The usefulness of 3-dimensional endoscope systems in endoscopic surgery.

    PubMed

    Egi, Hiroyuki; Hattori, Minoru; Suzuki, Takahisa; Sawada, Hiroyuki; Kurita, Yuichi; Ohdan, Hideki

    2016-10-01

    The image quality and performance of 3-dimensional video image systems has improved along with improvements in technology. However, objective evaluation on the usefulness of 3-dimensional video image systems is insufficient. Therefore, we decided to investigate the usefulness of 3-dimensional video image systems using the objective endoscopic surgery technology evaluating apparatus that we have developed, the Hiroshima University Endoscopic Surgical Assessment Device (HUESAD). The participants were 28 student volunteers enrolled in Hiroshima University (17 men and 11 women, age: median 22.5, range 20-25), with no one having experienced endoscopic surgery training. Testing was carried out by dividing the subjects into two groups to initially carry out HUESAD with 2-dimensional video imaging (N = 14) and with 3-dimensional video imaging (N = 14). Questionnaires were carried out along with the investigation regarding both 2-dimensional and 3-dimensional video imaging. The task was carried out for approximately 15 min regarding both 2-dimensional and 3-dimensional video imaging. Lastly, the Mental Rotation Test, which is a standard space perception ability test, was used to evaluate the space perception ability. No difference was observed in the nauseous and uncomfortable feeling of practitioners between the two groups. Regarding smoothness, no difference was observed between 2-dimensional and 3-dimensional video imaging (p = 0.8665). Deviation (space perception ability) and approaching time (accuracy) were significantly lower with 3-dimensional video imaging compared to 2-dimensional video imaging. Moreover, the approaching time (accuracy) significantly improved in 3-dimensional video imaging compared to 2-dimensional video imaging in the group with low space perception ability (p = 0.0085). Objective evaluation using HUESAD and subjective evaluation by questionnaire revealed that endoscopic surgery techniques significantly improved in 3-dimensional video

  2. Chondrocyte-Specific Inhibition of β-Catenin Signaling Leads to Dysplasia of the Caudal Vertebrae in Mice

    PubMed Central

    Shu, Bing; Li, Tian-Fang; Li, Xiao-Feng; Tang, De-Zhi; Zhang, Yejia; Shi, Qi

    2013-01-01

    Study Design. To inhibit β-catenin specifically signaling in chondrocytes Col2-ICAT transgenic mice were generated. Anomalies in caudal vertebrae were detected during embryonic and postnatal stages of Col2-ICAT transgenic mice. Objective. To determine the role of canonical β-catenin signaling in caudal vertebral development. Summary of Background Data. β-catenin signaling plays a critical role in skeletal development. Col2-ICAT transgenic mice were generated to selectively block β-catenin signaling by overexpression of the ICAT gene in chondrocytes. Methods. Tails of E16.5 transgenic embryos and adult Col2-ICAT transgenic mice and their wild-type littermates were collected and analyzed. Skeletal preparation, 3-dimensional micro-computed tomographic and histological analyses were performed to evaluate changes in the structure of caudal vertebrae. Bromodeoxyuridine labeling was performed to evaluate changes in chondrocyte proliferation in caudal vertebrae. Results. Skeletal preparation and 3-dimensional micro-computed tomographic analyses revealed bone deformation and angulated deformities in tail tissue in Col2-ICAT transgenic mice. Histological studies revealed abnormal bone development and dysplastic caudal vertebrae in Col2-ICAT transgenic mice. Inhibition of β-catenin signaling in cartilage resulted in vertebral dysplasia leading to aberrant resegmenting process. Thus, 2 poorly developed sclerotomes failed to fuse to form a complete vertebrae. BrdU labeling revealed a decreased chondrocyte proliferation in both cartilageous templates of transgenic embryos and the growth plate of adult Col2-ICAT transgenic mice. Conclusion. Wnt/β-catenin signaling plays an important role in vertebral development. Inhibition of β-catenin signaling in chondrocytes results in caudal vertebra deformity in mice, which may occur as early as in the stage of sclerotome formation. Level of Evidence: N/A PMID:24026150

  3. Optimization of 3-dimensional imaging of the breast region with 3-dimensional laser scanners.

    PubMed

    Kovacs, Laszlo; Yassouridis, Alexander; Zimmermann, Alexander; Brockmann, Gernot; Wöhnl, Antonia; Blaschke, Matthias; Eder, Maximilian; Schwenzer-Zimmerer, Katja; Rosenberg, Robert; Papadopulos, Nikolaos A; Biemer, Edgar

    2006-03-01

    The anatomic conditions of the female breast require imaging the breast region 3-dimensionally in a normal standing position for quality assurance and for surgery planning or surgery simulation. The goal of this work was to optimize the imaging technology for the mammary region with a 3-dimensional (3D) laser scanner, to evaluate the precision and accuracy of the method, and to allow optimum data reproducibility. Avoiding the influence of biotic factors, such as mobility, we tested the most favorable imaging technology on dummy models for scanner-related factors such as the scanner position in comparison with the torso and the number of scanners and single shots. The influence of different factors of the breast region, such as different breast shapes or premarking of anatomic landmarks, was also first investigated on dummies. The findings from the dummy models were then compared with investigations on test persons, and the accuracy of measurements on the virtual models was compared with a coincidence analysis of the manually measured values. The best precision and accuracy of breast region measurements were achieved when landmarks were marked before taking the shots and when shots at 30 degrees left and 30 degrees right, relative to the sagittal line, were taken with 2 connected scanners mounted with a +10-degree upward angle. However, the precision of the measurements on test persons was significantly lower than those measured on dummies. Our findings show that the correct settings for 3D imaging of the breast region with a laser scanner can achieve an acceptable degree of accuracy and reproducibility.

  4. Chondrocyte Viability After a Simulated Blast Exposure.

    PubMed

    Shaw, K Aaron; Johnson, Peter C; Williams, David; Zumbrun, Steven D; Topolski, Richard; Cameron, Craig D

    2017-07-01

    The effects of blast exposure have gained increasing interest in the military medical community with their continued occurrence on the battlefield. The impact of the direct and indirect energy imparted from blasts to hollow viscera, as well as closed head injuries, have been well studied. However, the injury to articular cartilage has not been investigated, despite previous correlations regarding the development of osteoarthritis. The purpose of this study was to assess the degree of injury to articular chondrocytes after exposure to a simulated blast overpressure wave. Fresh juvenile porcine stifle joints were subjected to a simulated blast overpressure wave utilizing a custom fabricated blast simulator with compressed gases, within the reported range of observed battlefield blasts. Chondrocyte viability was assessed with live/dead staining using ethidium homodimer-2 and calcien acetoxymethylester stain and confocal laser scanning microscopy, calculated as a ratio of dead chondrocytes to live chondrocytes. Testing was performed at time points of 2, 4, and 8 hours after blast exposure and was compared with unblasted control samples. Chondrocyte viability decreased after exposure to a blast overpressure wave when compared with control samples. The amount of death was greater closer to the articular surface and dissipated with increasing tissue depth. Chondrocyte death increased with time after exposure. Chondrocyte death is present after exposure to a simulated blast wave. There is an inverse relationship between chondrocyte viability and the depth from the articular surface. Additional studies are needed to further characterize dose and time effects of blast exposure. Reprint & Copyright © 2017 Association of Military Surgeons of the U.S.

  5. Passaged human chondrocytes accumulate extracellular matrix when induced by bovine chondrocytes.

    PubMed

    Ahmed, Nazish; Taylor, Drew W; Wunder, Jay; Nagy, Andras; Gross, Allan E; Kandel, Rita A

    2010-03-01

    A source of sufficient number of cells is a major limiting factor for cartilage tissue engineering. To circumvent this problem, we developed a co-culture method to induce redifferentiation in bovine articular chondrocytes, which had undergone dedifferentiation following serial passage in monolayer culture. In this study we determine whether human osteoarthritic (OA) and non-diseased passaged dedifferentiated chondrocytes will respond similarly. Human passaged chondrocytes were co-cultured for 4 weeks with primary bovine chondrocytes and their redifferentiation status was determined. Afterwards the cells were cultured either independently or in co-culture with cryopreserved passaged cells for functional analysis. The co-culture of passaged cells with primary chondrocytes resulted in reversion of their phenotype towards articular chondrocytes, as shown by increased gene expression of type II collagen and COMP, decreased type I collagen expression and extracellular matrix formation in vitro. Furthermore, this redifferentiation was stable, as those cells not only formed hyaline-like cartilage tissue when grown on their own but also they could induce redifferentiation of passaged chondrocytes in co-culture. These data suggest that it may be possible to use autologous chondrocytes obtained from osteoarthritic cartilage to form tissue suitable to use for cartilage repair. Copyright (c) 2009 John Wiley & Sons, Ltd.

  6. Effects of mechanical stress on chondrocyte phenotype and chondrocyte extracellular matrix expression

    PubMed Central

    Liu, Qiang; Hu, Xiaoqing; Zhang, Xin; Duan, Xiaoning; Yang, Peng; Zhao, Fengyuan; Ao, Yingfang

    2016-01-01

    Mechanical factors play a key role in regulating the development of cartilage degradation in osteoarthritis. This study aimed to identify the influence of mechanical stress in cartilage and chondrocytes. To explore the effects of mechanical stress on cartilage morphology, we observed cartilages in different regions by histological and microscopic examination. Nanoindentation was performed to assess cartilage biomechanics. To investigate the effects of mechanical stress on chondrocytes, cyclic tensile strain (CTS, 0.5 Hz, 10%) was applied to monolayer cultures of human articular chondrocytes by using Flexcell-5000. We quantified the mechanical properties of chondrocytes by atomic force microscopy. Chondrocytes were stained with Toluidine blue and Alcian blue after exposure to CTS. The expression of extracellular matrix (ECM) molecules was detected by qPCR and immunofluorescence analyses in chondrocytes after CTS. Our results demonstrated distinct morphologies and mechanical properties in different cartilage regions. In conclusion, mechanical stress can affect the chondrocyte phenotype, thereby altering the expression of chondrocyte ECM. PMID:27853300

  7. Collagen VI enhances cartilage tissue generation by stimulating chondrocyte proliferation.

    PubMed

    Smeriglio, Piera; Dhulipala, Lakshmi; Lai, Janice H; Goodman, Stuart B; Dragoo, Jason L; Smith, Robert L; Maloney, William J; Yang, Fan; Bhutani, Nidhi

    2015-02-01

    Regeneration of human cartilage is inherently inefficient. Current cell-based approaches for cartilage repair, including autologous chondrocytes, are limited by the paucity of cells, associated donor site morbidity, and generation of functionally inferior fibrocartilage rather than articular cartilage. Upon investigating the role of collagen VI (Col VI), a major component of the chondrocyte pericellular matrix (PCM), we observe that soluble Col VI stimulates chondrocyte proliferation. Interestingly, both adult and osteoarthritis chondrocytes respond to soluble Col VI in a similar manner. The proliferative effect is, however, strictly due to the soluble Col VI as no proliferation is observed upon exposure of chondrocytes to immobilized Col VI. Upon short Col VI treatment in 2D monolayer culture, chondrocytes maintain high expression of characteristic chondrocyte markers like Col2a1, agc, and Sox9 whereas the expression of the fibrocartilage marker Collagen I (Col I) and of the hypertrophy marker Collagen X (Col X) is minimal. Additionally, Col VI-expanded chondrocytes show a similar potential to untreated chondrocytes in engineering cartilage in 3D biomimetic hydrogel constructs. Our study has, therefore, identified soluble Col VI as a biologic that can be useful for the expansion and utilization of scarce sources of chondrocytes, potentially for autologous chondrocyte implantation. Additionally, our results underscore the importance of further investigating the changes in chondrocyte PCM with age and disease and the subsequent effects on chondrocyte growth and function.

  8. Sodium nitroprusside induces apoptosis of rabbit chondrocytes

    NASA Astrophysics Data System (ADS)

    Liang, Qian; Wang, Xiao-Ping; Chen, Tong-Sheng

    2013-02-01

    Osteoarthritis (OA) is characterized by a slowly progressing degradation of the matrix and destruction of articular cartilage. Apoptosis of chondrocyte is accounted for the mechanism of OA. Nitric oxide (NO), as a stimulus, has been shown to induce chondrocyte apoptosis by activating the matrix metalloproteinases (MMPs), increasing the expression of cyclooxygenase 2 (COX-2) and the level of prostaglandin E2 (PGE2), inhibiting the proteoglycan synthesis and type II collagen expression. In this study, sodium nitroprusside (SNP) was administered to be the NO donor to explore the mechanism of NO-induced apoptosis of rabbit chondrocytes obtained from six weeks old New Zealand rabbits. CCK-8 assay revealed the inhibitory effect of SNP on cell viability. We used flow cytometry (FCM) to assess the form of cell death by Annexin-V/propidium iodide (PI) double staining, and evaluate the change of mitochondrial membrane potential (ΔΨm). We found that the SNP induced chondrocyte apoptosis in a dose- and time-dependent manner and an observable reduction of ΔΨm. In conclusion, our findings indicate that SNP induces apoptosis of rabbit chondrocytes via a mitochondria-mediated pathway.

  9. Cell Death in Chondrocytes, Osteoblasts, and Osteocytes

    PubMed Central

    Komori, Toshihisa

    2016-01-01

    Cell death in skeletal component cells, including chondrocytes, osteoblasts, and osteocytes, plays roles in skeletal development, maintenance, and repair as well as in the pathogenesis of osteoarthritis and osteoporosis. Chondrocyte proliferation, differentiation, and apoptosis are important steps for endochondral ossification. Although the inactivation of P53 and RB is involved in the pathogenesis of osteosarcomas, the deletion of p53 and inactivation of Rb are insufficient to enhance chondrocyte proliferation, indicating the presence of multiple inhibitory mechanisms against sarcomagenesis in chondrocytes. The inflammatory processes induced by mechanical injury and chondrocyte death through the release of danger-associated molecular patterns (DAMPs) are involved in the pathogenesis of posttraumatic osteoarthritis. The overexpression of BCLXL increases bone volume with a normal structure and maintains bone during aging by inhibiting osteoblast apoptosis. p53 inhibits osteoblast proliferation and enhances osteoblast apoptosis, thereby reducing bone formation, but also exerts positive effects on osteoblast differentiation through the Akt–FoxOs pathway. Apoptotic osteocytes release ATP, which induces the receptor activator of nuclear factor κ-B ligand (Rankl) expression and osteoclastogenesis, from pannexin 1 channels. Osteocyte death ultimately results in necrosis; DAMPs are released to the bone surface and promote the production of proinflammatory cytokines, which induce Rankl expression, and osteoclastogenesis is further enhanced. PMID:27929439

  10. Combined 3D and hypoxic culture improves cartilage-specific gene expression in human chondrocytes.

    PubMed

    Foldager, Casper B; Nielsen, Anna B; Munir, Samir; Ulrich-Vinther, Michael; Søballe, Kjeld; Bünger, Cody; Lind, Martin

    2011-04-01

    In vitro expansion of autologous chondrocytes is an essential part of many clinically used cartilage repair treatments. Native chondrocytes reside in a 3-dimensional (3D) network and are exposed to low levels of oxygen. We compared monolayer culture to combined 3D and hypoxic culture using quantitative gene expression analysis. Cartilage biopsies were collected from the intercondylar groove in the distal femur from 12 patients with healthy cartilage. Cells were used for either monolayer or scaffold culture. The scaffolds were clinically available MPEG-PLGA scaffolds (ASEED). After harvesting of cells for baseline investigation, the remainder was divided into 3 groups for incubation in conditions of normoxia (21% oxygen), hypoxia (5% oxygen), or severe hypoxia (1% oxygen). RNA extractions were performed 1, 2, and 6 days after the baseline time point, respectively. Quantitative RT-PCR was performed using assays for RNA encoding collagen types 1 and 2, aggrecan, sox9, ankyrin repeat domain-37, and glyceraldehyde-3-phosphate dehydrogenase relative to 2 hypoxia-stable housekeeping genes. Sox9, aggrecan, and collagen type 2 RNA expression increased with reduced oxygen. On day 6, the expression of collagen type 2 and aggrecan RNA was higher in 3D culture than in monolayer culture. Our findings suggest that there was a combined positive effect of 3D culture and hypoxia on cartilage-specific gene expression. The positive effects of 3D culture alone were not detected until day 6, suggesting that seeding of chondrocytes onto a scaffold for matrix-assisted chondrocyte implantation should be performed earlier than 2 days before implantation.

  11. Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes

    PubMed Central

    Terpstra, Leonieke; Prud'homme, Josée; Arabian, Alice; Takeda, Shu; Karsenty, Gérard; Dedhar, Shoukat; St-Arnaud, René

    2003-01-01

    Chondrocyte proliferation and differentiation requires their attachment to the collagen type II–rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells. PMID:12835312

  12. Oxygen tension affects lubricin expression in chondrocytes.

    PubMed

    Hatta, Taku; Kishimoto, Koshi N; Okuno, Hiroshi; Itoi, Eiji

    2014-10-01

    We assessed the effects of oxygen tension on lubricin expression in bovine chondrocytes and cartilage explants and a role for hypoxia-inducible transcription factor (HIF)-1α in regulating lubricin expression was investigated using a murine chondroprogenitor cell line, ATDC5, and bovine chondrocytes isolated from superficial and middle/deep zones of femoral cartilage. ATDC5 cells and bovine chondrocytes were cultured in micromass under different oxygen tensions (21%, 5%, and 1%). ATDC5 cells and middle/deep zone chondrocytes that initially had low lubricin expression levels were also cultured with or without transforming growth factor (TGF)-β1. Quantitative reverse transcription (RT)-PCR was used to determine lubricin and chondrogenic marker gene mRNA levels and immunohistochemistry was used to assess lubricin protein expression. Explant cartilage plugs cultured under different oxygen tensions were also subjected to immunohistological analysis for lubricin. HIF-1α gene silencing was achieved by electroporatic transfer into ATDC5 cells. A low oxygen tension reduced lubricin gene expression levels in bovine superficial chondrocytes, TGF-β1-treated middle/deep zone chondrocytes, and TGF-β1-treated ATDC5 cells. Lubricin expression in explant cartilage was also suppressed under hypoxia. HIF-1α gene silencing in ATDC5 cells attenuated the lubricin expression response to the oxygen tension. These results corroborate with previous studies that the oxygen tension regulates lubricin gene expression and suggest that HIF-1α plays an important role in this regulation. The normal distribution of lubricin in articular cartilage may be due to the hypoxic oxygen environment of cartilage as it is an avascular tissue. An oxygen tension gradient may be a key factor for engineering cartilage tissue with a layered morphology.

  13. Bovine achondrogenesis: evidence for defective chondrocyte differentiation.

    PubMed

    Horton, W A; Jayo, M J; Leipold, H W; Machado, M A; Campbell, D; Ahmed, S

    1987-01-01

    A survey study of growth cartilage abnormalities in bovine bone dysplasias revealed that a disorder in Holstein cattle called bulldog calf closely resembles human achondrogenesis Type II. Substantial amounts of Type I collagen and other non Type II collagens were detected in the bulldog cartilage which was comprised primarily of extensive vascular canals and cells having the characteristics of hypertrophic and degenerative chondrocytes normally found in the growth plate. It is proposed that chondrocytes throughout the bulldog growth cartilage prematurely differentiate into hypertrophic cells that degenerate and predispose the cartilage to vascular invasion and the formation of cartilage canals. The presence of these canals probably accounts for most of the observed collagen abnormalities.

  14. 3-Dimensional wireless sensor network localization: A review

    NASA Astrophysics Data System (ADS)

    Najib, Yasmeen Nadhirah Ahmad; Daud, Hanita; Aziz, Azrina Abd; Razali, Radzuan

    2016-11-01

    The proliferation of wireless sensor network (WSN) has shifted the focus to 3-Dimensional geometry rather than 2-Dimensional geometry. Since exact location of sensors has been the fundamental issue in wireless sensor network, node localization is essential for any wireless sensor network applications. Most algorithms mainly focus on 2-Dimensional geometry, where the application of this algorithm will decrease the accuracy on 3-Dimensional geometry. The low rank attribute in WSN's node estimation makes the application of nuclear norm minimization as a viable solution for dimensionality reduction problems. This research proposes a novel localization algorithm for 3-Dimensional WSN which is nuclear norm minimization. The node localization is formulated via Euclidean Distance Matrix (EDM) and is then optimized using Nuclear-Norm Minimization (NNM).

  15. Autoimmune regulator, Aire, is a novel regulator of chondrocyte differentiation.

    PubMed

    Si, Yuan; Inoue, Kazuki; Igarashi, Katsuhide; Kanno, Jun; Imai, Yuuki

    2013-08-09

    Chondrocyte differentiation is controlled by various regulators, such as Sox9 and Runx2, but the process is complex. To further understand the precise underlying molecular mechanisms of chondrocyte differentiation, we aimed to identify a novel regulatory factor of chondrocyte differentiation using gene expression profiles of micromass-cultured chondrocytes at different differentiation stages. From the results of microarray analysis, the autoimmune regulator, Aire, was identified as a novel regulator. Aire stable knockdown cells, and primary cultured chondrocytes obtained from Aire(-/-) mice, showed reduced mRNA expression levels of chondrocyte-related genes. Over-expression of Aire induced the early stages of chondrocyte differentiation by facilitating expression of Bmp2. A ChIP assay revealed that Aire was recruited on an Airebinding site (T box) in the Bmp2 promoter region in the early stages of chondrocyte differentiation and histone methylation was modified. These results suggest that Aire can facilitate early chondrocyte differentiation by expression of Bmp2 through altering the histone modification status of the promoter region of Bmp2. Taken together, Aire might play a role as an active regulator of chondrocyte differentiation, which leads to new insights into the regulatory mechanisms of chondrocyte differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Prolactin inhibits the apoptosis of chondrocytes induced by serum starvation.

    PubMed

    Zermeño, C; Guzmán-Morales, J; Macotela, Y; Nava, G; López-Barrera, F; Kouri, J B; Lavalle, C; de la Escalera, G Martínez; Clapp, C

    2006-05-01

    The apoptosis of chondrocytes plays an important role in endochondral bone formation and in cartilage degradation during aging and disease. Prolactin (PRL) is produced in chondrocytes and is known to promote the survival of various cell types. Here we show that articular chondrocytes from rat postpubescent and adult cartilage express the long form of the PRL receptor as revealed by immunohistochemistry of cartilage sections and by RT-PCR and Western blot analyses of the isolated chondrocytes. Furthermore, we demonstrate that PRL inhibits the apoptosis of these same chondrocytes cultured in low-serum. Chondrocyte apoptosis was measured by hypodiploid DNA content determined by flow cytometry and by DNA fragmentation evaluated by the ELISA and the TUNEL methods. The anti-apoptotic effect of PRL was dose-dependent and was prevented by heat inactivation. These data demonstrate that PRL can act as a survival factor for chondrocytes and that it has potential preventive and therapeutic value in arthropathies characterized by cartilage degradation.

  17. Adipose-Derived Stem Cells Cocultured with Chondrocytes Promote the Proliferation of Chondrocytes

    PubMed Central

    2017-01-01

    Articular cartilage injury and defect caused by trauma and chronic osteoarthritis vascularity are very common, while the repair of injured cartilage remains a great challenge due to its limited healing capacity. Stem cell-based tissue engineering provides a promising treatment option for injured articular cartilage because of the cells potential for multiple differentiations. However, its application has been largely limited by stem cell type, number, source, proliferation, and differentiation. We hypothesized that (1) adipose-derived stem cells are ideal seed cells for articular cartilage repair because of their accessibility and abundance and (2) the microenvironment of articular cartilage could induce adipose-derived stem cells (ADSCs) to differentiate into chondrocytes. In order to test our hypotheses, we isolated stem cells from rabbit adipose tissues and cocultured these ADSCs with rabbit articular cartilage chondrocytes. We found that when ADSCs were cocultured with chondrocytes, the proliferation of articular cartilage chondrocytes was promoted, the apoptosis of chondrocytes was inhibited, and the osteogenic and chondrogenic differentiation of ADSCs was enhanced. The study on the mechanism of this coculture system indicated that the role of this coculture system is similar to the function of TGF-β1 in the promotion of chondrocytes. PMID:28133485

  18. Giant crystals inside mitochondria of equine chondrocytes.

    PubMed

    Nürnberger, S; Rentenberger, C; Thiel, K; Schädl, B; Grunwald, I; Ponomarev, I; Marlovits, St; Meyer, Ch; Barnewitz, D

    2017-05-01

    The present study reports for the first time the presence of giant crystals in mitochondria of equine chondrocytes. These structures show dark contrast in TEM images as well as a granular substructure of regularly aligned 1-2 nm small units. Different zone axes of the crystalline structure were analysed by means of Fourier transformation of lattice-resolution TEM images proving the crystalline nature of the structure. Elemental analysis reveals a high content of nitrogen referring to protein. The outer shape of the crystals is geometrical with an up to hexagonal profile in cross sections. It is elongated, spanning a length of several micrometres through the whole cell. In some chondrocytes, several crystals were found, sometimes combined in a single mitochondrion. Crystals were preferentially aligned along the long axis of the cells, thus appearing in the same orientation as the chondrocytes in the tissue. Although no similar structures have been found in the cartilage of any other species investigated, they have been found in cartilage repair tissue formed within a mechanically stimulated equine chondrocyte construct. Crystals were mainly located in superficial regions of cartilage, especially in joint regions of well-developed superficial layers, more often in yearlings than in adult horses. These results indicate that intramitochondrial crystals are related to the high mechanical stress in the horse joint and potentially also to the increased metabolic activity of immature individuals.

  19. Differential Cross Section Kinematics for 3-dimensional Transport Codes

    NASA Technical Reports Server (NTRS)

    Norbury, John W.; Dick, Frank

    2008-01-01

    In support of the development of 3-dimensional transport codes, this paper derives the relevant relativistic particle kinematic theory. Formulas are given for invariant, spectral and angular distributions in both the lab (spacecraft) and center of momentum frames, for collisions involving 2, 3 and n - body final states.

  20. Controlled teleportation of a 3-dimensional bipartite quantum state

    NASA Astrophysics Data System (ADS)

    Cao, Hai-Jing; Chen, Zhong-Hua; Song, He-Shan

    2008-07-01

    A controlled teleportation scheme of an unknown 3-dimensional (3D) two-particle quantum state is proposed, where a 3D Bell state and 3D GHZ state function as the quantum channel. This teleportation scheme can be directly generalized to teleport an unknown d-dimensional bipartite quantum state.

  1. Airway growth and development: a computerized 3-dimensional analysis.

    PubMed

    Schendel, Stephen A; Jacobson, Richard; Khalessi, Sadri

    2012-09-01

    The present study was undertaken to investigate the changes in the normal upper airway during growth and development using 3-dimensional computer analysis from cone-beam computed tomography (CBCT) data to provide a normative reference. The airway size and respiratory mode are known to have a relationship to facial morphology and the development of a malocclusion. The use of CBCT, 3-dimensional imaging, and automated computer analysis in treatment planning allows the upper airway to be precisely evaluated. In the present study, we evaluated the growth of the airway using 3-dimensional analysis and CBCT data from age 6 through old age, in 1300 normal individuals. The airway size and length increase until age 20 at which time a variable period of stability occurs. Next, the airway at first decreases slowly in size and then, after age 40, more rapidly. Normative data are provided in the present study for age groups from 6 to 60 years in relation to the airway total volume, smallest cross-sectional area and vertical length of the airway. This 3-dimensional data of the upper airway will provide a normative reference as an aid in the early understanding of respiration and dentofacial anatomy, which will help in early treatment planning. Copyright © 2012 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

  2. 3-dimensional bundle adjustments in industrial metrology: A comparison

    SciTech Connect

    Gaydosh, M.; LeCocq, C.; Ruland, R.; Wand, B.

    1992-07-01

    Several theodolite measurement systems are available for use in the industrial metrology market. Many of them offer a rigorous 3-dimensional bundle adjustment routine. In this paper several systems in use and available for evaluation purposes at the Stanford Linear Accelerator Center will be tested and their results compared.

  3. [Toxicity of antiseptics on chondrocytes in vitro].

    PubMed

    Schaumburger, J; Beckmann, J; Springorum, H-R; Handel, M; Anders, S; Kalteis, T; Grifka, J; Rath, B

    2010-01-01

    Local antiseptics are commonly used for perioperative skin and wound disinfection and as solutions for joint lavage. Therefore, we examined if an intra-articular use of these antiseptics is possible by using an IN VITRO chondrocyte model. Articular chondrocytes harvested from 7 patients were cultured. After reaching 80% confluency different concentrations (0%, 1%, 10%, 50%, 100%) of polyhexanide, hydrogen peroxide and povidone-iodine were added for 5 minutes. Afterwards, the solution was removed and the chondrocytes were cultured for 24 hours. Subsequently the vitality and proliferation rate (DNA synthesis) were analysed with the WST-1 and BrdU tests. 1% povidone-iodine and 1% hydrogen peroxide solutions significantly (p=0.001) decreased the chondrocyte vitality as compared to our control group. There was no significant difference (p=0.71) after the application of 1% polyhexanide in the vitality ratios. A significant decrease in vitality was also observed after the application of 10% polyhexanide solution (p=0.001). Application of 1% povidone-iodine solution, 1% hydrogen peroxide solution and 10% polyhexanide revealed a decrease in the metabolic cell activity of 80% compared to our control group, whereas the activity was 65% (p=0.026) compared to the control group after application of 1% polyhexanide solution. Our results demonstrate the chondrotoxic effect of the tested antiseptic solutions in clinical used concentrations within short time points. Polyhexanide in a low concentrated solution (1%) was the antiseptic with the lowest influence on the vitality and the DNA synthesis of chondrocytes. Thus, this antiseptic solution seemed to be the best choice for intra-articular application. But overall, our study showed general limitations for the intra-articular use of local antiseptics. Copyright (c) Georg Thieme Verlag KG Stuttgart-New York.

  4. DNA Methylation Profiling in Chondrocyte Dedifferentiation In Vitro.

    PubMed

    Duan, Li; Liang, Yujie; Ma, Bin; Wang, Daming; Liu, Wei; Huang, Jianghong; Xiong, Jianyi; Peng, Liangquan; Chen, Jielin; Zhu, Weimin; Wang, Daping

    2017-07-01

    DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full-scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome-wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5-azacytidine (5-AzaC). This research revealed that the general methylation level of CpG was increased while the COL-1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5-AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL-1A1 promoter was increased after 5-AzaC treatment. The COL-1A1 expression level was increased while that of SOX-9 was decreased during the chondrocyte dedifferentiation. 5-AzaC treatment up-regulated the SOX-9 expression while down-regulated the COL-1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage-specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. J. Cell. Physiol. 232: 1708-1716, 2017. © 2016 Wiley Periodicals, Inc.

  5. Differences between chondrocytes and bone marrow-derived chondrogenic cells.

    PubMed

    Chiang, Hongsen; Hsieh, Chang-Hsun; Lin, Yun-Han; Lin, Shiming; Tsai-Wu, Jyy-Jih; Jiang, Ching-Chuan

    2011-12-01

    Implantation of autologous chondrogenic cells has become the mainstay strategy for repairing articular cartilage defects. Because the availability of autologous chondrocytes is extremely limited, many recent studies have used artificially induced mesenchymal stem cells (iMSCs) as substitutes for chondrocytes. In this study, we analyzed the differences between the iMSCs and chondrocytes, including their molecular biological and mechanical properties. Human bone marrow-derived MSCs were collected and induced to exhibit the chondrogenic phenotype by culturing the pelleted MSCs in a chemically defined culture medium supplemented with transforming growth factor-beta 1. The molecular biological properties of iMSCs and culture-expanded chondrocytes, including their mRNA profiles and surface proteomics, were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry, respectively. The biomechanical properties of iMSCs and native chondrocytes, including their surface topology, adhesion force, and membrane stiffness, were analyzed using atomic force microscopy (AFM). Both iMSCs and chondrocytes presented type II collagen and glycosaminoglycan, whereas only chondrocytes presented type X collagen. Flow cytometric assays showed that the expression of type II collagen and integrin-1 was higher in the chondrocytes than in the iMSCs. AFM revealed that the MSCs, iMSCs, and chondrocytes greatly differed in their shape. The MSCs were spindle shaped and easily distinguishable from the spherical chondrocytes. The iMSCs appeared round and resembled the spherical chondrocytes; however, the iMSCs were flatter with a central hump of condensed mass and a surrounding thin and broad pleat. The mean adhesion force and mean surface stiffness were significantly lower for the iMSCs (4.54 nN and 0.109 N/m, respectively) than for the chondrocytes (6.86 nN and 0.134 N/m, respectively). To conclude, although the iMSCs exhibited the chondrogenic phenotype, they differed

  6. 3-dimensional (3D) fabricated polymer based drug delivery systems.

    PubMed

    Moulton, Simon E; Wallace, Gordon G

    2014-11-10

    Drug delivery from 3-dimensional (3D) structures is a rapidly growing area of research. It is essential to achieve structures wherein drug stability is ensured, the drug loading capacity is appropriate and the desired controlled release profile can be attained. Attention must also be paid to the development of appropriate fabrication machinery that allows 3D drug delivery systems (DDS) to be produced in a simple, reliable and reproducible manner. The range of fabrication methods currently being used to form 3D DDSs include electrospinning (solution and melt), wet-spinning and printing (3-dimensional). The use of these techniques enables production of DDSs from the macro-scale down to the nano-scale. This article reviews progress in these fabrication techniques to form DDSs that possess desirable drug delivery kinetics for a wide range of applications.

  7. Wetting characteristics of 3-dimensional nanostructured fractal surfaces

    NASA Astrophysics Data System (ADS)

    Davis, Ethan; Liu, Ying; Jiang, Lijia; Lu, Yongfeng; Ndao, Sidy

    2017-01-01

    This article reports the fabrication and wetting characteristics of 3-dimensional nanostructured fractal surfaces (3DNFS). Three distinct 3DNFS surfaces, namely cubic, Romanesco broccoli, and sphereflake were fabricated using two-photon direct laser writing. Contact angle measurements were performed on the multiscale fractal surfaces to characterize their wetting properties. Average contact angles ranged from 66.8° for the smooth control surface to 0° for one of the fractal surfaces. The change in wetting behavior was attributed to modification of the interfacial surface properties due to the inclusion of 3-dimensional hierarchical fractal nanostructures. However, this behavior does not exactly obey existing surface wetting models in the literature. Potential applications for these types of surfaces in physical and biological sciences are also discussed.

  8. Cohomological rigidity of manifolds defined by 3-dimensional polytopes

    NASA Astrophysics Data System (ADS)

    Buchstaber, V. M.; Erokhovets, N. Yu.; Masuda, M.; Panov, T. E.; Park, S.

    2017-04-01

    A family of closed manifolds is said to be cohomologically rigid if a cohomology ring isomorphism implies a diffeomorphism for any two manifolds in the family. Cohomological rigidity is established here for large families of 3-dimensional and 6-dimensional manifolds defined by 3-dimensional polytopes. The class \\mathscr{P} of 3-dimensional combinatorial simple polytopes P different from tetrahedra and without facets forming 3- and 4-belts is studied. This class includes mathematical fullerenes, that is, simple 3- polytopes with only 5-gonal and 6-gonal facets. By a theorem of Pogorelov, any polytope in \\mathscr{P} admits in Lobachevsky 3-space a right-angled realisation which is unique up to isometry. Our families of smooth manifolds are associated with polytopes in the class \\mathscr{P}. The first family consists of 3-dimensional small covers of polytopes in \\mathscr{P}, or equivalently, hyperbolic 3-manifolds of Löbell type. The second family consists of 6-dimensional quasitoric manifolds over polytopes in \\mathscr{P}. Our main result is that both families are cohomologically rigid, that is, two manifolds M and M' from either family are diffeomorphic if and only if their cohomology rings are isomorphic. It is also proved that if M and M' are diffeomorphic, then their corresponding polytopes P and P' are combinatorially equivalent. These results are intertwined with classical subjects in geometry and topology such as the combinatorics of 3-polytopes, the Four Colour Theorem, aspherical manifolds, a diffeomorphism classification of 6-manifolds, and invariance of Pontryagin classes. The proofs use techniques of toric topology. Bibliography: 69 titles.

  9. Multimodality 3-Dimensional Image Integration for Congenital Cardiac Catheterization

    PubMed Central

    2014-01-01

    Cardiac catheterization procedures for patients with congenital and structural heart disease are becoming more complex. New imaging strategies involving integration of 3-dimensional images from rotational angiography, magnetic resonance imaging (MRI), computerized tomography (CT), and transesophageal echocardiography (TEE) are employed to facilitate these procedures. We discuss the current use of these new 3D imaging technologies and their advantages and challenges when used to guide complex diagnostic and interventional catheterization procedures in patients with congenital heart disease. PMID:25114757

  10. [Interaction between human chondrocytes and extracellular matrix in vitro: a contribution to autologous chondrocyte transplantation].

    PubMed

    Shakibaei, M; Csaki, C; Rahmanzadeh, M; Putz, R

    2008-05-01

    Autologous chondrocyte transplantation (ACT) has had reasonable success for repairing small articular cartilage defects. A limiting factor for ACT is, however, the in vitro cultivation of chondrocytes because it leads to dedifferentiation. Therefore, the goal of this work was to optimize the monolayer culture of chondrocytes in vitro. Human articular chondrocytes were plated on either collagen type II or untreated surfaces. The cells were evaluated morphologically and with immunoblotting. On collagen type II surfaces, a stable chondrogenic phenotype, expression of beta1-integrin, and a significant activation of phosphorylated intracellular proteins and the adaptor protein Shc could be observed up to day 20 in culture. Treatment with beta1 integrin antibody led to a loss of cell adhesion (82%). The results indicate that on collagen type II, beta1-integrin receptors are activated. Through the activation of Shc, these stimulate the Ras-MAPK pathway, which stabilizes the chondrogenic phenotype. Our results provide a practical and low-cost solution for improved long-term chondrocyte cultivation, thus providing a new perspective for using ACT on larger or arthrotic cartilage defects.

  11. Targeted Deletion of Capn4 in Cells of the Chondrocyte Lineage Impairs Chondrocyte Proliferation and Differentiation▿

    PubMed Central

    Kashiwagi, Aki; Schipani, Ernestina; Fein, Mikaela J.; Greer, Peter A.; Shimada, Masako

    2010-01-01

    Calpains are calcium-dependent intracellular cysteine proteases, which include ubiquitously expressed μ- and m-calpains. Both calpains are heterodimers consisting of a large catalytic subunit and a small regulatory subunit. The calpain small subunit encoded by the gene Capn4 directly binds to the intracellular C-terminal tail of the receptor for the parathyroid hormone (PTH) and PTH-related peptide and modulates cellular functions in cells of the osteoblast lineage in vitro and in vivo. To investigate a physiological role of the calpain small subunit in cells of the chondrocyte lineage, we generated chondrocyte-specific Capn4 knockout mice. Mutant embryos had reduced chondrocyte proliferation and differentiation in embryonic growth plates compared with control littermates. In vitro analysis further revealed that deletion of Capn4 in cells of the chondrocyte lineage correlated with impaired cell cycle progression at the G1/S transition, reduced cyclin D gene transcription, and accumulated cell cycle proteins known as calpain substrates. Moreover, silencing of p27Kip1 rescued an impaired cell growth phenotype in Capn4 knockdown cells, and reintroducing the calpain small subunit partially normalized cell growth and accumulated cyclin D protein levels in a dose-dependent manner. Collectively, our findings suggest that the calpain small subunit is essential for proper chondrocyte functions in embryonic growth plates. PMID:20368361

  12. Chondrocyte hypertrophy in skeletal development, growth, and disease.

    PubMed

    Sun, Margaret Man-Ger; Beier, Frank

    2014-03-01

    Most of our bones form through the process of endochondral ossification, which is tightly regulated by the activity of the cartilage growth plate. Chondrocyte maturation through the various stages of growth plate physiology ultimately results in hypertrophy. Chondrocyte hypertrophy is an essential contributor to longitudinal bone growth, but recent data suggest that these cells also play fundamental roles in signaling to other skeletal cells, thus coordinating endochondral ossification. On the other hand, ectopic hypertrophy of articular chondrocytes has been implicated in the pathogenesis of osteoarthritis. Thus, a better understanding of the processes that control chondrocyte hypertrophy in the growth plate as well as in articular cartilage is required for improved management of both skeletal growth disorders and osteoarthritis. This review summarizes recent findings on the regulation of hypertrophic chondrocyte differentiation, the cellular mechanisms involved in hypertrophy, and the role of chondrocyte hypertrophy in skeletal physiology and pathophysiology.

  13. [Cartilage biopsy for autologous chondrocyte implantation (ACI)].

    PubMed

    Pestka, J M; Salzmann, G M; Südkamp, N P; Niemeyer, P

    2013-06-01

    Autologous chondrocyte implantation (ACI) is an established two-step procedure for the treatment of full-thickness cartilage defects of the knee. Cartilage harvest from the affected knee joint represents the first step of this procedure and is essential for further in vitro expansion of autologous chondrocytes. Nevertheless, the cartilage biopsy process itself is underrepresented in the scientific literature and currently there is only a limited amount of data available addressing this process. Biopsy location as well as the technique itself and instruments used for cartilage collection are not well defined and only little standardisation can be found. The article describes the relevant aspects of the biopsy in the context of ACI with regard to the literature available. Follow-up studies to better define and standardise the cartilage biopsy process are thus required.

  14. Increased chondrocyte death after steroid and local anesthetic combination.

    PubMed

    Farkas, Boglárka; Kvell, Krisztián; Czömpöly, Tamás; Illés, Tamás; Bárdos, Tamás

    2010-11-01

    Hyaline articular cartilage has limited repair and regeneration capacity. Intraarticular administration of glucocorticoid and local anesthetic injections play an important role in the therapy of osteoarthritis. Glucocorticoids and anesthetics reportedly enhance apoptosis in chondrocytes, but effects of the combined use of glucocorticoids and local anesthetics are unknown. We asked whether glucocorticoid and local anesthetic agents combined had any synergistic effects on chondrocyte apoptosis. Cell viability and apoptosis/necrosis assessment of human articular chondrocytes were performed in vitro (chondrocyte cell cultures) and ex vivo (osteochondral specimens) using flow cytometry and TUNEL analysis, respectively. Glucocorticoids and local anesthetics induce apoptosis in chondrocytes at various rates. When used in combination, the percentage of dead chondrocytes was increased in in vitro chondrocyte cell cultures and osteochondral ex vivo specimens. We observed a time-dependent decrease in chondrocyte viability after concurrent steroid and local anesthetic exposure. The combination of glucocorticoids and local anesthetics has an adverse effect on articular chondrocytes, and it raises a question regarding whether concomitant administration should be used in treating osteoarthritis.

  15. Smad4 regulates growth plate matrix production and chondrocyte polarity

    PubMed Central

    Whitaker, Amanda T.; Berthet, Ellora; Cantu, Andrea; Laird, Diana J.

    2017-01-01

    ABSTRACT Smad4 is an intracellular effector of the TGFβ family that has been implicated in Myhre syndrome, a skeletal dysplasia characterized by short stature, brachydactyly and stiff joints. The TGFβ pathway also plays a critical role in the development, organization and proliferation of the growth plate, although the exact mechanisms remain unclear. Skeletal phenotypes in Myhre syndrome overlap with processes regulated by the TGFβ pathway, including organization and proliferation of the growth plate and polarity of the chondrocyte. We used in vitro and in vivo models of Smad4 deficiency in chondrocytes to test the hypothesis that deregulated TGFβ signaling leads to aberrant extracellular matrix production and loss of chondrocyte polarity. Specifically, we evaluated growth plate chondrocyte polarity in tibiae of Col2-Cre+/−;Smad4fl/fl mice and in chondrocyte pellet cultures. In vitro and in vivo, Smad4 deficiency decreased aggrecan expression and increased MMP13 expression. Smad4 deficiency disrupted the balance of cartilage matrix synthesis and degradation, even though the sequential expression of growth plate chondrocyte markers was intact. Chondrocytes in Smad4-deficient growth plates also showed evidence of polarity defects, with impaired proliferation and ability to undergo the characteristic changes in shape, size and orientation as they differentiated from resting to hypertrophic chondrocytes. Therefore, we show that Smad4 controls chondrocyte proliferation, orientation, and hypertrophy and is important in regulating the extracellular matrix composition of the growth plate. PMID:28167493

  16. Inhibition of phosphate-induced apoptosis in resting zone chondrocytes by thrombin peptide 508.

    PubMed

    Zhong, Ming; Carney, Darrell H; Ryaby, James T; Schwartz, Zvi; Boyan, Barbara D

    2009-01-01

    Growth plate chondrocytes are susceptible to apoptosis. Terminally differentiated chondrocytes are deleted via apoptosis, which primes the growth plate to vascular invasion and subsequent bone formation. Whether less differentiated resting zone chondrocytes are subject to the same mechanism that governs the apoptotic pathway of more differentiated growth zone chondrocytes is not known. In our current study, we demonstrated that inorganic phosphate, a key inducer of growth plate chondrocyte apoptosis, also causes apoptosis in resting zone chondrocytes, via a pathway similar to the one in growth zone chondrocytes. Our results demonstrated that the conditions that cause growth plate chondrocyte apoptosis lie in the external environment, instead of the differences in differentiation state.

  17. Cellular Changes of Stem Cells in 3-Dimensional Culture.

    PubMed

    Green, Matthew P; Hou, Bo

    2017-06-12

    During various operations and procedures, such as distraction osteogenesis and orthodontics, skeletal tissues use mechanotransduction. Mechanotransduction is important for maintaining bone health and converting mechanical forces into biochemical signals. We hypothesized that cells put under mechanical stress would adapt and change morphologically and respond with a decrease in cellular proliferation to accommodate the stress differences. These differences will be measured at the molecular and genetic level. We also wanted to test the practicality of an in vitro 3-dimensional gel model system. We implemented a 3-dimensional cell culture model. The sample was composed of isolated mouse mesenchymal prefibroblast bone marrow cells from the femurs and tibias of 6- to 8-week-old wild-type C57BL6 mice. The cells were seeded on fibronectin-coated hydrogels along with fibrin and nodulin growth factors. The variables tested were a no-force model (control) and a force model. The force model required two 0.1-mm suture pins put through one 0.25-cm length of cell-gel matrix. After the experiments were run to completion, the samples were fixed with 4% paraformaldehyde and embedded in paraffin. Serial sections were cut at a thickness of 5 μm along the long axis for the force construct and encompassing the entire circular area of the control construct. Descriptive and bivariate statistics were computed, and the P value was set at 5%. There was a statistically significant difference between the 2 models. The force model had longer and straighter primary cilia, less apoptosis, and an increase in cell proliferation. In addition, the shape of the cells was markedly different after the experiment. The results of the study suggest cells put under tensile stress have the ability to mechanically sense the environment to provide improved adaptation. Our work also confirms the usefulness of the in vitro 3-dimensional gel model system to mimic in vivo applications. Published by Elsevier

  18. [Bile duct reconstruction using 3-dimensional collagen tubes].

    PubMed

    Pérez Alonso, Alejandro José; del Olmo Rivas, Carlos; Machado Romero, Ignacio; Pérez Cabrera, Beatriz; Cañizares Garcia, Francisco Javier; Torne Poyatos, Pablo

    2013-11-01

    In recent years, with widespread laparoscopic cholecystectomy and liver transplantation, complications involving the biliary system are increasing. All current techniques have a high risk of recurrence or high-morbidity. A 3-dimensional collagen bile duct modified with agarose hydrogel was developed to substitute the affected extrahepatic bile duct. It was used in 40 guinea pigs and the histology and physiology was studied at 4 weeks, 3 and 6 months after transplantation. The graft shows to have a high potential in applications to treat hepatobiliary diseases which require surgery. Copyright © 2012 AEC. Published by Elsevier Espana. All rights reserved.

  19. 3-dimensional electronic structures of CaC6

    NASA Astrophysics Data System (ADS)

    Kyung, Wonshik; Kim, Yeongkwan; Han, Garam; Leem, Choonshik; Kim, Junsung; Kim, Yeongwook; Kim, Keunsu; Rotenberg, Eli; Kim, Changyoung; Postech Collaboration; Advanced Light Source Collaboration; Yonsei University Team

    2014-03-01

    There is still remaining issues on origin of superconductivity in graphite intercalation compounds, especially CaC6 because of its relatively high transition temperature than other GICs. There are two competing theories on where the superconductivity occurs in this material; intercalant metal or charge doped graphene layer. To elucidate this issue, it is necessary to confirm existence of intercalant driven band. Therefore, we performed 3 dimensional electronic structure studies with ARPES to find out 3d dispersive intercalant band. However, we could not observe it, instead observed 3d dispersive carbon band. This support the aspect of charge doped graphene superconductivity more than intercalant driving aspect.

  20. The 3-dimensional cellular automata for HIV infection

    NASA Astrophysics Data System (ADS)

    Mo, Youbin; Ren, Bin; Yang, Wencao; Shuai, Jianwei

    2014-04-01

    The HIV infection dynamics is discussed in detail with a 3-dimensional cellular automata model in this paper. The model can reproduce the three-phase development, i.e., the acute period, the asymptotic period and the AIDS period, observed in the HIV-infected patients in a clinic. We show that the 3D HIV model performs a better robustness on the model parameters than the 2D cellular automata. Furthermore, we reveal that the occurrence of a perpetual source to successively generate infectious waves to spread to the whole system drives the model from the asymptotic state to the AIDS state.

  1. Chondrocyte-specific ablation of Osterix leads to impaired endochondral ossification

    SciTech Connect

    Oh, Jung-Hoon; Park, Seung-Yoon; Crombrugghe, Benoit de; Kim, Jung-Eun

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer Conditional ablation of Osterix (Osx) in chondrocytes leads to skeletal defects. Black-Right-Pointing-Pointer Osx regulates chondrocyte differentiation and bone growth in growth plate chondrocytes. Black-Right-Pointing-Pointer Osx has an autonomous function in chondrocytes during endochondral ossification. -- Abstract: Osterix (Osx) is an essential transcription factor required for osteoblast differentiation during both intramembranous and endochondral ossification. Endochondral ossification, a process in which bone formation initiates from a cartilage intermediate, is crucial for skeletal development and growth. Osx is expressed in differentiating chondrocytes as well as osteoblasts during mouse development, but its role in chondrocytes has not been well studied. Here, the in vivo function of Osx in chondrocytes was examined in a chondrocyte-specific Osx conditional knockout model using Col2a1-Cre. Chondrocyte-specific Osx deficiency resulted in a weak and bent skeleton which was evident in newborn by radiographic analysis and skeletal preparation. To further understand the skeletal deformity of the chondrocyte-specific Osx conditional knockout, histological analysis was performed on developing long bones during embryogenesis. Hypertrophic chondrocytes were expanded, the formation of bone trabeculae and marrow cavities was remarkably delayed, and subsequent skeletal growth was reduced. The expression of several chondrocyte differentiation markers was reduced, indicating the impairment of chondrocyte differentiation and endochondral ossification in the chondrocyte-specific Osx conditional knockout. Taken together, Osx regulates chondrocyte differentiation and bone growth in growth plate chondrocytes, suggesting an autonomous function of Osx in chondrocytes during endochondral ossification.

  2. Applications of Chondrocyte-Based Cartilage Engineering: An Overview

    PubMed Central

    Eo, Seong-Hui; Abbas, Qamar; Ahmed, Madiha

    2016-01-01

    Chondrocytes are the exclusive cells residing in cartilage and maintain the functionality of cartilage tissue. Series of biocomponents such as different growth factors, cytokines, and transcriptional factors regulate the mesenchymal stem cells (MSCs) differentiation to chondrocytes. The number of chondrocytes and dedifferentiation are the key limitations in subsequent clinical application of the chondrocytes. Different culture methods are being developed to overcome such issues. Using tissue engineering and cell based approaches, chondrocytes offer prominent therapeutic option specifically in orthopedics for cartilage repair and to treat ailments such as tracheal defects, facial reconstruction, and urinary incontinence. Matrix-assisted autologous chondrocyte transplantation/implantation is an improved version of traditional autologous chondrocyte transplantation (ACT) method. An increasing number of studies show the clinical significance of this technique for the chondral lesions treatment. Literature survey was carried out to address clinical and functional findings by using various ACT procedures. The current study was conducted to study the pharmacological significance and biomedical application of chondrocytes. Furthermore, it is inferred from the present study that long term follow-up studies are required to evaluate the potential of these methods and specific positive outcomes. PMID:27631002

  3. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation.

    PubMed

    Kobayashi, Tatsuya; Lu, Jun; Cobb, Bradley S; Rodda, Stephen J; McMahon, Andrew P; Schipani, Ernestina; Merkenschlager, Matthias; Kronenberg, Henry M

    2008-02-12

    Small noncoding RNAs, microRNAs (miRNAs), bind to messenger RNAs through base pairing to suppress gene expression. Despite accumulating evidence that miRNAs play critical roles in various biological processes across diverse organisms, their roles in mammalian skeletal development have not been demonstrated. Here, we show that Dicer, an essential component for biogenesis of miRNAs, is essential for normal skeletal development. Dicer-null growth plates show a progressive reduction in the proliferating pool of chondrocytes, leading to severe skeletal growth defects and premature death of mice. The reduction of proliferating chondrocytes in Dicer-null growth plates is caused by two distinct mechanisms: decreased chondrocyte proliferation and accelerated differentiation into postmitotic hypertrophic chondrocytes. These defects appear to be caused by mechanisms downstream or independent of the Ihh-PTHrP signaling pathway, a pivotal signaling system that regulates chondrocyte proliferation and differentiation. Microarray analysis of Dicer-null chondrocytes showed limited expression changes in miRNA-target genes, suggesting that, in the majority of cases, chondrocytic miRNAs do not directly regulate target RNA abundance. Our results demonstrate the critical role of the Dicer-dependent pathway in the regulation of chondrocyte proliferation and differentiation during skeletal development.

  4. A theoretical analysis of water transport through chondrocytes.

    PubMed

    Ateshian, G A; Costa, K D; Hung, C T

    2007-01-01

    Because of the avascular nature of adult cartilage, nutrients and waste products are transported to and from the chondrocytes by diffusion and convection through the extracellular matrix. The convective interstitial fluid flow within and around chondrocytes is poorly understood. This theoretical study demonstrates that the incorporation of a semi-permeable membrane when modeling the chondrocyte leads to the following findings: under mechanical loading of an isolated chondrocyte the intracellular fluid pressure is on the order of tens of Pascals and the transmembrane fluid outflow, on the order of picometers per second, takes several days to subside; consequently, the chondrocyte behaves practically as an incompressible solid whenever the loading duration is on the order of minutes or hours. When embedded in its extracellular matrix (ECM), the chondrocyte response is substantially different. Mechanical loading of the tissue leads to a fluid pressure difference between intracellular and extracellular compartments on the order of tens of kilopascals and the transmembrane outflow, on the order of a nanometer per second, subsides in about 1 h. The volume of the chondrocyte decreases concomitantly with that of the ECM. The interstitial fluid flow in the extracellular matrix is directed around the cell, with peak values on the order of tens of nanometers per second. The viscous fluid shear stress acting on the cell surface is several orders of magnitude smaller than the solid matrix shear stresses resulting from the ECM deformation. These results provide new insight toward our understanding of water transport in chondrocytes.

  5. The effect of matrix stiffness on biomechanical properties of chondrocytes.

    PubMed

    Zhang, Quanyou; Yu, Yang; Zhao, Hucheng

    2016-10-01

    The behavior of chondrocytes is regulated by multiple mechanical microenvironmental cues. During development and degenerative disease of articular cartilage, as an external signal, the extracellular matrix stiffness of chondrocytes changes significantly, but whether and how this biophysical cue affects biomechanical properties of chondrocytes remain elusive. In the present study, we designed supporting-biomaterials as  mimics of native pericellular matrix to study the effect of matrix stiffness on chondrocyte morphology and F-actin distribution. Furthermore, the active mechanical behavior of chondrocytes during sensing and responding to different matrix stiffness was quantitatively investigated using atom force microscope technique and theoretical model. Our results indicated that stiffer matrix tends to increase the cell spreading area, the percentage of irregular cell shape distribution and mechanical parameters including elastic modulus (Eelastic), instantaneous modulus (E0), relaxed modulus (ER) and apparent viscosity (μ) of chondrocytes. Knowledge of matrix stiffness-dependent biomechanical behaviors of chondrocytes has important implications for optimizing matrix material and advancing chondrocyte-based applications for functional tissue engineering. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Automated feature extraction for 3-dimensional point clouds

    NASA Astrophysics Data System (ADS)

    Magruder, Lori A.; Leigh, Holly W.; Soderlund, Alexander; Clymer, Bradley; Baer, Jessica; Neuenschwander, Amy L.

    2016-05-01

    Light detection and ranging (LIDAR) technology offers the capability to rapidly capture high-resolution, 3-dimensional surface data with centimeter-level accuracy for a large variety of applications. Due to the foliage-penetrating properties of LIDAR systems, these geospatial data sets can detect ground surfaces beneath trees, enabling the production of highfidelity bare earth elevation models. Precise characterization of the ground surface allows for identification of terrain and non-terrain points within the point cloud, and facilitates further discernment between natural and man-made objects based solely on structural aspects and relative neighboring parameterizations. A framework is presented here for automated extraction of natural and man-made features that does not rely on coincident ortho-imagery or point RGB attributes. The TEXAS (Terrain EXtraction And Segmentation) algorithm is used first to generate a bare earth surface from a lidar survey, which is then used to classify points as terrain or non-terrain. Further classifications are assigned at the point level by leveraging local spatial information. Similarly classed points are then clustered together into regions to identify individual features. Descriptions of the spatial attributes of each region are generated, resulting in the identification of individual tree locations, forest extents, building footprints, and 3-dimensional building shapes, among others. Results of the fully-automated feature extraction algorithm are then compared to ground truth to assess completeness and accuracy of the methodology.

  7. Osteoarthritic articular chondrocytes stimulate autologous T cell responses in vitro.

    PubMed

    Sakata, M; Masuko-Hongo, K; Nakamura, H; Onuma, H; Tsuruha, J I; Aoki, H; Nishioka, K; Kato, T

    2003-01-01

    To clarify the presence of specific T cell immune response to autologous chondrocytes in patients with osteoarthritis (OA). Peripheral blood mononuclear cells obtained from OA or post-traumatic patients were co-cultured with irradiated autologous chondrocytes, and their proliferative response was assessed using 3H-thymidine incorporation. Expression of HLA-class II molecules was also assessed on chondrocytes by immunohistochemistry or flow cytometry. T cell responses to autologous chondrocytes in OA yielded a significantly greater mean stimulation index (6.35 +/- 1.63) compared to controls (1.21 +/- 0.09, p < 0.01). This response was partially blocked by antibodies against HLA class I, class II, CD4 or CD8. Increased expression of HLA-DP, -DQ, and -DR was observed. This study showed the autologous T cell-stimulating property of OA chondrocytes in vitro. The elucidation of the autoimmune responses may contribute to the understanding of immune-mediated mechanisms in OA.

  8. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis

    PubMed Central

    Musumeci, Giuseppe; Castrogiovanni, Paola; Trovato, Francesca Maria; Weinberg, Annelie Martina; Al-Wasiyah, Mohammad K.; Alqahtani, Mohammed H.; Mobasheri, Ali

    2015-01-01

    Cell death with morphological and molecular features of apoptosis has been detected in osteoarthritic (OA) cartilage, which suggests a key role for chondrocyte death/survival in the pathogenesis of OA. Identification of biomarkers of chondrocyte apoptosis may facilitate the development of novel therapies that may eliminate the cause or, at least, slow down the degenerative processes in OA. The aim of this review was to explore the molecular markers and signals that induce chondrocyte apoptosis in OA. A literature search was conducted in PubMed, Scopus, Web of Science and Google Scholar using the keywords chondrocyte death, apoptosis, osteoarthritis, autophagy and biomarker. Several molecules considered to be markers of chondrocyte apoptosis will be discussed in this brief review. Molecular markers and signalling pathways associated with chondroycte apoptosis may turn out to be therapeutic targets in OA and approaches aimed at neutralizing apoptosis-inducing molecules may at least delay the progression of cartilage degeneration in OA. PMID:26334269

  9. Selenium effect on selenoprotein transcriptome in chondrocytes.

    PubMed

    Yan, Jidong; Zheng, Yuewen; Min, Zixin; Ning, Qilan; Lu, Shemin

    2013-04-01

    Selenium is an essential micronutrient and exerts its biological functions predominantly through selenoproteins. Selenium deficiency is associated with cartilage function. This study demonstrated that all 24 selenoprotein transcripts in mouse genome were detectable in ATDC5 chondrocytes except deiodinase 1 (DIO1), DIO2, and selenoprotein V (Sel V), while all 25 selenoprotein transcripts in human genome were detectable in C28/I2 chondrocytes except glutathione peroxidase 6 (GPx6) and DIO1. In addition, gene expression of five selenoproteins (GPx1, Sel H, Sel N, Sel P, and Sel W) was up-regulated and two selenoproteins (SPS2 and Sel O) was down-regulated by sodium selenite (Se) in both ATDC5 and C28/I2 cells. Gene expression of six selenoproteins (TrxR1, Sel I, Sel M, Sel R, Sel S, Sel T) and one selenoprotein (GPx3) was up-regulated by Se in ATDC5 and C28/I2 cells, respectively. Gene expression of one selenoprotein (TrxR2) was down-regulated by Se only in ATDC5 cells. Further transcription inhibition assay showed that both transcriptional and posttranscriptional mechanisms involved in Se-regulated gene expression of GPx1, TrxR1, TrxR2, SPS2, Sel O, and Sel S. However, Se-regulated gene expression of Sel H, Sel I, Sel M, Sel N, Sel P, Sel R, Sel T, and Sel W mainly at posttranscriptional level. Moreover, new protein synthesis inhibition assay indicated that Se-mediated new protein synthesis also played roles in Se-regulated gene expression of GPx1, TrxR1, TrxR2, Sel H, Sel O, Sel P, Sel R, and Sel W. In summary, this study described the selenoprotein transcriptome, Se-regulated selenoproteins and possible mechanisms involved in chondrocytes.

  10. 5-Aza-2'-deoxycytidine acts as a modulator of chondrocyte hypertrophy and maturation in chick caudal region chondrocytes in culture

    PubMed Central

    2016-01-01

    This study was carried out to explore the effect of DNA hypomethylation on chondrocytes phenotype, in particular the effect on chondrocyte hypertrophy, maturation, and apoptosis. Chondrocytes derived from caudal region of day 17 embryonic chick sterna were pretreated with hypomethylating drug 5-aza-2'-deoxycytidine for 48 hours and then maintained in the normal culture medium for up to 14 days. Histological studies showed distinct morphological changes occurred in the pretreated cultures when compared to the control cultures. The pretreated chondrocytes after 7 days in culture became bigger in size and acquired more flattened fibroblastic phenotype as well as a loss of cartilage specific extracellular matrix. Scanning electron microscopy at day 7 showed chondrocytes to have increased in cell volume and at day 14 in culture the extracellular matrix of the pretreated cultures showed regular fibrillar structure heavily embedded with matrix vesicles, which is the characteristic feature of chondrocyte hypertrophy. Transmission electron microscopic studies indicated the terminal fate of the hypertrophic cells in culture. The pretreated chondrocytes grown for 14 days in culture showed two types of cells: dark cells which had condense chromatin in dark patches and dark cytoplasm. The other light chondrocytes appeared to be heavily loaded with endoplasmic reticulum indicative of very active protein and secretory activity; their cytoplasm had large vacuoles and disintegrating cytoplasm. The biosynthetic profile showed that the pretreated cultures were actively synthesizing and secreting type X collagen and alkaline phosphatase as a major biosynthetic product. PMID:27382512

  11. [Chondrocytes - one cell type, different subpopulations : characteristics and behavior of different types of chondrocytes and implications for tissue engineering applications].

    PubMed

    Grad, S; Salzmann, G M

    2009-11-01

    Chondrocytes represent the most important cell source for engineering of cartilaginous tissues. Depending on the tissue type and the localization within the tissue, these cells may behave differently. Numerous studies have been done to compare articular, nasal, auricular, and costal chondrocytes in order to evaluate differences between knee and ankle joint cartilage and to investigate topographical variations within an articular joint. Moreover, the zonal structure of articular cartilage needs to be considered because it leads to phenotypical differences between chondrocytes of the superficial and the deeper zones. Several studies indicate, however, that even differentiated chondrocytes demonstrate a certain plasticity and strive to adapt their phenotypes to a new mechanical and biochemical environment. The aim of this review is to report on similarities and differences of chondrocytes from different tissues, zones, and topographical locations. In particular, an overview of recent results from comparative studies is presented, and possible consequences for the design of tissue engineering models are discussed.

  12. Scientific visualization of 3-dimensional optimized stellarator configurations

    SciTech Connect

    Spong, D.A.

    1998-01-01

    The design techniques and physics analysis of modern stellarator configurations for magnetic fusion research rely heavily on high performance computing and simulation. Stellarators, which are fundamentally 3-dimensional in nature, offer significantly more design flexibility than more symmetric devices such as the tokamak. By varying the outer boundary shape of the plasma, a variety of physics features, such as transport, stability, and heating efficiency can be optimized. Scientific visualization techniques are an important adjunct to this effort as they provide a necessary ergonomic link between the numerical results and the intuition of the human researcher. The authors have developed a variety of visualization techniques for stellarators which both facilitate the design optimization process and allow the physics simulations to be more readily understood.

  13. [3-Dimensional model reconstruction of penis and surrounding tissue].

    PubMed

    Wang, Rui-Heng; Cao, Chuan; Mei, Wen-Ming; Wang, Wen-Xian; Tan, Li-Wen; Li, Shi-Rong

    2012-07-01

    To evaluate the feasibility of 3-Dimensional (3-D) model reconstruction of penis and surrounding structures based on magnetic resonance images, which may provide the model building method for modeling surgery of individual penoplasty. Magnetic resonance (MR) images of penis with different imaging parameters were evaluated. With the surface rendering construction, the 3D virtual model was established by Amira software. The anatomical details imaging is better in T2-weighted fast spin-echo images with 3.0 mm slice thickness. The established model based on the MR images can show the soft-tissue, suspensory ligament of the penis. The suspensory ligament stretches between the pubic symphysis and the corpora cavernosa. The penile roots attach to inferior ramus of pubis. MR imaging provides enough anatomical information for modeling. It can be used for the development of model surgery system of individual penoplasty.

  14. Quantitative 3-dimensional computed tomography analysis of olecranon fractures.

    PubMed

    Lubberts, Bart; Janssen, Stein; Mellema, Jos; Ring, David

    2016-05-01

    Olecranon fractures have variable size of the proximal fragment, patterns of fragmentation, and subluxation of the ulnohumeral joint that might be better understood and categorized on the basis of quantitative 3-dimensional computed tomography analysis. Mayo type I fractures are undisplaced, Mayo type II are displaced and stable, and Mayo type III are displaced and unstable. The last is categorized into anterior and posterior dislocations. The purpose of this study was to further clarify fracture morphology between Mayo type I, II, and III fractures. Three-dimensional models were created for a consecutive series of 78 patients with olecranon fractures that were evaluated with computed tomography. We determined the total number of fracture fragments, the volume and articular surface area of each fracture fragment, and the degree of displacement of the most proximal olecranon fracture fragment. Displaced olecranon fractures were more comminuted than nondisplaced fractures (P = .02). Displaced fractures without ulnohumeral subluxation were smallest in terms of both volume (P < .001) and articular surface involvement (P < .001) of the most proximal olecranon fracture fragment. There was no difference in average displacement of the proximal fragment between displaced fractures with and without ulnohumeral subluxation (P = .74). Anterior olecranon fracture-dislocations created more displaced (P = .04) and smaller proximal fragments than posterior fracture-dislocations (P = .005), with comparable fragmentation on average (P = .60). The ability to quantify volume, articular surface area, displacement, and fragmentation using quantitative 3-dimensional computed tomography should be considered when increased knowledge of fracture morphology and fracture patterns might be useful. Copyright © 2016 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

  15. 3-dimensional analysis of regenerative endodontic treatment outcome.

    PubMed

    EzEldeen, Mostafa; Van Gorp, Gertrude; Van Dessel, Jeroen; Vandermeulen, Dirk; Jacobs, Reinhilde

    2015-03-01

    A growing body of evidence supports the regeneration potential of dental tissues after regenerative endodontic treatment (RET). Nevertheless, a standard method for the evaluation of RET outcome is lacking. The aim of this study was to develop a standardized quantitative method for RET outcome analysis based on cone-beam computed tomographic (CBCT) volumetric measurements. Five human teeth embedded in mandibular bone samples were scanned using both an Accuitomo 170 CBCT machine (Morita, Kyoto, Japan) and a SkyScan 1174 micro-computed tomographic (μCT) system (SkyScan, Antwerp, Belgium). For subsequent clinical application, clinical data and low-dose CBCT scans (preoperatively and follow-up) from 5 immature permanent teeth treated with RET were retrieved. In vitro and clinical 3-dimensional image data sets were imported into a dedicated software tool. Two segmentation steps were applied to extract the teeth of interest from the surrounding tissue (livewire) and to separate tooth hard tissue and root canal space (level set methods). In vitro and clinical volumetric measurements were assessed separately for differences using Wilcoxon matched pairs test. Pearson correlation analysis and Bland-Altman plots were used to evaluate the relation and agreement between the segmented CBCT and μCT volumes. The results showed no statistical differences and strong agreement between CBCT and μCT volumetric measurements. Volumetric comparison of the root hard tissue showed significant hard tissue formation. (The mean volume of newly formed hard tissue was 27.9 [±10.5] mm(3) [P < .05]). Analysis of 3-dimensional data for teeth treated with RET offers valuable insights into the treatment outcome and patterns of hard tissue formation. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  16. Review of 3-Dimensional Printing on Cranial Neurosurgery Simulation Training.

    PubMed

    Vakharia, Vejay N; Vakharia, Nilesh N; Hill, Ciaran S

    2016-04-01

    Shorter working times, reduced operative exposure to complex procedures, and increased subspecialization have resulted in training constraints within most surgical fields. Simulation has been suggested as a possible means of acquiring new surgical skills without exposing patients to the surgeon's operative "learning curve." Here we review the potential impact of 3-dimensional printing on simulation and training within cranial neurosurgery and its implications for the future. In accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines, a comprehensive search of PubMed, OVID MEDLINE, Embase, and the Cochrane Database of Systematic Reviews was performed. In total, 31 studies relating to the use of 3-dimensional (3D) printing within neurosurgery, of which 16 were specifically related to simulation and training, were identified. The main impact of 3D printing on neurosurgical simulation training was within vascular surgery, where patient-specific replication of vascular anatomy and pathologies can aid surgeons in operative planning and clip placement for reconstruction of vascular anatomy. Models containing replicas of brain tumors have also been reconstructed and used for training purposes, with some providing realistic representations of skin, subcutaneous tissue, bone, dura, normal brain, and tumor tissue. 3D printing provides a unique means of directly replicating patient-specific pathologies. It can identify anatomic variation and provide a medium in which training models can be generated rapidly, allowing the trainee and experienced neurosurgeon to practice parts of operations preoperatively. Future studies are required to validate this technology in comparison with current simulators and show improved patient outcomes. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Autologous chondrocyte implantation. Culture in a TGF-beta-containing medium enhances the re-expression of a chondrocytic phenotype in passaged human chondrocytes in pellet culture.

    PubMed

    Goldberg, A J; Lee, D A; Bader, D L; Bentley, G

    2005-01-01

    An increasing number of patients are treated by autologous chondrocyte implantation (ACI). This study tests the hypothesis that culture within a defined chondrogenic medium containing TGF-beta enhances the re-expression of a chondrocytic phenotype and the subsequent production of cartilaginous extracellular matrix by human chondrocytes used in ACI. Chondrocytes surplus to clinical requirements for ACI from 24 patients were pelleted and cultured in either DMEM (Dulbecco's modified eagles medium)/ITS+Premix/TGF-beta1 or DMEM/10%FCS (fetal calf serum) and were subsequently analysed biochemically and morphologically. Pellets cultured in DMEM/ITS+/TGF-beta1 stained positively for type-II collagen, while those maintained in DMEM/10%FCS expressed type-I collagen. The pellets cultured in DMEM/ITS+/TGF-beta1 were larger and contained significantly greater amounts of DNA and glycosaminoglycans. This study suggests that the use of a defined medium containing TGF-beta is necessary to induce the re-expression of a differentiated chondrocytic phenotype and the subsequent stimulation of glycosaminoglycan and type-II collagen production by human monolayer expanded chondrocytes.

  18. Induced superficial chondrocyte death reduces catabolic cartilage damage in murine posttraumatic osteoarthritis.

    PubMed

    Zhang, Minjie; Mani, Sriniwasan B; He, Yao; Hall, Amber M; Xu, Lin; Li, Yefu; Zurakowski, David; Jay, Gregory D; Warman, Matthew L

    2016-08-01

    Joints that have degenerated as a result of aging or injury contain dead chondrocytes and damaged cartilage. Some studies have suggested that chondrocyte death precedes cartilage damage, but how the loss of chondrocytes affects cartilage integrity is not clear. In this study, we examined whether chondrocyte death undermines cartilage integrity in aging and injury using a rapid 3D confocal cartilage imaging technique coupled with standard histology. We induced autonomous expression of diphtheria toxin to kill articular surface chondrocytes in mice and determined that chondrocyte death did not lead to cartilage damage. Moreover, cartilage damage after surgical destabilization of the medial meniscus of the knee was increased in mice with intact chondrocytes compared with animals whose chondrocytes had been killed, suggesting that chondrocyte death does not drive cartilage damage in response to injury. These data imply that chondrocyte catabolism, not death, contributes to articular cartilage damage following injury. Therefore, therapies targeted at reducing the catabolic phenotype may protect against degenerative joint disease.

  19. Cryoprotectant agent toxicity in porcine articular chondrocytes.

    PubMed

    Jomha, Nadr M; Weiss, Andrew D H; Fraser Forbes, J; Law, Garson K; Elliott, Janet A W; McGann, Locksley E

    2010-12-01

    Large articular cartilage defects have proven difficult to treat and often result in osteoarthritis of the affected joint. Cryopreservation of articular cartilage can provide an increased supply of tissues for osteochondral allograft but cryoprotective agents are required; however, few studies have been performed on the toxicity of these agents. This study was designed to determine the order of toxicity of five commonly used cryoprotectant agents as well as interactions that occur between them. Isolated porcine articular chondrocytes were exposed to individual cryoprotectant agents and combinations of these agents at 1M and 3M concentrations for 5 min and 120 min. Cell viability was determined using membrane integrity dyes and a metabolic activity assay. Subsequently, a regression analysis based study was undertaken to extract the maximum amount of information from this data. Results of this study demonstrated that all 1M solutions were minimally toxic. The 3M solutions demonstrated varying toxicity after 120 min. Ethylene glycol and glycerol were less toxic than propylene glycol, dimethyl sulfoxide, and formamide. Combinations of cryoprotectant agents were less toxic than single cryoprotectant agents at the same concentration. This is the most comprehensive study investigating cryoprotectant agent toxicity in articular chondrocytes and has resulted in important information regarding the order of toxicity and interactions that occur between these agents. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. Characterization of auricular chondrocytes and auricular/articular chondrocyte co-cultures in terms of an application in articular cartilage repair.

    PubMed

    Kuhne, Maren; John, Thilo; El-Sayed, Karym; Marzahn, Ulrike; Aue, Annekatrin; Kohl, Benjamin; Stoelzel, Katharina; Ertel, Wolfgang; Blottner, Dieter; Haisch, Andreas; Schulze-Tanzil, Gundula

    2010-05-01

    Cartilage injury remains a challenge in orthopedic surgery as articular cartilage only has a limited capacity for intrinsic healing. Autologous chondrocyte transplantation (ACT) is a suitable technique for cartilage repair, but requires articular cartilage biopsies for autologous chondrocyte expansion. The use of heterotopic chondrocytes derived from non-articular cartilage sources such as auricular chondrocytes may be a novel approach for ACT. The aim of the study is to evaluate whether co-cultured articular/auricular chondrocytes exhibit characteristics comparable to articular chondrocytes. Analysis of the proliferation rate, extracellular cartilage matrix (ECM) gene and protein expression (type II and I collagen, elastin, lubricin), beta1-integrins and the chondrogenic transcription factor sox9 in articular/auricular chondrocytes was performed using RTD-PCR, flow cytometry, immunofluorescence microscopy and Western blot analysis. Additionally, three-dimensional (3D) chondrocyte mono- and co-cultures were established. The proliferative activity and elastin gene expression were lower and that of type II collagen and lubricin was higher in articular compared with auricular chondrocytes. The species generally did not influence the chondrocyte characteristics, with the exception of type I collagen and sox9 expression, which was higher in porcine but not in human articular chondrocytes compared with both types of auricular chondrocytes. beta1-integrin gene expression did not differ significantly between the chondrocyte types. The type II collagen gene and protein expression was higher in articular chondrocyte monocultures and was slightly higher in co-cultures compared with monocultured auricular chondrocytes. Both chondrocyte types survived in co-culture. Despite their differing expression profiles, co-cultures revealed some adjustment in the ECM expression of both chondrocyte types.

  1. Adenoviral transduction supports matrix expression of alginate cultured articular chondrocytes.

    PubMed

    Pohle, D; Kasch, R; Herlyn, P; Bader, R; Mittlmeier, T; Pützer, B M; Müller-Hilke, B

    2012-09-01

    The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins. Copyright © 2012 Wiley Periodicals, Inc.

  2. In vitro cartilage regeneration from proliferated adult elastic chondrocytes.

    PubMed

    Terada, Shinichi; Fuchs, Julie R; Yoshimoto, Hiroshi; Fauza, Dario O; Vacanti, Joseph P

    2005-08-01

    The purpose of this study was to investigate cellular feasibility in the proliferation and differentiation status of adult chondrocytes for cartilage regeneration in comparison to fetal chondrocytes. Primary cells were isolated from adult (n = 6) and fetal (n = 6) sheep ear cartilages and expanded in 10% fetal bovine serum (FBS) containing Ham's F12 medium, in which adult and fetal cell proliferation rates were compared using a WST-1 assay kit. Approximately 4 million cells were seeded onto each 1 x 1 x 0.2-cm (200 microL) nonwoven fabric scaffold made from polyglycolic acid. Cell/polymer constructs were cultured in serum-free DMEM/F12 medium supplemented with 5 ng/mL TGF-beta2 and 5 ng/mL des(1-3)IGF-I (adult chondrocytes, group A) or in 10% FBS containing Ham's F12 medium (adult chondrocytes, group B, and fetal chondrocytes, group C) as controls in a rotating bioreactor for 6 weeks. The proliferation assay showed that fetal cells had a significantly better growth potential than did adult cells. Histology and extracellular matrix analyses revealed that groups A and C qualitatively displayed better matrix deposition than did group B. In conclusion, although adult sheep elastic chondrocytes had less growth potential than did fetal cells, the serum-free medium supplemented with growth factors significantly enhanced the production of cartilage matrix secreted from proliferated adult sheep elastic chondrocytes.

  3. Influence of ion channels on the proliferation of human chondrocytes.

    PubMed

    Wohlrab, David; Lebek, Susanne; Krüger, Thomas; Reichel, Heiko

    2002-01-01

    The goal of the study was to examine connections between ion channel activity and the proliferation of human chondrocytes. Chondrocytes were isolated form human osteoarthritic knee joint cartilage. In this study the concentration-dependent influence of the ion channel modulators tetraethylammonium (TEA), 4-aminopyridine (4-AP), 4',4' diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS), verapamil (vp) and lidocaine (lido) on the membrane potential and the proliferation of human chondrocytes was investigated using flow cytometry and the measurement of (3)H-thymidine incorporation as measure for the cell proliferation. The results show an effect of the used ion channel modulators causing a change of the membrane potential of human chondrocytes. The maximal measurable effects of the membrane potential were listed with 0.25 mmol/l verapamil (-18%) and 0.1 mmol/l lidocaine (+20%). When measuring DNA distribution, it became apparent that the human chondrocytes are diploid cells with a very low proliferation tendency. After 12 days culture duration, lidocaine and 4-AP cause an increase of the DNA synthesis rate being a limited effect. These results allow the conclusion of an influence of ion channel modulators on chondrocyte proliferation. To gain knowledge of the regulation of chondrocyte proliferation via ion channel modulators could serve the research of new osteoarthritis treatment concepts.

  4. Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation

    SciTech Connect

    Kato, Y.; Iwamoto, M. )

    1990-04-05

    The effects of basic fibroblast growth factor (bFGF) on terminal differentiation of chondrocytes and cartilage-matrix calcification were investigated. Rabbit growth-plate chondrocytes maintained as a pelleted mass in a centrifuge tube produced an abundant proteoglycan matrix during the matrix-maturation stage, yielding a cartilage-like tissue. Thereafter, they terminally differentiated to hypertrophic chondrocytes which produced high levels of alkaline phosphatase. These cells induced extensive calcification of the matrix in the absence of additional phosphate. Addition of bFGF to the chondrocyte cultures abolished the increases in alkaline phosphatase activity, {sup 45}Ca deposition, and the calcium content. These effects were dose-dependent, reversible, and observed in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. The inhibitory effects could be observed only when chondrocytes were exposed to bFGF in a transition period between the matrix-maturation and hypertrophic stages. As chondrocytes differentiated to hypertrophic cells, bFGF became less effective in inhibiting the expression of the mineralization-related phenotypes. The present study also shows that although the rate of ({sup 35}S)sulfate incorporation into large, chondroitin sulfate proteoglycan in the cell-matrix fraction is very high during the matrix-maturation stage, it abruptly decreases by 90% after terminal differentiation. Furthermore, the terminal differentiation-associated decrease in proteoglycan synthesis was delayed by bFGF. These results provide evidence that bFGF inhibits terminal differentiation of chondrocytes and calcification.

  5. Serum-free media for articular chondrocytes in vitro expansion.

    PubMed

    Shao, Xin-xin; Duncan, Neil A; Lin, Lin; Fu, Xin; Zhang, Ji-ying; Yu, Chang-long

    2013-07-01

    In vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair. Classical culture conditions usually use animal serum as a medium supplement, which raises a number of undesirable questions. In the present study, two kinds of defined, serum-free media were developed to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering. Bovine chondrocytes were expanded in serum-free media supplemented with fibroblast growth factor-2 and platelet-derived growth factor or fibroblast growth factor-2 and insulin-like growth factor. Expansion culture in a conventional 10% fetal bovine serum (FBS) medium served as control. Fibronectin coating was used to help cell adhesion in serum-free medium. Next, in vitro three-dimensional pellet culture was used to evaluate the chondrocyte capacity. Cell pellets were expanded in different media to re-express the differentiated phenotype (re-differentiation) and to form cartilaginous tissue. The pellets were assessed by glycosaminoglycans contents, collagen II, collagen I and collagen X immunohistological staining. Chondrocytes cultured in serum-free media showed no proliferation difference than cells grown with 10% FBS medium. In addition, chondrocytes expanded in both serum-free media expressed more differentiated phenotypes at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I. Pellets derived from chondrocytes cultured in both serum-free media displayed comparable chondrogenic capacities to pellets from cells expanded in 10% FBS medium. These findings provide alternative culture approaches for chondrocytes in vitro expansion, which may benefit the clinical use of autologous chondrocytes implantation.

  6. Response of zonal chondrocytes to extracellular matrix-hydrogels.

    PubMed

    Hwang, Nathaniel S; Varghese, Shyni; Lee, H Janice; Theprungsirikul, Parnduangjai; Canver, Adam; Sharma, Blanka; Elisseeff, Jennifer

    2007-09-04

    We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)-based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS-based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen-based hydrogels demonstrated zonal-dependent cellular responses.

  7. RESPONSE OF ZONAL CHONDROCYTES TO EXTRACELLULAR MATRIX-HYDROGELS

    PubMed Central

    Hwang, Nathaniel S.; Varghese, Shyni; Lee, H. Janice; Theprungsirikul, Parnduangjai; Canver, Adam; Sharma, Blanka; Elisseeff, Jennifer

    2009-01-01

    We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)-based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS-based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen-based hydrogels demonstrated zonal-dependent cellular responses. PMID:17692846

  8. Calcium flux and endogenous calcium content in isolated mammalian growth-plate chondrocytes, hyaline-cartilage chondrocytes, and hepatocytes.

    PubMed

    Iannotti, J P; Brighton, C T; Stambough, J L; Storey, B T

    1985-01-01

    The role of chondrocyte mitochondria in endochondral ossification has been the subject of intensive investigation and controversy. The purpose of this study was to quantitate the endogenous calcium content and the maximum capacity for calcium accumulation and release in isolated mammalian growth-plate chondrocytes and hyaline-cartilage chondrocytes. The results indicated that the mitochondria of the isolated growth-plate and hyaline-cartilage chondrocytes possess a greater endogenous calcium content, a greater capacity for calcium accumulation, and a larger labile Ca+2 pool than do the mitochondria of hepatocytes. Growth-plate and hyaline-cartilage mitochondria had an endogenous calcium content of 908 and 142 nanomoles of Ca+2 per milligram of mitochondrial protein. The growth-plate mitochondria had a maximum calcium capacity of 5249 nanomoles of Ca+2 per milligram of mitochondrial protein. In comparison, the mitochondria of hepatocytes had a much smaller endogenous-calcium content and a smaller maximum Ca+2 capacity: twenty-one and 3262 nanomoles of Ca+2 per milligram of mitochondrial protein, respectively. The mitochondrial labile-calcium pool in both growth-plate and hyaline-cartilage chondrocytes was twofold greater than that in the mitochondria of hepatocytes. Chondrocyte mitochondria released approximately 2400 nanomoles of Ca+2 per milligram of mitochondrial protein, whereas hepatocyte mitochondria released 1200 nanomoles of Ca+2 per milligram. These results suggest that the chondrocyte mitochondria are specialized for calcium transport and are important in the calcification of the extracellular matrix of the growth plate.

  9. Runx1 Activities in Superficial Zone Chondrocytes, Osteoarthritic Chondrocyte Clones and Response to Mechanical Loading

    PubMed Central

    LeBlanc, Kimberly T.; Walcott, Marie E.; Gaur, Tripti; O’Connell, Shannon L.; Basil, Kirti; Tadiri, Christina P.; Mason-Savas, April; Silva, Jason A.; van Wijnen, Andre J.; Stein, Janet L.; Stein, Gary S; Ayers, David C.; Lian, Jane B.; Fanning, Paul J.

    2015-01-01

    Objective Runx1, the hematopoietic lineage determining transcription factor, is present in perichondrium and chondrocytes. Here we addressed Runx1 functions, by examining expression in cartilage during mouse and human osteoarthritis (OA) progression and in response to mechanical loading. Methods Spared and diseased compartments in knees of OA patients and in mice with surgical destabilization of the medial meniscus were examined for changes in expression of Runx1 mRNA (Q-PCR) and protein (immunoblot, immunohistochemistry). Runx1 levels were quantified in response to static mechanical compression of bovine articular cartilage. Runx1 function was assessed by cell proliferation (Ki67, PCNA) and cell type phenotypic markers. Results Runx1 is enriched in superficial zone (SZ) chondrocytes of normal bovine, mouse, and human tissues. Increasing loading conditions in bovine cartilage revealed a positive correlation with a significant elevation of Runx1. Runx1 becomes highly expressed at the periphery of mouse OA lesions and in human OA chondrocyte ‘clones’ where Runx1 co-localizes with Vcam1, the mesenchymal stem cell (MSC) marker and lubricin (Prg4), a cartilage chondroprotective protein. These OA induced cells represent a proliferative cell population, Runx1 depletion in MPCs decreases cell growth, supporting Runx1 contribution to cell expansion. Conclusion The highest Runx1 levels in SZC of normal cartilage suggest a function that supports the unique phenotype of articular chondrocytes, reflected by upregulation under conditions of compression. We propose Runx1 co-expression with Vcam1 and lubricin in murine cell clusters and human ‘clones’ of OA cartilage, participate in a cooperative mechanism for a compensatory anabolic function. PMID:25078095

  10. [Reformatting 3-dimensional medical images. Application to MRI and scanners].

    PubMed

    Cuchet, E; Lambert, F; Derosier, C

    1994-04-01

    Several kinds of images, each giving a different information, are now available to radiologists. The MRI images have excellent contrast resolution and enable soft tissues to be differentiated, but they do not distinguish structures with low water content, notably air and bone, whereas these are easily recognized by CT. The aim of this study is to present a simple, entirely radiologist-supervised method to examine the radiological data of any patient, obtained from several kinds of images. MRI is performed using a GEMS Signa, 1.5 Tesla, 4.9 version magnet. Acquisitions are T1- or T2-weighted spin-echo or gradient sequences, with a 256 or 512 matrix, on axial sections, with of without contrast injection. CT is performed using a GEMS Hi Speed scanner. Acquisitions are obtained on a 512 matrix and with a "Soft" or "Bone" filter, without contrast injection. The two series of sections are transmitted, through an Etherne network, to a Sun console where the two corresponding volumes are reconstructed on a GEMS Voxtol by means of a 3-dimensional soft ware for image treatment. At least 3 couples define the rotation and translation required for one of the two volumes to reset it in the guide mark of the other. The soft ware then looks for the best transformation, in terms of least square, between the two 3-dimensional volumes. The calculation demands only a few seconds. One of the two objects is then recalculated in the guide mark of the other. The cursor positioned by the user on any point of the object is linked to a second cursor which will automatically position itself on the corresponding point of the other object. The accuracy obtained (about one millimeter) is specified by the soft ware which indicates how to improve resetting. In addition to its teaching value, this superimposition image can help in the diagnosis and can be used for surgical stimulation because it is possible to mix the images. This mixing gives access to a new type of imaging, since the images spared

  11. A Qualitative Model of the Differentiation Network in Chondrocyte Maturation: A Holistic View of Chondrocyte Hypertrophy

    PubMed Central

    Kerkhofs, Johan; Leijten, Jeroen; Bolander, Johanna; Luyten, Frank P.; Post, Janine N.; Geris, Liesbet

    2016-01-01

    Differentiation of chondrocytes towards hypertrophy is a natural process whose control is essential in endochondral bone formation. It is additionally thought to play a role in several pathophysiological processes, with osteoarthritis being a prominent example. We perform a dynamic analysis of a qualitative mathematical model of the regulatory network that directs this phenotypic switch to investigate the influence of the individual factors holistically. To estimate the stability of a SOX9 positive state (associated with resting/proliferation chondrocytes) versus a RUNX2 positive one (associated with hypertrophy) we employ two measures. The robustness of the state in canalisation (size of the attractor basin) is assessed by a Monte Carlo analysis and the sensitivity to perturbations is assessed by a perturbational analysis of the attractor. Through qualitative predictions, these measures allow for an in silico screening of the effect of the modelled factors on chondrocyte maintenance and hypertrophy. We show how discrepancies between experimental data and the model’s results can be resolved by evaluating the dynamic plausibility of alternative network topologies. The findings are further supported by a literature study of proposed therapeutic targets in the case of osteoarthritis. PMID:27579819

  12. Chondrogenic differentiation potential of osteoarthritic chondrocytes and their possible use in matrix-associated autologous chondrocyte transplantation.

    PubMed

    Dehne, Tilo; Karlsson, Camilla; Ringe, Jochen; Sittinger, Michael; Lindahl, Anders

    2009-01-01

    Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score > 3, Ahlbäck Score > 2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using real-time PCR. Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (ACAN, COL2A1, COMP, CRTL1, SOX9) and genes involved in matrix synthesis (BGN, CILP2, COL9A2, COL11A1, TIMP4) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (ALPL, COL1A1, COL3A1, COL10A1, MMP13, POSTN, PTH1R, RUNX2) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, was differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The

  13. Chondrogenic differentiation potential of osteoarthritic chondrocytes and their possible use in matrix-associated autologous chondrocyte transplantation

    PubMed Central

    2009-01-01

    Introduction Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Methods Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score > 3, Ahlbäck Score > 2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using real-time PCR. Results Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (ACAN, COL2A1, COMP, CRTL1, SOX9) and genes involved in matrix synthesis (BGN, CILP2, COL9A2, COL11A1, TIMP4) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (ALPL, COL1A1, COL3A1, COL10A1, MMP13, POSTN, PTH1R, RUNX2) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, was differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Conclusions Only few genes were differentially expressed between OA and

  14. In vitro measurement of muscle volume with 3-dimensional ultrasound.

    PubMed

    Delcker, A; Walker, F; Caress, J; Hunt, C; Tegeler, C

    1999-05-01

    The aim was to test the accuracy of muscle volume measurements with a new 3-dimensional (3-D) ultrasound system, which allows a freehand scanning of the transducer with an improved quality of the ultrasound images and therefore the outlines of the muscles. Five resected cadaveric hand muscles were insonated and the muscle volumes calculated by 3-D reconstructions of the acquired 2-D ultrasound sections. Intra-reader, inter-reader and follow-up variability were calculated, as well as the volume of the muscle tissue measured by water displacement. In the results, 3-D ultrasound and water displacement measurements showed an average deviation of 10.1%; Data of 3-D ultrasound measurements were: intra-reader variability 2.8%; inter-reader variability 2.4% and follow-up variability 2.3%. 3-D measurements of muscle volume are valid and reliable. Serial sonographic measurements of muscle may be able to quantitate changes in muscle volume that occur in disease and recovery.

  15. A 3-Dimensional Atlas of Human Tongue Muscles

    PubMed Central

    SANDERS, IRA; MU, LIANCAI

    2013-01-01

    The human tongue is one of the most important yet least understood structures of the body. One reason for the relative lack of research on the human tongue is its complex anatomy. This is a real barrier to investigators as there are few anatomical resources in the literature that show this complex anatomy clearly. As a result, the diagnosis and treatment of tongue disorders lags behind that for other structures of the head and neck. This report intended to fill this gap by displaying the tongue’s anatomy in multiple ways. The primary material used in this study was serial axial images of the male and female human tongue from the Visible Human (VH) Project of the National Library of Medicine. In addition, thick serial coronal sections of three human tongues were rendered translucent. The VH axial images were computer reconstructed into serial coronal sections and each tongue muscle was outlined. These outlines were used to construct a 3-dimensional computer model of the tongue that allows each muscle to be seen in its in vivo anatomical position. The thick coronal sections supplement the 3-D model by showing details of the complex interweaving of tongue muscles throughout the tongue. The graphics are perhaps the clearest guide to date to aid clinical or basic science investigators in identifying each tongue muscle in any part of the human tongue. PMID:23650264

  16. Thermal crosstalk in 3-dimensional RRAM crossbar array.

    PubMed

    Sun, Pengxiao; Lu, Nianduan; Li, Ling; Li, Yingtao; Wang, Hong; Lv, Hangbing; Liu, Qi; Long, Shibing; Liu, Su; Liu, Ming

    2015-08-27

    High density 3-dimensional (3D) crossbar resistive random access memory (RRAM) is one of the major focus of the new age technologies. To compete with the ultra-high density NAND and NOR memories, understanding of reliability mechanisms and scaling potential of 3D RRAM crossbar array is needed. Thermal crosstalk is one of the most critical effects that should be considered in 3D crossbar array application. The Joule heat generated inside the RRAM device will determine the switching behavior itself, and for dense memory arrays, the temperature surrounding may lead to a consequent resistance degradation of neighboring devices. In this work, thermal crosstalk effect and scaling potential under thermal effect in 3D RRAM crossbar array are systematically investigated. It is revealed that the reset process is dominated by transient thermal effect in 3D RRAM array. More importantly, thermal crosstalk phenomena could deteriorate device retention performance and even lead to data storage state failure from LRS (low resistance state) to HRS (high resistance state) of the disturbed RRAM cell. In addition, the resistance state degradation will be more serious with continuously scaling down the feature size. Possible methods for alleviating thermal crosstalk effect while further advancing the scaling potential are also provided and verified by numerical simulation.

  17. Mandibular reconstruction using stereolithographic 3-dimensional printing modeling technology.

    PubMed

    Cohen, Adir; Laviv, Amir; Berman, Phillip; Nashef, Rizan; Abu-Tair, Jawad

    2009-11-01

    Mandibular reconstruction can be challenging for the surgeon wishing to restore its unique geometry. Reconstruction can be achieved with titanium bone plates followed by autogenous bone grafting. Incorporation of the bone graft into the mandible provides continuity and strength required for proper esthetics and function and permitting dental implant rehabilitation at a later stage. Precious time in the operating room is invested in plate contouring to reconstruct the mandible. Rapid prototyping technologies can construct physical models from computer-aided design via 3-dimensional (3D) printers. A prefabricated 3D model is achieved, which assists in accurate contouring of plates and/or planning of bone graft harvest geometry before surgery. The 2 most commonly used rapid prototyping technologies are stereolithography and 3D printing (3DP). Three-dimensional printing is advantageous to stereolithography for better accuracy, quicker printing time, and lower cost. We present 3 clinical cases based on 3DP modeling technology. Models were fabricated before the resection of mandibular ameloblastoma and were used to prepare bridging plates before the first stage of reconstruction. In 1 case, another model was fabricated and used as a template for iliac crest bone graft in the second stage of reconstruction. The 3DP technology provided a precise, fast, and cheap mandibular reconstruction, which aids in shortened operation time (and therefore decreased exposure time to general anesthesia, decreased blood loss, and shorter wound exposure time) and easier surgical procedure.

  18. Thermal crosstalk in 3-dimensional RRAM crossbar array

    NASA Astrophysics Data System (ADS)

    Sun, Pengxiao; Lu, Nianduan; Li, Ling; Li, Yingtao; Wang, Hong; Lv, Hangbing; Liu, Qi; Long, Shibing; Liu, Su; Liu, Ming

    2015-08-01

    High density 3-dimensional (3D) crossbar resistive random access memory (RRAM) is one of the major focus of the new age technologies. To compete with the ultra-high density NAND and NOR memories, understanding of reliability mechanisms and scaling potential of 3D RRAM crossbar array is needed. Thermal crosstalk is one of the most critical effects that should be considered in 3D crossbar array application. The Joule heat generated inside the RRAM device will determine the switching behavior itself, and for dense memory arrays, the temperature surrounding may lead to a consequent resistance degradation of neighboring devices. In this work, thermal crosstalk effect and scaling potential under thermal effect in 3D RRAM crossbar array are systematically investigated. It is revealed that the reset process is dominated by transient thermal effect in 3D RRAM array. More importantly, thermal crosstalk phenomena could deteriorate device retention performance and even lead to data storage state failure from LRS (low resistance state) to HRS (high resistance state) of the disturbed RRAM cell. In addition, the resistance state degradation will be more serious with continuously scaling down the feature size. Possible methods for alleviating thermal crosstalk effect while further advancing the scaling potential are also provided and verified by numerical simulation.

  19. Thermal crosstalk in 3-dimensional RRAM crossbar array

    PubMed Central

    Sun, Pengxiao; Lu, Nianduan; Li, Ling; Li, Yingtao; Wang, Hong; Lv, Hangbing; Liu, Qi; Long, Shibing; Liu, Su; Liu, Ming

    2015-01-01

    High density 3-dimensional (3D) crossbar resistive random access memory (RRAM) is one of the major focus of the new age technologies. To compete with the ultra-high density NAND and NOR memories, understanding of reliability mechanisms and scaling potential of 3D RRAM crossbar array is needed. Thermal crosstalk is one of the most critical effects that should be considered in 3D crossbar array application. The Joule heat generated inside the RRAM device will determine the switching behavior itself, and for dense memory arrays, the temperature surrounding may lead to a consequent resistance degradation of neighboring devices. In this work, thermal crosstalk effect and scaling potential under thermal effect in 3D RRAM crossbar array are systematically investigated. It is revealed that the reset process is dominated by transient thermal effect in 3D RRAM array. More importantly, thermal crosstalk phenomena could deteriorate device retention performance and even lead to data storage state failure from LRS (low resistance state) to HRS (high resistance state) of the disturbed RRAM cell. In addition, the resistance state degradation will be more serious with continuously scaling down the feature size. Possible methods for alleviating thermal crosstalk effect while further advancing the scaling potential are also provided and verified by numerical simulation. PMID:26310537

  20. A 3-Dimensional Printed Ultrasound Probe Visuospatial Trainer.

    PubMed

    McKenna, Ryan T; Dove, Jesse C; Ratzlaff, Robert A; Diaz-Gomez, Jose L; Cox, Daniel J; Simon, Leslie V

    2017-09-04

    Training adult learners to use ultrasound in clinical practice relies on the ability of the learner to apply visuospatial concepts to the anatomy of the human body. We describe a visuospatial trainer that replicates the housing of an ultrasound transducer, through which a linear laser projects light in the same plane and orientation as the ultrasonic sound waves. We use this trainer in combination with a porcine heart dissection laboratory to teach bedside cardiac ultrasound and transthoracic echocardiography (TTE). Off-the-shelf components, including an on/off switch, a laser, and 2 ampere batteries are connected in series and placed inside the 3-dimensional (3D)-printed housing. The trainer's laser emission projects a red line that visually represents the ultrasound's field. Learners project the laser against a porcine or human heart in the orientation of the TTE window they wish to obtain and then dissect the heart in that plane, allowing for visualization of how grayscale images are obtained from 3D structures. Previous research has demonstrated that visuospatial aptitude is correlated with ultrasound procedural performance. We present this trainer and educational method as a specific training intervention that could enhance the visuospatial ability of the ultrasound learner. This visuospatial trainer and educational method present a novel process for enhancing learner understanding of 2-dimensional ultrasound images as they relate to 3D structures. Having a clear understanding of how images are generated in cross section may translate into more proficient adaptation of cardiac ultrasound and TTE.

  1. The first 3-dimensional assemblies of organotin-functionalized polyanions.

    PubMed

    Piedra-Garza, Luis Fernando; Reinoso, Santiago; Dickman, Michael H; Sanguineti, Michael M; Kortz, Ulrich

    2009-08-21

    Reaction of the (CH(3))(2)Sn(2+) electrophile toward trilacunary [A-alpha-XW(9)O(34)](n-) Keggin polytungstates (X = P(V), As(V), Si(IV)) with guanidinium as templating-cation resulted in the isostructural compounds Na[C(NH(2))(3)](2)[{(CH(3))(2)Sn(H(2)O)}(3)(A-alpha-PW(9)O(34))] x 9 H(2)O (1), Na[C(NH(2))(3)](2)[{(CH(3))(2)Sn(H(2)O)}(3)(A-alpha-AsW(9)O(34))] x 8 H(2)O (2) and Na(2)[C(NH(2))(3)](2)[{(CH(3))(2)Sn(H(2)O)}(3)(A-alpha-SiW(9)O(34))] x 10 H(2)O (3). Compounds 1-3 constitute the first 3-dimensional assemblies of organotin-functionalized polyanions, as well as the first example of a dimethyltin-containing tungstosilicate in the case of 3, and they show a similar chiral architecture based on tetrahedrally-arranged {(CH(3))(2)Sn}(3)(A-alpha-XW(9)O(34)) monomeric building-blocks connected via intermolecular Sn-O=W bridges regardless of the size and/or charge of the heteroatom.

  2. 3-Dimensional simulation of the grain formation in investment castings

    SciTech Connect

    Gandin, C.A.; Rappaz, M. ); Tintillier, R. . Dept. Materiaux et Procedes-Direction Technique)

    1994-03-01

    A 3-dimensional (3-D) probabilistic model which has been developed previously for the prediction of grain structure formation during solidification is applied to thin superalloy plates produced using the investment-casting process. This model considers the random nucleation and orientation of nuclei formed at the mold surface and in the bulk of the liquid, the growth kinetics of the dendrite tips, and the preferential growth directions of the dendrite trunks and arms. In the present study, the grains are assumed to nucleate at the surface of the mold only. The computed grain structures, as observed in 2-dimensional (2-D) sections made parallel to the mold surface, are compared with experimental micrographs. The grain densities are then deduced as a function of the distance from the mold surface for both the experiment and the simulation. It is shown that these values are in good agreement, thus, providing validation of the grain formation mechanisms built into the 3-D probabilistic model. Finally, this model is further extended to more complex geometries and the 3-D computed grain structure of an equiaxed turbine-blade airfoil is compared with the experimental transverse section micrograph.

  3. Finger Character Recognition Using 3-Dimensional Template Matching

    NASA Astrophysics Data System (ADS)

    Higashiyama, Kazuhiro; Ono, Satoshi; Wang, Yu; Nakayama, Shigeru

    This paper proposes a method for Japanese finger character recognition, using a 3-dimensional (3D) scanner. A hand is a complex dexterous manipulator, evolved to be more complex than any other animals. The hand, being capable of making many different complex shapes, it is ideal for communicating using gestures. The recognition of a whole language, such as the Japanese finger characters, requires the differentiation of subtle similar positioning of each digit. To know the exact 3D position of the hand's digits and overall shape, data gloves had been developed, but these are inconvenient to use. 2D image recognition systems struggle with recreating the 3D information. To capture the 3D information, the proposed method uses a 3D scanner, and then makes matches with 3D templates representing each unique character. Experimental results show that the proposed method recognizes a greater number of characters than existing 2D-based systems with recognition accuracy, on average of 93% for 9 testees, and a peak of over 98% for 4 of them.

  4. The International Intercomparison of 3-Dimensional Radiation Codes

    NASA Technical Reports Server (NTRS)

    Cahalan, R. F.; Lau, William K. M. (Technical Monitor)

    2002-01-01

    I3RC (International Intercomparison of 3-dimensional Radiation Codes) has as its primary goal to compare a wide variety of three-dimensional (3D) radiative transfer methods applied to Earth's atmosphere, with a few selected cloud fields as input, and a few selected radiative quantities as output. Phases 1 and 2 are now complete, and participants represented institutions in Canada, France, Germany, Russia, the United Kingdom, and the USA, who met for two workshops in Tucson, Arizona USA, and compared results from 5 cloud fields of varying complexity, beginning with simplified atmosphere and surface, and proceeding to more realistic cases. Phase 3 is now underway, focusing on improvement and sharing of 3D radiation code, aided by working groups on "Approximations" and "Open Source". The "Approximations" group has so far focused on diffusive approximate methods in an attempt to gain advantages in execution time, and also to advance the understanding of 3D radiation processes. The "Open Source" subgroup is developing a Monte Carlo radiative transfer toolkit that makes state-of-the-art techniques available to a wide range of users. Activities of both subgroups are further explained at the I3RC website http://i3rc.gsfc.nasa.gov. Participants in 13RC are forming a 3D Working Group under the auspices of the International Radiation Commission, and will meet for this and related activities at a workshop in Tucson in November 2002.

  5. 3-Dimensional Facial Analysis—Facing Precision Public Health

    PubMed Central

    Baynam, Gareth; Bauskis, Alicia; Pachter, Nicholas; Schofield, Lyn; Verhoef, Hedwig; Palmer, Richard L.; Kung, Stefanie; Helmholz, Petra; Ridout, Michael; Walker, Caroline E.; Hawkins, Anne; Goldblatt, Jack; Weeramanthri, Tarun S.; Dawkins, Hugh J. S.; Molster, Caron M.

    2017-01-01

    Precision public health is a new field driven by technological advances that enable more precise descriptions and analyses of individuals and population groups, with a view to improving the overall health of populations. This promises to lead to more precise clinical and public health practices, across the continuum of prevention, screening, diagnosis, and treatment. A phenotype is the set of observable characteristics of an individual resulting from the interaction of a genotype with the environment. Precision (deep) phenotyping applies innovative technologies to exhaustively and more precisely examine the discrete components of a phenotype and goes beyond the information usually included in medical charts. This form of phenotyping is a critical component of more precise diagnostic capability and 3-dimensional facial analysis (3DFA) is a key technological enabler in this domain. In this paper, we examine the potential of 3DFA as a public health tool, by viewing it against the 10 essential public health services of the “public health wheel,” developed by the US Centers for Disease Control. This provides an illustrative framework to gage current and emergent applications of genomic technologies for implementing precision public health. PMID:28443272

  6. Invasive 3-Dimensional Organotypic Neoplasia from Multiple Normal Human Epithelia

    PubMed Central

    Ridky, Todd W.; Chow, Jennifer M.; Wong, David J.; Khavari, Paul A.

    2013-01-01

    Refined cancer models are required to assess the burgeoning number of potential targets for cancer therapeutics within a rapid and clinically relevant context. Here we utilize tumor-associated genetic pathways to transform primary human epithelial cells from epidermis, oropharynx, esophagus, and cervix into genetically defined tumors within a human 3-dimensional (3-D) tissue environment incorporating cell-populated stroma and intact basement membrane. These engineered organotypic tissues recapitulated natural features of tumor progression, including epithelial invasion through basement membrane, a complex process critically required for biologic malignancy in 90% of human cancers. Invasion was rapid, and potentiated by stromal cells. Oncogenic signals in 3-D tissue, but not 2-D culture, resembled gene expression profiles from spontaneous human cancers. Screening well-characterized signaling pathway inhibitors in 3-D organotypic neoplasia helped distil a clinically faithful cancer gene signature. Multi-tissue 3-D human tissue cancer models may provide an efficient and relevant complement to current approaches to characterize cancer progression. PMID:21102459

  7. Autologous Chondrocyte Implantation: Past, Present, and Future.

    PubMed

    Welch, Tyler; Mandelbaum, Bert; Tom, Minas

    2016-06-01

    Focal cartilage defects of the knee are relatively common and may increase the risk of developing osteoarthritis. Autologous chondrocyte implantation (ACI) aims to restore the integrity of isolated cartilage lesions through the induction of hyaline-like cartilage formation. Although ACI has traditionally been used as a second-line treatment, recent evidence suggests that ACI should be considered as a first-line treatment option in certain patients. Recent controlled trials also suggest that there are improved clinical outcomes among those patients who undergo ACI over the mid-term and long-term compared with those treated with microfracture or osteochondral autograft/mosaicplasty, regardless of lesion size. Recent literature also indicates that arthroscopic, second-generation and third-generation techniques are associated with better outcomes and fewer complications than first-generation ACI. In summary, ACI is an effective tool for cartilage restoration that may be more efficacious and durable than other cartilage restoration techniques for appropriate candidates.

  8. Catabolic effects of muramyl dipeptide on rabbit chondrocytes

    SciTech Connect

    Ikebe, T.; Iribe, H.; Hirata, M.; Yanaga, F.; Koga, T. )

    1990-12-01

    Muramyl dipeptide, an essential structure for the diverse biologic activities of bacterial cell wall peptidoglycan, inhibited the synthesis of glycosaminoglycan/proteoglycan in cultured rabbit costal chondrocytes in a dose-dependent manner. Muramyl dipeptide, as well as lipopolysaccharide and interleukin-1 alpha, also enhanced the release of 35S-sulfate-prelabeled glycosaminoglycan/proteoglycan from the cell layer, which seems to reflect, at least partially, the increasing degradation of glycosaminoglycan/proteoglycan. Five synthetic analogs of muramyl dipeptide known to be adjuvant active or adjuvant inactive were tested for their potential to inhibit synthesis of glycosaminoglycan/proteoglycan and to enhance the release of glycosaminoglycan/proteoglycan in chondrocytes. The structural dependence of these synthetic analogs on chondrocytes was found to parallel that of immunoadjuvant activity. These results suggest that muramyl dipeptide is a potent mediator of catabolism in chondrocytes.

  9. Vascular endothelial growth factor activities on osteoarthritic chondrocytes.

    PubMed

    Pulsatelli, L; Dolzani, P; Silvestri, T; Frizziero, L; Facchini, A; Meliconi, R

    2005-01-01

    Evaluation of the role of VEGF in cartilage pathophysiology. VEGF release from chondrocytes in the presence of IL-1beta, TGFbeta and IL-10 was detected by immunoassay. VEGF receptor -1 and -2 expression and VEGF ability to modulate caspase -3 and cathepsin B expression were detected by immunohistochemistry on cartilage biopsies and cartilage explants. VEGF effects on chondrocyte proliferation was analysed by a fluorescent dye that binds nucleic acids. VEGF production by osteoartritis (OA) chondrocytes was significantly reduced by IL-1beta while it was increased in the presence of TGFbeta. Cartilage VEGFR-1 immunostaining was significantly downregulated in 'early' OA patients compared to normal controls (NC). VEGFR-2 expression was negligible both in OA and in NC. VEGF decreased the expression of caspase-3 and cathepsin B, whereas it did not affect proliferation. VEGF is able to down-modulate chondrocyte activities related to catabolic events involved in OA cartilage degradation.

  10. Recombinant human midkine stimulates proliferation and decreases dedifferentiation of auricular chondrocytes in vitro.

    PubMed

    Xu, Chuanying; Zhang, Zhonghui; Wu, Mingyuan; Zhu, Shunying; Gao, Jin; Zhang, Jing; Yuan, Yunsheng; Zhang, Kejian; Yu, Yan; Han, Wei

    2011-11-01

    Autologous chondrocyte implantation (ACI) is widely used for the repair of cartilage defects. However, due to the lack of chondrocyte growth factor and dedifferentiation of the cultured primary chondrocytes, cell source has limited the clinical potential of ACI. Auricular cartilage is an attractive potential source of cells for cartilage tissue engineering. Here we demonstrated that recombinant human midkine (rhMK) significantly promoted proliferation of rat primary auricular chondrocytes cultured and passaged in monolayer, which was mediated by the activation of mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. Furthermore, rhMK attenuated the dedifferentiation of cultured chondrocytes by maintaining the expression of chondrocyte-specific matrix proteins during culture expansion and passage. Importantly, rhMK-expanded chondrocytes reserved their full chondrogenic potential and redifferentiated into elastic chondrocytes after being cultured in high density. The results suggest that rhMK may be used for the preparation of chondrocytes in cartilage tissue engineering.

  11. Telomerase Activity in Articular Chondrocytes Is Lost after Puberty

    PubMed Central

    Wilson, Brooke; Novakofski, Kira D.; Donocoff, Rachel Sacher; Liang, Yan-Xiang Amber

    2014-01-01

    Objective: Telomere length and telomerase activity are important indicators of cellular senescence and replicative ability. Loss of telomerase is associated with ageing and the development of osteoarthritis. Implantation of telomerase-positive cells, chondrocytes, or stem cells expressing a normal chondrocyte phenotype is desired for cartilage repair procedures. The objective of this study was to identify at what age chondrocytes and at what passage bone marrow–derived mesenchymal stem cells (MSCs) become senescent based on telomerase activity. The effect of osteogenic protein–1 (OP-1) or interleukin-1α (IL-1α) treatment on telomerase activity in chondrocytes was also measured to determine the response to anabolic or catabolic stimuli. Methods: Articular cartilage was collected from horses (n = 12) aged 1 month to 18 years. Chondrocytes from prepubescent horses (<15 months) were treated with OP-1 or IL-1α. Bone marrow aspirate from adult horses was collected and cultured for up to 10 days to isolate MSCs. Telomerase activity was measured using the TeloTAGGG Telomerase PCR ELISA kit. Results: Chondrocytes from prepubescent horses were positive for telomerase activity. Treatment with IL-1α resulted in a decrease in chondrocyte telomerase activity; however, treatment with OP-1 did not change telomerase activity. One MSC culture sample was positive for telomerase activity on day 2; all samples were negative for telomerase activity on day 10. Conclusions: These results suggest that chondrocytes from prepubescent donors are potentially more suitable for cartilage repair procedures and that telomerase activity is diminished by anabolic and catabolic cytokine stimulation. If MSCs are utilized in cartilage repair, minimal passaging should be performed prior to implantation. PMID:26069700

  12. Haploinsufficiency of osterix in chondrocytes impairs skeletal growth in mice.

    PubMed

    Cheng, Shaohong; Xing, Weirong; Zhou, Xin; Mohan, Subburaman

    2013-10-01

    Osterix (Osx) is essential for both intramembranous or endochondral bone formation. Osteoblast-specific ablation of Osx using Col1α1-Cre resulted in osteopenia, because of impaired osteoblast differentiation in adult mice. Since Osx is also known to be expressed in chondrocytes, we evaluated the role of Osx expressed in chondrocytes by examining the skeletal phenotype of mice with conditional disruption of Osx in Col2α1-expressing chondrocytes. Surprisingly, Cre-positive mice that were homozygous for Osx floxed alleles died after birth. Alcian blue and alizarin red staining revealed that the lengths of skeleton, femur, and vertebrae were reduced by 21, 26, and 14% (P < 0.01), respectively, in the knockout (KO) compared with wild-type mice. To determine if haploid insufficiency of Osx in chondrocytes influenced postnatal skeletal growth, we compared skeletal phenotype of floxed heterozygous mice that were Cre-positive or Cre-negative. Body length was reduced by 8% (P < 0.001), and areal BMD of total body, femur, and tibia was reduced by 5, 7, and 8% (P < 0.05), respectively, in mice with conditional disruption of one allele of Osx in chondrocytes. Micro-CT showed reduced cortical volumetric bone mineral density and trabecular bone volume to total volume in the femurs of Osx(flox/+);col2α1-Cre mice. Histological analysis revealed that the impairment of longitudinal growth was associated with disrupted growth plates in the Osx(flox/+);col2α1-Cre mice. Primary chondrocytes isolated from KO embryos showed reduced expression of chondral ossification markers but elevated expression of chondrogenesis markers. Our findings indicate that Osx expressed in chondrocytes regulates bone growth in part by regulating chondrocyte hypertrophy.

  13. Membrane channel gene expression in human costal and articular chondrocytes

    PubMed Central

    Asmar, A.; Barrett-Jolley, R.; Werner, A.; Kelly, R.; Stacey, M.

    2016-01-01

    ABSTRACT Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca2+ activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought. PMID:27116676

  14. Geranylgeranylacetone suppresses hydrogen peroxide-induced apoptosis of osteoarthritic chondrocytes.

    PubMed

    Yoda, Masaki; Sakai, Tadahiro; Mitsuyama, Hirohito; Hiraiwa, Hideki; Ishiguro, Naoki

    2011-11-01

    Osteoarthritis (OA) is a common disease, afflicting many sufferers with both pain and functional disorders. Various therapies have been attempted for OA, but no fully effective treatment has been established yet. Apoptosis of chondrocytes caused by reactive oxygen species (ROS) has been considered important in the pathogenesis of OA. The progression of OA may be prevented by suppressing apoptosis of chondrocytes. Geranylgeranylacetone (GGA) has been used as an anti-ulcer drug in Japan for more than 20 years. Several recent studies have shown that GGA can induce heat shock protein (HSP) and exert cytoprotective actions on a large variety of cells and tissues. In this study, we investigated the effects of GGA on the apoptosis of OA chondrocytes induced by hydrogen peroxide (H(2)O(2)). Human isolated OA chondrocytes were cultured in the absence or presence of GGA. Cell viability, caspase 3/7 and 9 activities, HSP70 mRNA and protein expressions were examined, and morphological analyses were conducted after exposure of cells to H(2)O(2) to induce apoptosis. Geranylgeranylacetone dose-dependently reversed the H(2)O(2)-induced decrease in cell viability. It was recognized that GGA rendered OA chondrocytes resistant to H(2)O(2)-induced apoptosis from Hoechst 33342 staining and TUNEL staining. Caspases 3 and 9 were activated by addition of H(2)O(2), and GGA suppressed this H(2)O(2)-induced activation of both caspases. H(2)O(2)-induced induction of HSP70 was enhanced in OA chondrocytes by pretreatment with GGA. The results showed that GGA can suppress apoptosis of chondrocytes and enhance production of HSP70. This study is the first, to our knowledge, to demonstrate that GGA protects OA chondrocytes from H(2)O(2)-induced apoptosis, at least in part by enhancing HSP70 production. These results indicate that GGA is a potentially useful drug for the treatment of OA.

  15. CCN1 Regulates Chondrocyte Maturation and Cartilage Development

    PubMed Central

    Zhang, Yongchun; Sheu, Tzong-jen; Hoak, Donna; Shen, Jie; Hilton, Matthew J; Zuscik, Michael J; Jonason, Jennifer H; O’Keefe, Regis J

    2016-01-01

    WNT/β-CATENIN signaling is involved in multiple aspects of skeletal development, including chondrocyte differentiation and maturation. Although the functions of β-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which β-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/β-CATENIN signaling in chondrocytes. Specifically, we found that β-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation, whereas inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues after meniscal/ligamentous injury (MLI) and in human cartilage after meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis. PMID:26363286

  16. Antioxidant effect of bisphosphonates and simvastatin on chondrocyte lipid peroxidation

    SciTech Connect

    Dombrecht, E.J.; De Tollenaere, C.B.; Aerts, K.; Cos, P.; Schuerwegh, A.J.; Bridts, C.H.; Van Offel, J.F.; Ebo, D.G.; Stevens, W.J. . E-mail: immuno@ua.ac.be; De Clerck, L.S.

    2006-09-22

    The objective of this study was to evaluate the effect of bisphosphonates (BPs) and simvastatin on chondrocyte lipid peroxidation. For this purpose, a flow cytometrical method using C11-BODIPY{sup 581/591} was developed to detect hydroperoxide-induced lipid peroxidation in chondrocytes. Tertiary butylhydroperoxide (t-BHP) induced a time and concentration dependent increase in chondrocyte lipid peroxidation. Addition of a Fe{sup 2+}/EDTA complex to t-BHP or hydrogen peroxide (H{sub 2}O{sub 2}) clearly enhanced lipid peroxidation. The lipophilic simvastatin demonstrated a small inhibition in the chondrocyte lipid peroxidation. None of three tested BPs (clodronate, pamidronate, and risedronate) had an effect on chondrocyte lipid peroxidation induced by t-BHP. However, when Fe{sup 2+}/EDTA complex was added to t-BHP or H{sub 2}O{sub 2}, BPs inhibited the lipid peroxidation process varying from 25% to 58%. This study demonstrates that BPs have antioxidant properties as iron chelators, thereby inhibiting the chondrocyte lipid peroxidation. These findings add evidence to the therapeutic potential of bisphosphonates and statins in rheumatoid arthritis.

  17. Endogenous versus Exogenous Growth Factor Regulation of Articular Chondrocytes

    PubMed Central

    Shi, Shuiliang; Chan, Albert G.; Mercer, Scott; Eckert, George J.; Trippel, Stephen B.

    2014-01-01

    Anabolic growth factors that regulate the function of articular chondrocytes are candidates for articular cartilage repair. Such factors may be delivered by pharmacotherapy in the form of exogenous proteins, or by gene therapy as endogenous proteins. It is unknown whether delivery method influences growth factor effectiveness in regulating articular chondrocyte reparative functions. We treated adult bovine articular chondrocytes with exogenous recombinant insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-β1), or with the genes encoding these growth factors for endogenous production. Treatment effects were measured as change in chondrocyte DNA content, glycosaminoglycan production, and aggrecan gene expression. We found that IGF-I stimulated chondrocyte biosynthesis similarly when delivered by either exogenous or endogenous means. In contrast, exogenous TGF-ß1 stimulated these reparative functions, while endogenous TGF-ß1 had little effect. Endogenous TGF-ß1 became more bioactive following activation of the transgene protein product. These data indicate that effective mechanisms of growth factor delivery for articular cartilage repair may differ for different growth factors. In the case of IGF-I, gene therapy or protein therapy appear to be viable options. In contrast, TGF-ß1 gene therapy may be constrained by a limited ability of chondrocytes to convert latent complexes to an active form. PMID:24105960

  18. Sphingosine-1-phosphate stimulates rat primary chondrocyte proliferation

    SciTech Connect

    Kim, Mi-Kyoung; Lee, Ha Young; Kwak, Jong-Young; Park, Joo-In; Yun, Jeanho; Bae, Yoe-Sik . E-mail: yoesik@donga.ac.kr

    2006-06-23

    Rat primary chondrocytes express the sphingosine-1-phosphate (S1P) receptor, S1P{sub 2}, S1P{sub 3}, S1P{sub 4}, but not S1P{sub 1}. When chondrocytes were stimulated with S1P or phytosphingosine-1-phosphate (PhS1P, an S1P{sub 1}- and S1P{sub 4}-selective agonist), phospholipase C-mediated cytosolic calcium increase was dramatically induced. S1P and PhS1P also stimulated two kinds of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) and p38 kinase in chondrocytes. In terms of the two phospholipids-mediated functional modulation of chondrocytes, S1P and PhS1P stimulated cellular proliferation. The two phospholipids-induced chondrocyte proliferations were almost completely blocked by PD98059 but not by SB203580, suggesting that ERK but not p38 kinase is essentially required for the proliferation. Pertussis toxin almost completely inhibited the two phospholipids-induced cellular proliferation and ERK activation, indicating the crucial role of G{sub i} protein. This study demonstrates the physiological role of two important phospholipids (S1P and PhS1P) on the modulation of rat primary chondrocyte proliferation, and the crucial role played by ERK in the process.

  19. Multi-membrane chitosan hydrogels as chondrocytic cell bioreactors.

    PubMed

    Ladet, S G; Tahiri, K; Montembault, A S; Domard, A J; Corvol, M-T M

    2011-08-01

    We investigated the bioactivity of new chitosan-based multi-membrane hydrogel (MMH) architectures towards chondrocyte-like cells. The microstructure of the hydrogels constituting the membranes precludes any living cell penetration, whereas their lower scale architecture allows the protein diffusion. The biological behavior of chondrocytes implanted within the MMH inter-membrane spaces was studied for 45 days in culture. Chondrocytes formed cell aggregates and proliferated without loosing their chondrogenic phenotype as illustrated by collagen II and aggrecan expressions at the mRNA and protein levels. Cells produced neo-formed alcyan blue matrix proteins filling MMH interspaces. The HiF-2α/SOX9 pattern of expression suggested that the elevated chondrocytic phenotype in MMH could be related to a better hypoxic local environment than in classical culture conditions. Pro-inflammatory markers were not expressed during the period of culture. The low level of nitric oxide accumulation within the inter-membrane spaces and in the incubation medium implied that chitosan consumed nitrites produced by entrapped chondrocytes, in relation with the decrease of its molecular weight of 50%. Our data suggest that MMH structures may be considered as complex chondrocytic cell bioreactors; "active decoys of biological media", potentially promising for various biomedical applications like the inter-vertebral disk replacement. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Effect of autophagy induced by dexamethasone on senescence in chondrocytes.

    PubMed

    Xue, Enxing; Zhang, Yu; Song, Bing; Xiao, Jun; Shi, Zhanjun

    2016-10-01

    The aim of the current study was to explore the effects of dexamethasone (DXM) on autophagy and senescence in chondrocytes. Collagen II and aggrecan were examined in normal chondrocytes isolated from Sprague‑Dawley rats. Following stimulation with DXM, LysoTracker Red staining, monodansylcadaverine (MDC) staining, green fluorescent protein‑red fluorescent protein‑light chain 3 (LC3) and western blotting were used to detect autophagy levels in the chondrocytes. Mechanistic target of rapamycin (mTOR) pathway‑associated molecules were investigated by western blotting. Cell senescence was analyzed by senescence‑associated (SA)‑β‑galactosidase (β‑gal) staining. A dose‑dependent increase in the number of autophagic vacuoles was observed in the DXM‑treated chondrocytes, as demonstrated by LysoTracker Red and MDC staining. A dose‑dependent increase in autophagosome formation was observed in the DXM‑treated chondrocytes. Expression of LC3‑II and beclin‑1 was increased by DXM, in particular in the cells treated with DXM for 4 days. However, P62 expression was reduced as a result of treatment. SA‑β‑gal staining indicated that DXM increased cell senescence. Notably, DXM‑induced cell senescence was exacerbated by the autophagic inhibitor 3‑MA. Autophagy induced by DXM protected chondrocytes from senescence, and it is suggested that the mTOR pathway may be involved in the activation of DXM‑induced autophagy.

  1. CCN1 Regulates Chondrocyte Maturation and Cartilage Development.

    PubMed

    Zhang, Yongchun; Sheu, Tzong-jen; Hoak, Donna; Shen, Jie; Hilton, Matthew J; Zuscik, Michael J; Jonason, Jennifer H; O'Keefe, Regis J

    2016-03-01

    WNT/β-CATENIN signaling is involved in multiple aspects of skeletal development, including chondrocyte differentiation and maturation. Although the functions of β-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which β-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/β-CATENIN signaling in chondrocytes. Specifically, we found that β-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation, whereas inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues after meniscal/ligamentous injury (MLI) and in human cartilage after meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis.

  2. ATF3 deficiency in chondrocytes alleviates osteoarthritis development.

    PubMed

    Iezaki, Takashi; Ozaki, Kakeru; Fukasawa, Kazuya; Inoue, Makoto; Kitajima, Shigetaka; Muneta, Takeshi; Takeda, Shu; Fujita, Hiroyuki; Onishi, Yuki; Horie, Tetsuhiro; Yoneda, Yukio; Takarada, Takeshi; Hinoi, Eiichi

    2016-08-01

    Activating transcription factor 3 (Atf3) has been implicated in the pathogenesis of various diseases, including cancer and inflammation, as well as in the regulation of cell proliferation and differentiation. However, the involvement of Atf3 in developmental skeletogenesis and joint disease has not been well studied to date. Here, we show that Atf3 is a critical mediator of osteoarthritis (OA) development through its expression in chondrocytes. ATF3 expression was markedly up-regulated in the OA cartilage of both mice and humans. Conditional deletion of Atf3 in chondrocytes did not result in skeletal abnormalities or affect the chondrogenesis, but alleviated the development of OA generated by surgically inducing knee joint instability in mice. Inflammatory cytokines significantly up-regulated Atf3 expression through the nuclear factor-kB (NF-kB) pathway, while cytokine-induced interleukin-6 (Il6) expression was repressed, in ATF3-deleted murine and human chondrocytes. Mechanistically, Atf3 deficiency decreased cytokine-induced Il6 transcription in chondrocytes through repressing NF-kB signalling by the attenuation of the phosphorylation status of IkB and p65. These findings suggest that Atf3 is implicated in the pathogenesis of OA through modulation of inflammatory cytokine expression in chondrocytes, and the feed-forward loop of inflammatory cytokines/NF-kB/Atf3 in chondrocytes may be a novel therapeutic target for the treatment for OA. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  3. Statins do not inhibit the FGFR signaling in chondrocytes.

    PubMed

    Fafilek, B; Hampl, M; Ricankova, N; Vesela, I; Balek, L; Kunova Bosakova, M; Gudernova, I; Varecha, M; Buchtova, M; Krejci, P

    2017-09-01

    Statins are widely used drugs for cholesterol lowering, which were recently found to counteract the effects of aberrant fibroblast growth factor receptor (FGFR3) signaling in cell and animal models of FGFR3-related chondrodysplasia. This opened an intriguing therapeutic possibility for human dwarfing conditions caused by gain-of-function mutations in FGFR3, although the mechanism of statin action on FGFR3 remains unclear. Here, we determine the effect of statins on FGFR signaling in chondrocytes. Cultured chondrocyte cell lines, mouse embryonic tibia cultures and limb bud micromasses were treated with FGF2 to activate FGFR signaling. The effects of atorvastatin, fluvastatin, lovastatin and pravastatin on FGFR3 protein stability and on FGFR-mediated chondrocyte growth-arrest, loss of extracellular matrix (ECM), induction of premature senescence and hypertrophic differentiation were evaluated. Statins did not alter the level of FGFR3 protein expression nor produce any effect on FGFR-mediated inhibition of chondrocyte proliferation and hypertrophic differentiation in cultured chondrocyte cell lines, mouse tibia cultures or limb bud micromasses. We conclude that statins do not inhibit the FGFR signaling in chondrocytes. Therefore the statin-mediated rescue of FGFR3-related chondrodysplasia, described before, is likely not intrinsic to the growth plate cartilage. Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  4. Effect of autophagy induced by dexamethasone on senescence in chondrocytes

    PubMed Central

    Xue, Enxing; Zhang, Yu; Song, Bing; Xiao, Jun; Shi, Zhanjun

    2016-01-01

    The aim of the current study was to explore the effects of dexamethasone (DXM) on autophagy and senescence in chondrocytes. Collagen II and aggrecan were examined in normal chondrocytes isolated from Sprague-Dawley rats. Following stimulation with DXM, LysoTracker Red staining, monodansylcadaverine (MDC) staining, green fluorescent protein-red fluorescent protein-light chain 3 (LC3) and western blotting were used to detect autophagy levels in the chondrocytes. Mechanistic target of rapamycin (mTOR) pathway-associated molecules were investigated by western blotting. Cell senescence was analyzed by senescence-associated (SA)-β-galactosidase (β-gal) staining. A dose-dependent increase in the number of autophagic vacuoles was observed in the DXM-treated chondrocytes, as demonstrated by LysoTracker Red and MDC staining. A dose-dependent increase in autophagosome formation was observed in the DXM-treated chondrocytes. Expression of LC3-II and beclin-1 was increased by DXM, in particular in the cells treated with DXM for 4 days. However, P62 expression was reduced as a result of treatment. SA-β-gal staining indicated that DXM increased cell senescence. Notably, DXM-induced cell senescence was exacerbated by the autophagic inhibitor 3-MA. Autophagy induced by DXM protected chondrocytes from senescence, and it is suggested that the mTOR pathway may be involved in the activation of DXM-induced autophagy. PMID:27572674

  5. Video Based Sensor for Tracking 3-Dimensional Targets

    NASA Technical Reports Server (NTRS)

    Howard, R. T.; Book, Michael L.; Bryan, Thomas C.

    2000-01-01

    Video-Based Sensor for Tracking 3-Dimensional Targets The National Aeronautics and Space Administration's (NASAs) Marshall Space Flight Center (MSFC) has been developing and testing video-based sensors for automated spacecraft guidance for several years, and the next generation of video sensor will have tracking rates up to 100 Hz and will be able to track multiple reflectors and targets. The Video Guidance Sensor (VGS) developed over the past several years has performed well in testing and met the objective of being used as the terminal guidance sensor for an automated rendezvous and capture system. The first VGS was successfully tested in closed-loop 3-degree-of-freedom (3- DOF) tests in 1989 and then in 6-DOF open-loop tests in 1992 and closed-loop tests in 1993-4. Development and testing continued, and in 1995 approval was given to test the VGS in an experiment on the Space Shuttle. The VGS flew in 1997 and in 1998, performing well for both flights. During the development and testing before, during, and after the flight experiments, numerous areas for improvement were found. The VGS was developed with a sensor head and an electronics box, connected by cables. The VGS was used in conjunction with a target that had wavelength-filtered retro-reflectors in a specific pattern, The sensor head contained the laser diodes, video camera, and heaters and coolers. The electronics box contained a frame grabber, image processor, the electronics to control the components in the sensor head, the communications electronics, and the power supply. The system works by sequentially firing two different wavelengths of laser diodes at the target and processing the two images. Since the target only reflects one wavelength, it shows up well in one image and not at all in the other. Because the target's dimensions are known, the relative positions and attitudes of the target and the sensor can be computed from the spots reflected from the target. The system was designed to work from I

  6. The 3-dimensional grid: a novel approach to stereoelectroencephalography.

    PubMed

    Munyon, Charles; Sweet, Jennifer; Luders, Hans; Lhatoo, Samden; Miller, Jonathan

    2015-03-01

    Successful surgical treatment of epilepsy requires accurate definition of areas of ictal onset and eloquent brain. Although invasive monitoring can help, subdural grids cannot sample sulci or subcortical tissue; traditional stereoelectroencephalography depth electrodes are usually placed too far apart to provide sufficient resolution for mapping. To report a strategy of depth electrode placement in a dense array to allow precise anatomic localization of epileptic and eloquent cortex. Twenty patients with medically intractable epilepsy either poorly localized or found to arise adjacent to eloquent areas underwent placement of arrays of depth electrodes into and around the putative area of seizure onset with the use of framed stereotaxy. Each array consisted of a "grid" of parallel electrodes in a rectangular pattern with 1 cm between entry sites. In a subset of patients, a few electrodes were placed initially, with additional electrodes placed in a second stage. Trajectories were modified to avoid cortical vessels defined on magnetic resonance imaging. Patients were monitored for 4 to 21 days to establish the precise location of seizure onset. Stimulation was performed to map cortical and subcortical eloquent regions. Electrode locations were coregistered for frameless stereotaxy during subsequent resection of seizure focus. Two hundred fifty-four electrodes were implanted. Discrete regions of seizure onset and functional cortex were identified, which were used during resection to remove epileptogenic tissue while preserving eloquent areas. There were no hemorrhagic or infectious complications; no patient suffered permanent neurological deficit. The 3-dimensional intraparenchymal grid is useful for identifying the location and extent of epileptic and eloquent brain.

  7. Development and Validation of a 3-Dimensional CFB Furnace Model

    NASA Astrophysics Data System (ADS)

    Vepsäläinen, Arl; Myöhänen, Karl; Hyppäneni, Timo; Leino, Timo; Tourunen, Antti

    At Foster Wheeler, a three-dimensional CFB furnace model is essential part of knowledge development of CFB furnace process regarding solid mixing, combustion, emission formation and heat transfer. Results of laboratory and pilot scale phenomenon research are utilized in development of sub-models. Analyses of field-test results in industrial-scale CFB boilers including furnace profile measurements are simultaneously carried out with development of 3-dimensional process modeling, which provides a chain of knowledge that is utilized as feedback for phenomenon research. Knowledge gathered by model validation studies and up-to-date parameter databases are utilized in performance prediction and design development of CFB boiler furnaces. This paper reports recent development steps related to modeling of combustion and formation of char and volatiles of various fuel types in CFB conditions. Also a new model for predicting the formation of nitrogen oxides is presented. Validation of mixing and combustion parameters for solids and gases are based on test balances at several large-scale CFB boilers combusting coal, peat and bio-fuels. Field-tests including lateral and vertical furnace profile measurements and characterization of solid materials provides a window for characterization of fuel specific mixing and combustion behavior in CFB furnace at different loads and operation conditions. Measured horizontal gas profiles are projection of balance between fuel mixing and reactions at lower part of furnace and are used together with both lateral temperature profiles at bed and upper parts of furnace for determination of solid mixing and combustion model parameters. Modeling of char and volatile based formation of NO profiles is followed by analysis of oxidizing and reducing regions formed due lower furnace design and mixing characteristics of fuel and combustion airs effecting to formation ofNO furnace profile by reduction and volatile-nitrogen reactions. This paper presents

  8. A new preclinical 3-dimensional agarose colony formation assay.

    PubMed

    Kajiwara, Yoshinori; Panchabhai, Sonali; Levin, Victor A

    2008-08-01

    The evaluation of new drug treatments and combination treatments for gliomas and other cancers requires a robust means to interrogate wide dose ranges and varying times of drug exposure without stain-inactivation of the cells (colonies). To this end, we developed a 3-dimensional (3D) colony formation assay that makes use of GelCount technology, a new cell colony counter for gels and soft agars. We used U251MG, SNB19, and LNZ308 glioma cell lines and MiaPaCa pancreas adenocarcinoma and SW480 colon adenocarcinoma cell lines. Colonies were grown in a two-tiered agarose that had 0.7% agarose on the bottom and 0.3% agarose on top. We then studied the effects of DFMO, carboplatin, and SAHA over a 3-log dose range and over multiple days of drug exposure. Using GelCount we approximated the area under the curve (AUC) of colony volumes as the sum of colony volumes (microm2xOD) in each plate to calculate IC50 values. Adenocarcinoma colonies were recognized by GelCount scanning at 3-4 days, while it took 6-7 days to detect glioma colonies. The growth rate of MiaPaCa and SW480 cells was rapid, with 100 colonies counted in 5-6 days; glioma cells grew more slowly, with 100 colonies counted in 9-10 days. Reliable log dose versus AUC curves were observed for all drugs studied. In conclusion, the GelCount method that we describe is more quantitative than traditional colony assays and allows precise study of drug effects with respect to both dose and time of exposure using fewer culture plates.

  9. Distance stereotest using a 3-dimensional monitor for adult subjects.

    PubMed

    Kim, Jongshin; Yang, Hee Kyung; Kim, Youngmin; Lee, Byoungho; Hwang, Jeong-Min

    2011-06-01

    To evaluate the validity and test-retest reliability of a contour-based 3-dimensional (3-D) monitor distance stereotest (distance 3-D stereotest) and to measure the maximum horizontal disparity that can be fused with disparity vergence for determining the largest measurable disparity of true stereopsis. Observational case series. Sixty-four normal adult subjects (age range, 23 to 39 years) were recruited. Contour-based circles (crossed disparity, 5000 to 20 seconds of arc; Microsoft Visual Studio C(++) 6.0; Microsoft, Inc, Seattle, Washington, USA) were generated on a 3-D monitor (46-inch stereoscopic display) using polarization glasses and were presented to subjects with normal binocularity at 3 m. While the position of the stimulus changed among 4 possible locations, the subjects were instructed to press the corresponding position of the stimulus on a keypad. The results with the new distance 3-D stereotest were compared with those from the distance Randot stereotest. The results of the distance 3-D stereotest and the distance Randot stereotests were identical in 64% and within 1 disparity level in 97% of normal adults. Scores obtained with the 2 tests showed a statistically significant correlation (r = 0.324, P = .009). The half-width of the 95% limit of agreement was 0.47 log seconds of arc (1.55 octaves) using the distance 3-D stereotest--similar to or better than that obtained with conventional distance stereotests. The maximum binocular disparity that can be fused with vergence was 1828 ± 794 seconds of arc (range, 4000 to 500). The distance 3-D stereotest showed good concordance with the distance Randot stereotest and relatively good test-retest reliability, supporting the validity of the distance 3-D stereotest. The normative data set obtained from the present study can serve as a useful reference for quantitative assessment of a wide range of binocular sensory abnormalities. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. The 3-dimensional construction of the Rae craton, central Canada

    NASA Astrophysics Data System (ADS)

    Snyder, David B.; Craven, James A.; Pilkington, Mark; Hillier, Michael J.

    2015-10-01

    Reconstruction of the 3-dimensional tectonic assembly of early continents, first as Archean cratons and then Proterozoic shields, remains poorly understood. In this paper, all readily available geophysical and geochemical data are assembled in a 3-D model with the most accurate bedrock geology in order to understand better the geometry of major structures within the Rae craton of central Canada. Analysis of geophysical observations of gravity and seismic wave speed variations revealed several lithospheric-scale discontinuities in physical properties. Where these discontinuities project upward to correlate with mapped upper crustal geological structures, the discontinuities can be interpreted as shear zones. Radiometric dating of xenoliths provides estimates of rock types and ages at depth beneath sparse kimberlite occurrences. These ages can also be correlated to surface rocks. The 3.6-2.6 Ga Rae craton comprises at least three smaller continental terranes, which "cratonized" during a granitic bloom. Cratonization probably represents final differentiation of early crust into a relatively homogeneous, uniformly thin (35-42 km), tonalite-trondhjemite-granodiorite crust with pyroxenite layers near the Moho. The peak thermotectonic event at 1.86-1.7 Ga was associated with the Hudsonian orogeny that assembled several cratons and lesser continental blocks into the Canadian Shield using a number of southeast-dipping megathrusts. This orogeny metasomatized, mineralized, and recrystallized mantle and lower crustal rocks, apparently making them more conductive by introducing or concentrating sulfides or graphite. Little evidence exists of thin slabs similar to modern oceanic lithosphere in this Precambrian construction history whereas underthrusting and wedging of continental lithosphere is inferred from multiple dipping discontinuities.

  11. Quantitative 3-dimensional computed tomography measurements of coronoid fractures.

    PubMed

    Mellema, Jos J; Janssen, Stein J; Guitton, Thierry G; Ring, David

    2015-03-01

    Using quantitative 3-dimensional computed tomography (Q3DCT) modeling, we tested the null hypothesis that there was no difference in fracture fragment volume, articular surface involvement, and number of fracture fragments between coronoid fracture types and patterns of traumatic elbow instability. We studied 82 patients with a computed tomography scan of a coronoid fracture using Q3DCT modeling. Fracture fragments were identified and fragment volume and articular surface involvement were measured within fracture types and injury patterns. Kruskal-Wallis test was used to evaluate the Q3DCT data of the coronoid fractures. Fractures of the coronoid tip (n = 45) were less fragmented and had the smallest fragment volume and articular surface area involvement compared with anteromedial facet fractures (n = 20) and base fractures (n = 17). Anteromedial facet and base fractures were more fragmented than tip fractures, and base fractures had the largest fragment volume and articular surface area involvement compared with tip and anteromedial facet fractures. We found similar differences between fracture types described by Regan and Morrey. Furthermore, fractures associated with terrible triad fracture dislocation (n = 42) had the smallest fragment volume, and fractures associated with olecranon fracture dislocations (n = 17) had the largest fragment volume and articular surface area involvement compared with the other injury patterns. Analyzing fractures of the coronoid using Q3DCT modeling demonstrated that fracture fragment characteristics differ significantly between fracture types and injury patterns. Detailed knowledge of fracture characteristics and their association with specific patterns of traumatic elbow instability may assist decision making and preoperative planning. Quantitative 3DCT modeling can provide a more detailed understanding of fracture morphology, which might guide decision making and implant development. Copyright © 2015 American Society for

  12. Brain tumor surgery with 3-dimensional surface navigation.

    PubMed

    Mert, Ayguel; Buehler, Katja; Sutherland, Garnette R; Tomanek, Boguslaw; Widhalm, Georg; Kasprian, Gregor; Knosp, Engelbert; Wolfsberger, Stefan

    2012-12-01

    Precise lesion localization is necessary for neurosurgical procedures not only during the operative approach, but also during the preoperative planning phase. To evaluate the advantages of 3-dimensional (3-D) brain surface visualization over conventional 2-dimensional (2-D) magnetic resonance images for surgical planning and intraoperative guidance in brain tumor surgery. Preoperative 3-D brain surface visualization was performed with neurosurgical planning software in 77 cases (58 gliomas, 7 cavernomas, 6 meningiomas, and 6 metastasis). Direct intraoperative navigation on the 3-D brain surface was additionally performed in the last 20 cases with a neurosurgical navigation system. For brain surface reconstruction, patient-specific anatomy was obtained from MR imaging and brain volume was extracted with skull stripping or watershed algorithms, respectively. Three-dimensional visualization was performed by direct volume rendering in both systems. To assess the value of 3-D brain surface visualization for topographic lesion localization, a multiple-choice test was developed. To assess accuracy and reliability of 3-D brain surface visualization for intraoperative orientation, we topographically correlated superficial vessels and gyral anatomy on 3-D brain models with intraoperative images. The rate of correct lesion localization with 3-D was significantly higher (P = .001, χ), while being significantly less time consuming (P < .001, χ) compared with 2-D images. Intraoperatively, visual correlation was found between the 3-D images, superficial vessels, and gyral anatomy. The proposed method of 3-D brain surface visualization is fast, clinically reliable for preoperative anatomic lesion localization and patient-specific planning, and, together with navigation, improves intraoperative orientation in brain tumor surgery and is relatively independent of brain shift.

  13. A 3-Dimensional Anatomic Study of the Distal Biceps Tendon

    PubMed Central

    Walton, Christine; Li, Zhi; Pennings, Amanda; Agur, Anne; Elmaraghy, Amr

    2015-01-01

    Background Complete rupture of the distal biceps tendon from its osseous attachment is most often treated with operative intervention. Knowledge of the overall tendon morphology as well as the orientation of the collagenous fibers throughout the musculotendinous junction are key to intraoperative decision making and surgical technique in both the acute and chronic setting. Unfortunately, there is little information available in the literature. Purpose To comprehensively describe the morphology of the distal biceps tendon. Study Design Descriptive laboratory study. Methods The distal biceps terminal musculature, musculotendinous junction, and tendon were digitized in 10 cadaveric specimens and data reconstructed using 3-dimensional modeling. Results The average length, width, and thickness of the external distal biceps tendon were found to be 63.0, 6.0, and 3.0 mm, respectively. A unique expansion of the tendon fibers within the distal muscle was characterized, creating a thick collagenous network along the central component between the long and short heads. Conclusion This study documents the morphologic parameters of the native distal biceps tendon. Reconstruction may be necessary, especially in chronic distal biceps tendon ruptures, if the remaining tendon morphology is significantly compromised compared with the native distal biceps tendon. Knowledge of normal anatomical distal biceps tendon parameters may also guide the selection of a substitute graft with similar morphological characteristics. Clinical Relevance A thorough description of distal biceps tendon morphology is important to guide intraoperative decision making between primary repair and reconstruction and to better select the most appropriate graft. The detailed description of the tendinous expansion into the muscle may provide insight into better graft-weaving and suture-grasping techniques to maximize proximal graft incorporation. PMID:26665092

  14. 3-Dimensional shear wave elastography of breast lesions

    PubMed Central

    Chen, Ya-ling; Chang, Cai; Zeng, Wei; Wang, Fen; Chen, Jia-jian; Qu, Ning

    2016-01-01

    Abstract Color patterns of 3-dimensional (3D) shear wave elastography (SWE) is a promising method in differentiating tumoral nodules recently. This study was to evaluate the diagnostic accuracy of color patterns of 3D SWE in breast lesions, with special emphasis on coronal planes. A total of 198 consecutive women with 198 breast lesions (125 malignant and 73 benign) were included, who underwent conventional ultrasound (US), 3D B-mode, and 3D SWE before surgical excision. SWE color patterns of Views A (transverse), T (sagittal), and C (coronal) were determined. Sensitivity, specificity, and the area under the receiver operating characteristic curve (AUC) were calculated. Distribution of SWE color patterns was significantly different between malignant and benign lesions (P = 0.001). In malignant lesions, “Stiff Rim” was significantly more frequent in View C (crater sign, 60.8%) than in View A (51.2%, P = 0.013) and View T (54.1%, P = 0.035). AUC for combination of “Crater Sign” and conventional US was significantly higher than View A (0.929 vs 0.902, P = 0.004) and View T (0.929 vs 0.907, P = 0.009), and specificity significantly increased (90.4% vs 78.1%, P = 0.013) without significant change in sensitivity (85.6% vs 88.0%, P = 0.664) as compared with conventional US. In conclusion, combination of conventional US with 3D SWE color patterns significantly increased diagnostic accuracy, with “Crater Sign” in coronal plane of the highest value. PMID:27684820

  15. Real-time 3-dimensional echocardiography for prosthetic valve endocarditis: initial experience.

    PubMed

    Kort, Smadar

    2006-02-01

    Real-time 3-dimensional echocardiography is a relatively new technology with rapidly growing potential applications. Prosthetic valve endocarditis is still a challenging diagnosis despite improvements in image qualities obtained by both transthoracic and transesophageal echocardiograms. The purpose of this article is to present 4 cases of suggested prosthetic valve endocarditis, in which real-time 3-dimensional echocardiography was performed, and to discuss the potential use of real-time 3-dimensional echocardiography for this application.

  16. The mechanical microenvironment of high concentration agarose for applying deformation to primary chondrocytes.

    PubMed

    Zignego, Donald L; Jutila, Aaron A; Gelbke, Martin K; Gannon, Daniel M; June, Ronald K

    2014-06-27

    Cartilage and chondrocytes experience loading that causes alterations in chondrocyte biological activity. In vivo chondrocytes are surrounded by a pericellular matrix with a stiffness of ~25-200kPa. Understanding the mechanical loading environment of the chondrocyte is of substantial interest for understanding chondrocyte mechanotransduction. The first objective of this study was to analyze the spatial variability of applied mechanical deformations in physiologically stiff agarose on cellular and sub-cellular length scales. Fluorescent microspheres were embedded in physiologically stiff agarose hydrogels. Microsphere positions were measured via confocal microscopy and used to calculate displacement and strain fields as a function of spatial position. The second objective was to assess the feasibility of encapsulating primary human chondrocytes in physiologically stiff agarose. The third objective was to determine if primary human chondrocytes could deform in high-stiffness agarose gels. Primary human chondrocyte viability was assessed using live-dead imaging following 24 and 72h in tissue culture. Chondrocyte shape was measured before and after application of 10% compression. These data indicate that (1) displacement and strain precision are ~1% and 6.5% respectively, (2) high-stiffness agarose gels can maintain primary human chondrocyte viability of >95%, and (3) compression of chondrocytes in 4.5% agarose can induce shape changes indicative of cellular compression. Overall, these results demonstrate the feasibility of using high-concentration agarose for applying in vitro compression to chondrocytes as a model for understanding how chondrocytes respond to in vivo loading.

  17. Formation of Hyaline Cartilage Tissue by Passaged Human Osteoarthritic Chondrocytes.

    PubMed

    Bianchi, Vanessa J; Weber, Joanna F; Waldman, Stephen D; Backstein, David; Kandel, Rita A

    2017-02-01

    When serially passaged in standard monolayer culture to expand cell number, articular chondrocytes lose their phenotype. This results in the formation of fibrocartilage when they are used clinically, thus limiting their use for cartilage repair therapies. Identifying a way to redifferentiate these cells in vitro is critical if they are to be used successfully. Transforming growth factor beta (TGFβ) family members are known to be crucial for regulating differentiation of fetal limb mesenchymal cells and mesenchymal stromal cells to chondrocytes. As passaged chondrocytes acquire a progenitor-like phenotype, the hypothesis of this study was that TGFβ supplementation will stimulate chondrocyte redifferentiation in vitro in serum-free three-dimensional (3D) culture. Human articular chondrocytes were serially passaged twice (P2) in monolayer culture. P2 cells were then placed in high-density (3D) culture on top of membranes (Millipore) and cultured for up to 6 weeks in chemically defined serum-free redifferentiation media (SFRM) in the presence or absence of TGFβ. The tissues were evaluated histologically, biochemically, by immunohistochemical staining, and biomechanically. Passaged human chondrocytes cultured in SFRM supplemented with 10 ng/mL TGFβ3 consistently formed a continuous layer of articular-like cartilage tissue rich in collagen type 2 and aggrecan and lacking collagen type 1 and X in the absence of a scaffold. The tissue developed a superficial zone characterized by expression of lubricin and clusterin with horizontally aligned collagen fibers. This study suggests that passaged human chondrocytes can be used to bioengineer a continuous layer of articular cartilage-like tissue in vitro scaffold free. Further study is required to evaluate their ability to repair cartilage defects in vivo.

  18. The chondrocytic journey in endochondral bone growth and skeletal dysplasia.

    PubMed

    Yeung Tsang, Kwok; Wa Tsang, Shun; Chan, Danny; Cheah, Kathryn S E

    2014-03-01

    The endochondral bones of the skeleton develop from a cartilage template and grow via a process involving a cascade of chondrocyte differentiation steps culminating in formation of a growth plate and the replacement of cartilage by bone. This process of endochondral ossification, driven by the generation of chondrocytes and their subsequent proliferation, differentiation, and production of extracellular matrix constitute a journey, deviation from which inevitably disrupts bone growth and development, and is the basis of human skeletal dysplasias with a wide range of phenotypic severity, from perinatal lethality to progressively deforming. This highly coordinated journey of chondrocyte specification and fate determination is controlled by a myriad of intrinsic and extrinsic factors. SOX9 is the master transcription factor that, in concert with varying partners along the way, directs the different phases of the journey from mesenchymal condensation, chondrogenesis, differentiation, proliferation, and maturation. Extracellular signals, including bone morphogenetic proteins, wingless-related MMTV integration site (WNT), fibroblast growth factor, Indian hedgehog, and parathyroid hormone-related peptide, are all indispensable for growth plate chondrocytes to align and organize into the appropriate columnar architecture and controls their maturation and transition to hypertrophy. Chondrocyte hypertrophy, marked by dramatic volume increase in phases, is controlled by transcription factors SOX9, Runt-related transcription factor, and FOXA2. Hypertrophic chondrocytes mediate the cartilage to bone transition and concomitantly face a live-or-die situation, a subject of much debate. We review recent insights into the coordination of the phases of the chondrocyte journey, and highlight the need for a systems level understanding of the regulatory networks that will facilitate the development of therapeutic approaches for skeletal dysplasia.

  19. Chondrocytic Atf4 regulates osteoblast differentiation and function via Ihh

    PubMed Central

    Wang, Weiguang; Lian, Na; Ma, Yun; Li, Lingzhen; Gallant, Richard C.; Elefteriou, Florent; Yang, Xiangli

    2012-01-01

    Atf4 is a leucine zipper-containing transcription factor that activates osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation of skeletal development and bone formation is poorly understood. Investigations of the Atf4–/–;Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteogenesis during development and bone remodeling postnatally. Atf4 overexpression in chondrocytes of the Atf4–/–;Col2a1-Atf4 double mutants corrects the reduction in stature and limb in Atf4–/– embryos and rectifies the decrease in Ihh expression, Hh signaling, proliferation and accelerated hypertrophy that characterize the Atf4–/– developing growth plate cartilages. Unexpectedly, this genetic manipulation also restores the expression of osteoblastic marker genes, namely Ocn and bone sialoprotein, in Atf4–/– developing bones. In Atf4–/–;Col2a1-Atf4 adult mice, all the defective bone parameters found in Atf4–/– mice, including bone volume, trabecular number and thickness, and bone formation rate, are rescued. In addition, the conditioned media of ex vivo cultures from wild-type or Atf4–/–;Col2a1-Atf4, but not Atf4–/– cartilage, corrects the differentiation defects of Atf4–/– bone marrow stromal cells and Ihh-blocking antibody eliminates this effect. Together, these data indicate that Atf4 in chondrocytes is required for normal Ihh expression and for its paracrine effect on osteoblast differentiation. Therefore, the cell-autonomous role of Atf4 in chondrocytes dominates the role of Atf4 in osteoblasts during development for the control of early osteogenesis and skeletal growth. PMID:22190639

  20. [Influence of BMP-7 on chondrocyte secretion and expression of Col-II,AGG and Sox9 mRNA in porous tantalum-chondrocyte composites in vitro].

    PubMed

    Zhang, H; Li, L; Wang, Q; Gan, H Q; Wang, H; Bi, C; Li, Q J; Wang, Z Q

    2015-04-18

    To study the influence of bone morphogenetic protein-7 (BMP-7) on chondrocyte secretion and expression of type II collagen (Col-II), aggrecan (AGG) and SRY-related high mobility group-box gene 9 (Sox9) mRNA in porous tantalum-chondrocyte composites. The articular chondrocytes were isolated from 3-week-old New Zealand immature rabbits and identified. The 2nd generation of chondrocytes with 1×10(6)/mL inoculate concentration was seeded in porous tantalum and divided into 4 groups, and control group (tantalum/chondrocyte), 50 μg/L BMP-7 group (50 μg/L BMP-7/tantalum/chondrocyte), 100 μg/L BMP-7 group (100 μg/L BMP-7/tantalum/chondrocyte), and 200 μg/L BMP-7 group (200 μg/L BMP-7/tantalum/chondrocyte). The proliferation of chondrocytes was measured by CCK-8 assay. The chondrocyte growth and morphology were observed by scanning electron microscopy (SEM). The synthesis of glycosaminoglycan (GAG) in chondrocytes was tested by dimethyl methylene blue (DMMB) colorimetric quantification method. Col-II, AGG and Sox9 mRNA in chondrocytes were detected by real-time PCR. The chondrocytes were spindle-shaped in 24 hours of primary cell culture and most cells became polygonal shaped in 4 days. The chondrocytes were affirmed by alcian blue, safranin O and Col-II immunocytochemistry staining. The result of CCK-8 assay showed that the level of cell proliferation in 100 μg/L BMP-7 groups were higher than those in the other groups (P<0.05). The chondrocytes implanted into porous tantalum scaffolds with BMP-7 had better functions, by which cytoplasmic processes developed and extended to the surface and inner of porous tantalum by SEM observation. DMMB quantitative determination of GAG showed that GAG amount of chondrocytes in 100 μg/L BMP-7 groups was significantly higher than those in the other groups (P<0.05). The expressions of Col-II, AGG and Sox9 mRNA in chondrocytes were up-regulated in the experimental groups, compared with the control group and the best effect appeared

  1. Quantitative comparison of operative skill using 2- and 3-dimensional monitors during laparoscopic phantom tasks.

    PubMed

    Nishi, Masayasu; Kanaji, Shingo; Otake, Yoshito; Harada, Hitoshi; Yamamoto, Masashi; Oshikiri, Taro; Nakamura, Tetsu; Suzuki, Satoshi; Suzuki, Yuki; Hiasa, Yuta; Sato, Yoshinobu; Kakeji, Yoshihiro

    2017-05-01

    The recent development of stereoscopic images using 3-dimensional monitors is expected to improve techniques for laparoscopic operation. Several studies have reported technical advantages in using 3-dimensional monitors with regard to operative accuracy and working speed, but there are few reports that analyze forceps motions by 3-dimensional optical tracking systems during standardized laparoscopic phantom tasks. We attempted to develop a 3-dimensional motion analysis system for assessing laparoscopic tasks and to clarify the efficacy of using stereoscopic images from a 3-dimensional monitor to track forceps movement during laparoscopy. Twenty surgeons performed 3 tasks (Task 1: a simple operation by the dominant hand, Task 2: a simple operation using both hands, Task 3: a complicated operation using both hands) under 2-dimensional and 3-dimensional systems. We tracked and recorded the motion of forceps tips with an optical marker captured by a 3-dimensional position tracker. We analyzed factors such as forceps path lengths, operation times, and technical errors for each task and compared the results of 2-dimensional and 3-dimensional monitors. Mean operation times and technical errors were improved significantly for all tasks performed under the 3-dimensional system compared with the 2-dimensional system; in addition, mean path lengths for the forceps tips were shorter for all tasks performed under the 3-dimensional system. We found that stereoscopic images using a 3-dimensional monitor improved operative techniques with regard to increased accuracy and shorter path lengths for forceps movement, which resulted in a shorter operation time for basic phantom laparoscopic tasks. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Protective effect of Capparis spinosa on chondrocytes.

    PubMed

    Panico, A M; Cardile, V; Garufi, F; Puglia, C; Bonina, F; Ronsisvalle, G

    2005-09-30

    The aim of the present study was to evaluate the in vitro chondroprotective effects of the lyophilised methanolic extract from flowering buds of Capparis Spinosa L (LECS). This plant, common to the Mediterranean basin, has been used by the traditional medicine for its diuretic and antihypertensive effects and also in certain pathological conditions related to uncontrolled lipid peroxidation. The extract contains many constituents, in particular some flavonoids (kaempferol and quercetin derivatives) and hydrocinammic acids with several known biological effects such as the anti-inflammatory and the antioxidant ones. In this study, we assayed the effect of LECS on human chondrocytes cultures stimulated by proinflammatory cytokine interleukin-1beta (IL-1beta) and we determined the production of key molecules released during chronic inflammatory events (nitric oxide, glycosaminoglycans, prostaglandins and reactive oxygen species). We observed that LECS was able to counteract the harmful effects induced by IL-1beta. This protection appeared to be greater than that elicited by indomethacin, which is usually employed in joint diseases. Since LECS possess a chondroprotective effect, it might be used in the management of cartilage damage during the inflammatory processes.

  3. Customized biomaterials to augment chondrocyte gene therapy.

    PubMed

    Aguilar, Izath Nizeet; Trippel, Stephen; Shi, Shuiliang; Bonassar, Lawrence J

    2017-02-07

    A persistent challenge in enhancing gene therapy is the transient availability of the target gene product. This is particularly true in tissue engineering applications. The transient exposure of cells to the product could be insufficient to promote tissue regeneration. Here we report the development of a new material engineered to have a high affinity for a therapeutic gene product. We focus on insulin-like growth factor-I (IGF-I) for its highly anabolic effects on many tissues such as spinal cord, heart, brain and cartilage. One of the ways that tissues store IGF-I is through a group of insulin like growth factor binding proteins (IGFBPs), such as IGFBP-5. We grafted the IGF-I binding peptide sequence from IGFBP-5 onto alginate in order to retain the endogenous IGF-I produced by transfected chondrocytes. This novel material bound IGF-I and released the growth factor for at least 30days in culture. We found that this binding enhanced the biosynthesis of transfected cells up to 19-fold. These data demonstrate the coordinated engineering of cell behavior and material chemistry to greatly enhance extracellular matrix synthesis and tissue assembly, and can serve as a template for the enhanced performance of other therapeutic proteins.

  4. Focal Adhesion Assembly Induces Phenotypic Changes and Dedifferentiation in Chondrocytes.

    PubMed

    Shin, Hyunjun; Lee, Mi Nam; Choung, Jin Seung; Kim, Sanghee; Choi, Byung Hyune; Noh, Minsoo; Shin, Jennifer H

    2016-08-01

    The expansion of autologous chondrocytes in vitro is used to generate sufficient populations for cell-based therapies. However, during monolayer culture, chondrocytes lose inherent characteristics and shift to fibroblast-like cells as passage number increase. Here, we investigated passage-dependent changes in cellular physiology, including cellular morphology, motility, and gene and protein expression, as well as the role of focal adhesion and cytoskeletal regulation in the dedifferentiation process. We found that the gene and protein expression levels of both the focal adhesion complex and small Rho GTPases are upregulated with increasing passage number and are closely linked to chondrocyte dedifferentiation. The inhibition of focal adhesion kinase (FAK) but not small Rho GTPases induced the loss of fibroblastic traits and the recovery of collagen type II, aggrecan, and SOX9 expression levels in dedifferentiated chondrocytes. Based on these findings, we propose a strategy to suppress chondrogenic dedifferentiation by inhibiting the identified FAK or Src pathways while maintaining the expansion capability of chondrocytes in a 2D environment. These results highlight a potential therapeutic target for the treatment of skeletal diseases and the generation of cartilage in tissue-engineering approaches. J. Cell. Physiol. 231: 1822-1831, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  5. Nasal chondrocytes and fibrin sealant for cartilage tissue engineering.

    PubMed

    Vinatier, C; Gauthier, O; Masson, M; Malard, O; Moreau, A; Fellah, B H; Bilban, M; Spaethe, R; Daculsi, G; Guicheux, J

    2009-04-01

    Hybrid constructs associating a biodegradable matrix and autologous chondrocytes hold promise for the treatment of articular cartilage defects. In this context, our objective was to investigate the potential use of nasal chondrocytes associated with a fibrin sealant for the treatment of articular cartilage defects. The phenotype of primary nasal chondrocytes (NC) from human (HNC) and rabbit (RNC) origin were characterized by RT-PCR. The ability of constructs associating fibrin sealant and NC to form a cartilaginous tissue in vivo was investigated, firstly in a subcutaneous site in nude mice and secondly in an articular cartilage defect in rabbit. HNC express type II collagen and aggrecan, the two major hallmarks of a chondrocytic phenotype. Furthermore, when injected subcutaneously into nude mice within a fibrin sealant, these chondrocytes were able to form a cartilage-like tissue. Our data indicate that RNC also express type II collagen and aggrecan and maintained their phenotype in three-dimensional culture within a fibrin sealant. Moreover, treatment of rabbit articular cartilage defects with autologous RNC embedded in a fibrin sealant led to the formation of a hyalin-like repair tissue. The use of fibrin sealant containing hybrid autologous NC therefore appears as a promising approach for cell-based therapy of articular cartilage. Copyright 2008 Wiley Periodicals, Inc.

  6. The Signaling Pathways Involved in Chondrocyte Differentiation and Hypertrophic Differentiation

    PubMed Central

    Li, Jianmei

    2016-01-01

    Chondrocytes communicate with each other mainly via diffusible signals rather than direct cell-to-cell contact. The chondrogenic differentiation of mesenchymal stem cells (MSCs) is well regulated by the interactions of varieties of growth factors, cytokines, and signaling molecules. A number of critical signaling molecules have been identified to regulate the differentiation of chondrocyte from mesenchymal progenitor cells to their terminal maturation of hypertrophic chondrocytes, including bone morphogenetic proteins (BMPs), SRY-related high-mobility group-box gene 9 (Sox9), parathyroid hormone-related peptide (PTHrP), Indian hedgehog (Ihh), fibroblast growth factor receptor 3 (FGFR3), and β-catenin. Except for these molecules, other factors such as adenosine, O2 tension, and reactive oxygen species (ROS) also have a vital role in cartilage formation and chondrocyte maturation. Here, we outlined the complex transcriptional network and the function of key factors in this network that determine and regulate the genetic program of chondrogenesis and chondrocyte differentiation. PMID:28074096

  7. Autologous chondrocyte implantation: superior biologic properties of hyaline cartilage repairs.

    PubMed

    Henderson, Ian; Lavigne, Patrick; Valenzuela, Herminio; Oakes, Barry

    2007-02-01

    Information regarding the quality of autologous chondrocyte implantation repair is needed to determine whether the current autologous chondrocyte implantation surgical technology and the subsequent biologic repair processes are capable of reliably forming durable hyaline or hyaline-like cartilage in vivo. We report and analyze the properties and qualities of autologous chondrocyte implantation repairs. We evaluated 66 autologous chondrocyte implantation repairs in 57 patients, 55 of whom had histology, indentometry, and International Cartilage Repair Society repair scoring at reoperation for mechanical symptoms or pain. International Knee Documentation Committee scores were used to address clinical outcome. Maximum stiffness, normalized stiffness, and International Cartilage Repair Society repair scoring were higher for hyaline articular cartilage repairs compared with fibrocartilage, with no difference in clinical outcome. Reoperations revealed 32 macroscopically abnormal repairs (Group B) and 23 knees with normal-looking repairs in which symptoms leading to arthroscopy were accounted for by other joint disorders (Group A). In Group A, 65% of repairs were either hyaline or hyaline-like cartilage compared with 28% in Group B. Autologous chondrocyte repairs composed of fibrocartilage showed more morphologic abnormalities and became symptomatic earlier than hyaline or hyaline-like cartilage repairs. The hyaline articular cartilage repairs had biomechanical properties comparable to surrounding cartilage and superior to those associated with fibrocartilage repairs.

  8. Roles of Chondrocytes in Endochondral Bone Formation and Fracture Repair.

    PubMed

    Hinton, R J; Jing, Y; Jing, J; Feng, J Q

    2017-01-01

    The formation of the mandibular condylar cartilage (MCC) and its subchondral bone is an important but understudied topic in dental research. The current concept regarding endochondral bone formation postulates that most hypertrophic chondrocytes undergo programmed cell death prior to bone formation. Under this paradigm, the MCC and its underlying bone are thought to result from 2 closely linked but separate processes: chondrogenesis and osteogenesis. However, recent investigations using cell lineage tracing techniques have demonstrated that many, perhaps the majority, of bone cells are derived via direct transformation from chondrocytes. In this review, the authors will briefly discuss the history of this idea and describe recent studies that clearly demonstrate that the direct transformation of chondrocytes into bone cells is common in both long bone and mandibular condyle development and during bone fracture repair. The authors will also provide new evidence of a distinct difference in ossification orientation in the condylar ramus (1 ossification center) versus long bone ossification formation (2 ossification centers). Based on our recent findings and those of other laboratories, we propose a new model that contrasts the mode of bone formation in much of the mandibular ramus (chondrocyte-derived) with intramembranous bone formation of the mandibular body (non-chondrocyte-derived).

  9. Osmotic Challenge Drives Rapid and Reversible Chromatin Condensation in Chondrocytes

    PubMed Central

    Irianto, Jerome; Swift, Joe; Martins, Rui P.; McPhail, Graham D.; Knight, Martin M.; Discher, Dennis E.; Lee, David A.

    2013-01-01

    Changes in extracellular osmolality have been shown to alter gene expression patterns and metabolic activity of various cell types, including chondrocytes. However, mechanisms by which physiological or pathological changes in osmolality impact chondrocyte function remain unclear. Here we use quantitative image analysis, electron microscopy, and a DNase I assay to show that hyperosmotic conditions (>400 mOsm/kg) induce chromatin condensation, while hypoosmotic conditions (100 mOsm/kg) cause decondensation. Large density changes (p < 0.001) occur over a very narrow range of physiological osmolalities, which suggests that chondrocytes likely experience chromatin condensation and decondensation during a daily loading cycle. The effect of changes in osmolality on nuclear morphology (p < 0.01) and chromatin condensation (p < 0.001) also differed between chondrocytes in monolayer culture and three-dimensional agarose, suggesting a role for cell adhesion. The relationship between condensation and osmolality was accurately modeled by a polymer gel model which, along with the rapid nature of the chromatin condensation (<20 s), reveals the basic physicochemical nature of the process. Alterations in chromatin structure are expected to influence gene expression and thereby regulate chondrocyte activity in response to osmotic changes. PMID:23442954

  10. Repair Mechanism of Osteochondral Defect Promoted by Bioengineered Chondrocyte Sheet

    PubMed Central

    Kamei, Naosuke; Adachi, Nobuo; Hamanishi, Michio; Kamei, Goki; Mahmoud, Elhussein Elbadry; Nakano, Tomohiro; Iwata, Takanori; Yamato, Masayuki; Okano, Teruo; Ochi, Mitsuo

    2015-01-01

    Cell sheet engineering has developed as a remarkable method for cell transplantation. In the field of cartilage regeneration, several studies previously reported that cartilage defects could be regenerated by transplantation of a chondrocyte sheet using cell sheet engineering. However, it remains unclear how such a thin cell sheet could repair a deep cartilage defect. We, therefore, focused on the mechanism of cartilage repair using cell sheet engineering in this study. Chondrocyte sheets and synovial cell sheets were fabricated using cell sheet engineering, and these allogenic cell sheets were transplanted to cover an osteochondral defect in a rat model. Macroscopic and histological evaluation was performed at 4 and 12 weeks after transplantation. Analysis of the gene expression of each cell sheet and of the regenerated tissue at 1 week after transplantation was performed. In addition, green fluorescent protein (GFP) transgenic rats were used as donors (transplanted chondrocyte sheets) or recipients (osteochondral defect models) to identify the cell origin of regenerated cartilage. Cartilage repair was significantly better in the group implanted with a chondrocyte sheet than in that with a synovial cell sheet. The results of gene expression analysis suggest that the possible factor contributing to cartilage repair might be TGFβ1. Cell tracking experiments using GFP transgenic rats showed that the regenerated cartilage was largely composed of cells derived from the transplanted chondrocyte sheets. PMID:25396711

  11. LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation.

    PubMed

    Asai, Nobuyuki; Ohkawara, Bisei; Ito, Mikako; Masuda, Akio; Ishiguro, Naoki; Ohno, Kinji

    2014-08-22

    Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes.

    PubMed

    Racz, Boglarka; Reglodi, Dora; Fodor, Barnabas; Gasz, Balazs; Lubics, Andrea; Gallyas, Ferenc; Roth, Erzsebet; Borsiczky, Balazs

    2007-06-01

    Articular chondrocytes have a well-developed osmoregulatory system that enables cells to survive in a constantly changing osmotic environment. However, osmotic loading exceeding that occurring under physiological conditions severely compromises chondrocyte function and leads to degenerative changes. The aim of the present study was to investigate the form of cell death and changes in apoptotic signaling pathways under hyperosmotic stress using a primary chondrocyte culture. Cell viability and apoptosis assays performed with annexin V and propidium iodide staining showed that a highly hyperosmotic medium (600 mOsm) severely reduced chondrocyte viability and led mainly to apoptotic cell death, while elevating osmotic pressure within the physiological range caused no changes compared to isosmotic conditions. Western blot analysis revealed that a 600 mOsm hyperosmotic environment induced the activation of proapoptotic members of the mitogen-activated protein kinase family such as c-Jun N-terminal kinase (JNK) and p38, and led to an increased level of extracellular signal regulated kinase (ERK1/2). Hyperosmotic stress also induced the activation of caspase-3. In summary, our results show that hyperosmotic stress leads to mainly apoptotic cell death via the involvement of proapoptotic signaling pathways in a primary chondrocyte culture.

  13. The influence of scaffold material on chondrocytes under inflammatory conditions.

    PubMed

    Kwon, Heenam; Sun, Lin; Cairns, Dana M; Rainbow, Roshni S; Preda, Rucsanda C; Kaplan, David L; Zeng, Li

    2013-05-01

    Cartilage tissue engineering aims to repair damaged cartilage tissue in arthritic joints. As arthritic joints have significantly higher levels of pro-inflammatory cytokines (such as IL-1β and TNFα that cause cartilage destruction, it is critical to engineer stable cartilage in an inflammatory environment. Biomaterial scaffolds constitute an important component of the microenvironment for chondrocytes in engineered cartilage. However, it remains unclear how the scaffold material influences the response of chondrocytes seeded in these scaffolds under inflammatory stimuli. Here we have compared the responses of articular chondrocytes seeded within three different polymeric scaffolding materials (silk, collagen and polylactic acid (PLA)) to IL-1β and TNFα. These scaffolds have different physical characteristics and yielded significant differences in the expression of genes associated with cartilage matrix production and degradation, cell adhesion and cell death. The silk and collagen scaffolds released pro-inflammatory cytokines faster and had higher uptake water abilities than PLA scaffolds. Correspondingly, chondrocytes cultured in silk and collagen scaffolds maintained higher levels of cartilage matrix than those in PLA, suggesting that these biophysical properties of scaffolds may regulate gene expression and the response to inflammatory stimuli in chondrocytes. Based on this study we conclude that selecting the proper scaffold material will aid in the engineering of more stable cartilage tissues for cartilage repair, and that silk and collagen are better scaffolds in terms of supporting the stability of three-dimensional cartilage under inflammatory conditions.

  14. Autophagy modulates articular cartilage vesicle formation in primary articular chondrocytes.

    PubMed

    Rosenthal, Ann K; Gohr, Claudia M; Mitton-Fitzgerald, Elizabeth; Grewal, Rupinder; Ninomiya, James; Coyne, Carolyn B; Jackson, William T

    2015-05-22

    Chondrocyte-derived extracellular organelles known as articular cartilage vesicles (ACVs) participate in non-classical protein secretion, intercellular communication, and pathologic calcification. Factors affecting ACV formation and release remain poorly characterized; although in some cell types, the generation of extracellular vesicles is associated with up-regulation of autophagy. We sought to determine the role of autophagy in ACV production by primary articular chondrocytes. Using an innovative dynamic model with a light scatter nanoparticle counting apparatus, we determined the effects of autophagy modulators on ACV number and content in conditioned medium from normal adult porcine and human osteoarthritic chondrocytes. Healthy articular chondrocytes release ACVs into conditioned medium and show significant levels of ongoing autophagy. Rapamycin, which promotes autophagy, increased ACV numbers in a dose- and time-dependent manner associated with increased levels of autophagy markers and autophagosome formation. These effects were suppressed by pharmacologic autophagy inhibitors and short interfering RNA for ATG5. Caspase-3 inhibition and a Rho/ROCK inhibitor prevented rapamycin-induced increases in ACV number. Osteoarthritic chondrocytes, which are deficient in autophagy, did not increase ACV number in response to rapamycin. SMER28, which induces autophagy via an mTOR-independent mechanism, also increased ACV number. ACVs induced under all conditions had similar ecto-enzyme specific activities and types of RNA, and all ACVs contained LC3, an autophagosome-resident protein. These findings identify autophagy as a critical participant in ACV formation, and augment our understanding of ACVs in cartilage disease and repair.

  15. Autophagy Modulates Articular Cartilage Vesicle Formation in Primary Articular Chondrocytes*

    PubMed Central

    Rosenthal, Ann K.; Gohr, Claudia M.; Mitton-Fitzgerald, Elizabeth; Grewal, Rupinder; Ninomiya, James; Coyne, Carolyn B.; Jackson, William T.

    2015-01-01

    Chondrocyte-derived extracellular organelles known as articular cartilage vesicles (ACVs) participate in non-classical protein secretion, intercellular communication, and pathologic calcification. Factors affecting ACV formation and release remain poorly characterized; although in some cell types, the generation of extracellular vesicles is associated with up-regulation of autophagy. We sought to determine the role of autophagy in ACV production by primary articular chondrocytes. Using an innovative dynamic model with a light scatter nanoparticle counting apparatus, we determined the effects of autophagy modulators on ACV number and content in conditioned medium from normal adult porcine and human osteoarthritic chondrocytes. Healthy articular chondrocytes release ACVs into conditioned medium and show significant levels of ongoing autophagy. Rapamycin, which promotes autophagy, increased ACV numbers in a dose- and time-dependent manner associated with increased levels of autophagy markers and autophagosome formation. These effects were suppressed by pharmacologic autophagy inhibitors and short interfering RNA for ATG5. Caspase-3 inhibition and a Rho/ROCK inhibitor prevented rapamycin-induced increases in ACV number. Osteoarthritic chondrocytes, which are deficient in autophagy, did not increase ACV number in response to rapamycin. SMER28, which induces autophagy via an mTOR-independent mechanism, also increased ACV number. ACVs induced under all conditions had similar ecto-enzyme specific activities and types of RNA, and all ACVs contained LC3, an autophagosome-resident protein. These findings identify autophagy as a critical participant in ACV formation, and augment our understanding of ACVs in cartilage disease and repair. PMID:25869133

  16. Method and apparatus for imaging through 3-dimensional tracking of protons

    NASA Technical Reports Server (NTRS)

    Ryan, James M. (Inventor); Macri, John R. (Inventor); McConnell, Mark L. (Inventor)

    2001-01-01

    A method and apparatus for creating density images of an object through the 3-dimensional tracking of protons that have passed through the object are provided. More specifically, the 3-dimensional tracking of the protons is accomplished by gathering and analyzing images of the ionization tracks of the protons in a closely packed stack of scintillating fibers.

  17. Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification.

    PubMed

    Ferrari, Deborah; Kosher, Robert A

    2002-12-15

    The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes. The expansion in the extent of hypertrophic maturation detectable histologically is accompanied by expanded and upregulated domains of expression of molecular markers of chondrocyte maturation, particularly type X collagen and osteopontin, and by expansion of mineralized cartilage matrix, which is characteristic of terminal hypertrophic differentiation. Furthermore, Dlx5 misexpression markedly reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together, these results indicate that Dlx5 is a positive regulator of chondrocyte maturation and suggest that it regulates the process at least in part by promoting conversion of immature proliferating chondrocytes into hypertrophying chondrocytes. Retroviral misexpression of Dlx5 also enhances formation of periosteal bone, which is derived from the Dlx5-expressing perichondrium that surrounds the diaphyses of the cartilage models. This suggests that Dlx5 may be involved in regulating osteoblast differentiation, as well as chondrocyte maturation, during endochondral ossification.

  18. Expression Pattern and Role of Chondrocyte Clusters in Osteoarthritic Human Knee Cartilage

    PubMed Central

    Hoshiyama, Yoshiaki; Otsuki, Shuhei; Oda, Shuhei; Kurokawa, Yoshitaka; Nakajima, Mikio; Jotoku, Tsuyoshi; Tamura, Ryuichi; Okamoto, Yoshinori; Lotz, Martin K.; Neo, Masashi

    2015-01-01

    The purpose of this study was to investigate the site-specific expression pattern and the role of chondrocyte clusters in human OA knee. Cartilage explants were obtained from 45 varus knees of medial and lateral femoral condyle undergoing total knee replacement surgery. Cartilage degeneration, number of chondrocytes, and the cell arrangement were evaluated by live/dead assay and immunohistochemical analyses with antibodies of STRO-1, FGF2, and Ki-67. Chondrocytes from medial and lateral femoral condyle were cultured to compare the potential of cell proliferation and production of cartilaginous nodules. Finally, cartilage tissue from medial femoral condyle, which included cartilage cleft with chondrocyte clusters, was observed the histological alternation. As the results, chondrocyte density adjacent to severe cartilage degeneration was highest, whereas chondrocytes in lateral femoral condyle displayed low density with single type of cells. Over 80% of these chondrocyte clusters were survived, expressing STRO-1, FGF2, and Ki-67. Furthermore, chondrocyte clusters proliferated faster and produced more cartilaginous nodules than single type of chondrocytes. Cartilage clefts involving numerous chondrocyte clusters were filled with extracellular matrix during organ culture. In conclusion, chondrocyte clusters adjacent to severe cartilage degeneration have shown completely specific characteristics with progenitor and proliferative potential. Regulating chondrocyte clusters may offer new approaches to cartilage repair and OA therapy in the future. PMID:25691232

  19. Deciphering chondrocyte behaviour in matrix-induced autologous chondrocyte implantation to undergo accurate cartilage repair with hyaline matrix.

    PubMed

    Demoor, M; Maneix, L; Ollitrault, D; Legendre, F; Duval, E; Claus, S; Mallein-Gerin, F; Moslemi, S; Boumediene, K; Galera, P

    2012-06-01

    Since the emergence in the 1990s of the autologous chondrocytes transplantation (ACT) in the treatment of cartilage defects, the technique, corresponding initially to implantation of chondrocytes, previously isolated and amplified in vitro, under a periosteal membrane, has greatly evolved. Indeed, the first generations of ACT showed their limits, with in particular the dedifferentiation of chondrocytes during the monolayer culture, inducing the synthesis of fibroblastic collagens, notably type I collagen to the detriment of type II collagen. Beyond the clinical aspect with its encouraging results, new biological substitutes must be tested to obtain a hyaline neocartilage. Therefore, the use of differentiated chondrocytes phenotypically stabilized is essential for the success of ACT at medium and long-term. That is why researchers try now to develop more reliable culture techniques, using among others, new types of biomaterials and molecules known for their chondrogenic activity, giving rise to the 4th generation of ACT. Other sources of cells, being able to follow chondrogenesis program, are also studied. The success of the cartilage regenerative medicine is based on the phenotypic status of the chondrocyte and on one of its essential component of the cartilage, type II collagen, the expression of which should be supported without induction of type I collagen. The knowledge accumulated by the scientific community and the experience of the clinicians will certainly allow to relief this technological challenge, which influence besides, the validation of such biological substitutes by the sanitary authorities. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  20. Astaxanthin reduces matrix metalloproteinase expression in human chondrocytes.

    PubMed

    Chen, Wei-Ping; Xiong, Yan; Shi, Yong-Xiang; Hu, Peng-Fei; Bao, Jia-Peng; Wu, Li-Dong

    2014-03-01

    Astaxanthin is a red carotenoid pigment which exerts multiple biological activities. However, little is known about the effects of astaxanthin on matrix metalloproteinases (MMPs) in OA. The present study investigated the effects of astaxanthin on MMPs in human chondrocytes. Human chondrocytes were pretreated with astaxanthin at 1, 10 or 50μM, then, cells were stimulated with IL-1β (10ng/ml) for 24h. MMP-1, MMP-3 and MMP-13 were observed. We found that astaxanthin reduced the expression of MMP-1, MMP-3 and MMP-13 as well as the phosphorylation of two mitogen-activated protein kinases (MAPK) (p38 and ERK1/2) in IL-1β-stimulated chondrocytes. Astaxanthin also blocked the IκB-α degradation. These results suggest that astaxanthin may be beneficial in the treatment of OA. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Epigenetic Regulation of Chondrocyte Catabolism and Anabolism in Osteoarthritis.

    PubMed

    Kim, Hyeonkyeong; Kang, Donghyun; Cho, Yongsik; Kim, Jin-Hong

    2015-08-01

    Osteoarthritis (OA) is one of the most prevalent forms of joint disorder, associated with a tremendous socioeconomic burden worldwide. Various non-genetic and lifestyle-related factors such as aging and obesity have been recognized as major risk factors for OA, underscoring the potential role for epigenetic regulation in the pathogenesis of the disease. OA-associated epigenetic aberrations have been noted at the level of DNA methylation and histone modification in chondrocytes. These epigenetic regulations are implicated in driving an imbalance between the expression of catabolic and anabolic factors, leading eventually to osteoarthritic cartilage destruction. Cellular senescence and metabolic abnormalities driven by OA-associated risk factors appear to accompany epigenetic drifts in chondrocytes. Notably, molecular events associated with metabolic disorders influence epigenetic regulation in chondrocytes, supporting the notion that OA is a metabolic disease. Here, we review accumulating evidence supporting a role for epigenetics in the regulation of cartilage homeostasis and OA pathogenesis.

  2. Chondrocytes Directly Transform into Bone Cells in Mandibular Condyle Growth

    PubMed Central

    Jing, Y.; Zhou, X.; Han, X.; Jing, J.; von der Mark, K.; Wang, J.; de Crombrugghe, B.; Hinton, R.J.; Feng, J.Q.

    2015-01-01

    For decades, it has been widely accepted that hypertrophic chondrocytes undergo apoptosis prior to endochondral bone formation. However, very recent studies in long bone suggest that chondrocytes can directly transform into bone cells. Our initial in vivo characterization of condylar hypertrophic chondrocytes revealed modest numbers of apoptotic cells but high levels of antiapoptotic Bcl-2 expression, some dividing cells, and clear alkaline phosphatase activity (early bone marker). Ex vivo culture of newborn condylar cartilage on a chick chorioallantoic membrane showed that after 5 d the cells on the periphery of the explants had begun to express Col1 (bone marker). The cartilage-specific cell lineage–tracing approach in triple mice containing Rosa 26tdTomato (tracing marker), 2.3 Col1GFP (bone cell marker), and aggrecan CreERT2 (onetime tamoxifen induced) or Col10-Cre (activated from E14.5 throughout adult stage) demonstrated the direct transformation of chondrocytes into bone cells in vivo. This transformation was initiated at the inferior portion of the condylar cartilage, in contrast to the initial ossification site in long bone, which is in the center. Quantitative data from the Col10-Cre compound mice showed that hypertrophic chondrocytes contributed to ~80% of bone cells in subchondral bone, ~70% in a somewhat more inferior region, and ~40% in the most inferior part of the condylar neck (n = 4, P < 0.01 for differences among regions). This multipronged approach clearly demonstrates that a majority of chondrocytes in the fibrocartilaginous condylar cartilage, similar to hyaline cartilage in long bones, directly transform into bone cells during endochondral bone formation. Moreover, ossification is initiated from the inferior portion of mandibular condylar cartilage with expansion in one direction. PMID:26341973

  3. Pericellular coat of chick embryo chondrocytes: structural role of hyaluronate

    PubMed Central

    1984-01-01

    Chondrocytes produce large pericellular coats in vitro that can be visualized by the exclusion of particles, e.g., fixed erythrocytes, and that are removed by treatment with Streptomyces hyaluronidase, which is specific for hyaluronate. In this study, we examined the kinetics of formation of these coats and the relationship of hyaluronate and proteoglycan to coat structure. Chondrocytes were isolated from chick tibia cartilage by collagenase-trypsin digestion and were characterized by their morphology and by their synthesis of both type II collagen and high molecular weight proteoglycans. The degree of spreading of the chondrocytes and the size of the coats were quantitated at various times subsequent to seeding by tracing phase-contrast photomicrographs of the cultures. After seeding, the chondrocytes attached themselves to the tissue culture dish and exhibited coats within 4 h. The coats reached a maximum size after 3-4 d and subsequently decreased over the next 2-3 d. Subcultured chondrocytes produced a large coat only if passaged before 4 d. Both primary and first passage cells, with or without coats, produced type II collagen but not type I collagen as determined by enzyme-linked immunosorbent assay. Treatment with Streptomyces hyaluronidase (1.0 mU/ml, 15 min), which completely removed the coat, released 58% of the chondroitin sulfate but only 9% of the proteins associated with the cell surface. The proteins released by hyaluronidase were not digestible by bacterial collagenase. Monensin and cycloheximide (0.01-10 microM, 48 h) caused a dose-dependent decrease in coat size that was linearly correlated to synthesis of cell surface hyaluronate (r = 0.98) but not chondroitin sulfate (r = 0.2). We conclude that the coat surrounding chondrocytes is dependent on hyaluronate for its structure and that hyaluronate retains a large proportion of the proteoglycan in the coat. PMID:6501414

  4. Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice.

    PubMed

    Zhou, Xin; von der Mark, Klaus; Henry, Stephen; Norton, William; Adams, Henry; de Crombrugghe, Benoit

    2014-12-01

    One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo.

  5. Should human chondrocytes fly? The impact of electromagnetic irradiation on chondrocyte viability and implications for their use in tissue engineering.

    PubMed

    Koehler, C; Niederbichler, A D; Scholz, T; Bode, B; Roos, J; Jung, F J; Hoerstrup, S P; Hellermann, J P; Wedler, V

    2006-12-01

    A significant logistic factor as to the successful clinical application of the autologous tissue engineering concept is efficient transportation: the donor cells need to be delivered to tissue processing facilities which in most cases requires air transportation. This study was designed to evaluate how human chondrocytes react to X-ray exposure. Primary cell cultures were established, cultured, incubated and exposed to different doses and time periods of radiation. Subsequently, quantitative cell proliferation assays were done and qualitative evaluation of cellular protein production were performed. Our results show that after irradiation of chondrocytes with different doses, no significant differences in terms of cellular viability occurred compared with the control group. These results were obtained when chondrocytes were exposed to luggage transillumination doses as well as exposure to clinically used radiation doses. Any damage affecting cell growth or quality was not observed in our study. However, information about damage of cellular DNA remains incomplete.

  6. Virtual 3-dimensional preoperative planning with the dextroscope for excision of a 4th ventricular ependymoma.

    PubMed

    Anil, S M; Kato, Y; Hayakawa, M; Yoshida, K; Nagahisha, S; Kanno, T

    2007-04-01

    Advances in computer imaging and technology have facilitated enhancement in surgical planning with a 3-dimensional model of the surgical plan of action utilizing advanced visualization tools in order to plan individual interactive operations with the aid of the dextroscope. This provides a proper 3-dimensional imaging insight to the pathological anatomy and sets a new dimension in collaboration for training and education. The case of a seventeen-year-old female, being operated with the aid of a preoperative 3-dimensional virtual reality planning and the practical application of the neurosurgical operation, is presented. This young lady presented with a two-year history of recurrent episodes of severe, global, throbbing headache with episodes of projectile vomiting associated with shoulder pain which progressively worsened. She had no obvious neurological deficits on clinical examination. CT and MRI showed a contrast-enhancing midline posterior fossa space-occupying lesion. Utilizing virtual imaging technology with the aid of a dextroscope which generates stereoscopic images, a 3-dimensional image was produced with the CT and MRI images. A preoperative planning for excision of the lesion was made and a real-time 3-dimensional volume was produced and surgical planning with the dextroscope was made and the lesion excised. Virtual reality has brought new proportions in 3-dimensional planning and management of various complex neuroanatomical problems that are faced during various operations. Integration of 3-dimensional imaging with stereoscopic vision makes understanding the complex anatomy easier and helps improve decision making in patient management.

  7. TNF-α Mediates Diabetes-Enhanced Chondrocyte Apoptosis During Fracture Healing and Stimulates Chondrocyte Apoptosis Through FOXO1

    PubMed Central

    Kayal, Rayyan A; Siqueira, Michelle; Alblowi, Jazia; McLean, Jody; Krothapalli, Nanarao; Faibish, Dan; Einhorn, Thomas A; Gerstenfeld, Louis C; Graves, Dana T

    2010-01-01

    To gain insight into the effect of diabetes on fracture healing, experiments were carried out focusing on chondrocyte apoptosis during the transition from cartilage to bone. Type 1 diabetes was induced in mice by multiple low-dose streptozotocin injections, and simple transverse fractures of the tibia or femur was carried out. Large-scale transcriptional profiling and gene set enrichment analysis were performed to examine apoptotic pathways on total RNA isolated from fracture calluses on days 12, 16, and 22, a period of endochondral bone formation when cartilage is resorbed and chondrocyte numbers decrease. Tumor necrosis factor α (TNF-α) protein levels were assessed by ELISA and caspase-3 by bioactivity assay. The role of TNF was examined by treating mice with the TNF-specific inhibitor pegsunercept. In vitro studies investigated the proapoptotic transcription factor FOXO1 in regulating TNF-induced apoptosis of chondrogenic ATDC5 and C3H10T1/2 cells as representative of differentiated chondrocytes, which are important during endochondral ossification. mRNA profiling revealed an upregulation of gene sets related to apoptosis in the diabetic group on day 16 when cartilage resorption is active but not day 12 or day 22. This coincided with elevated TNF-α protein levels, chondrocyte apoptosis, enhanced caspase-3 activity, and increased FOXO1 nuclear translocation (p < .05). Inhibition of TNF significantly reduced these parameters in the diabetic mice but not in normoglycemic control mice (p < .05). Silencing FOXO1 using siRNA in vitro significantly reduced TNF-induced apoptosis and caspase activity in differentiated chondrocytes. The mRNA levels of the proapoptotic genes caspase-3, caspase-8, caspase-9, and TRAIL were significantly reduced with silencing of FOXO1 in chondrocytic cells. Inhibiting caspase-8 and caspase-9 significantly reduced TNF-induced apoptosis in chondrogenic cells. These results suggest that diabetes causes an upregulation of proapoptotic genes

  8. Comparative study of the chondrogenic potential of human bone marrow stromal cells, neonatal chondrocytes and adult chondrocytes

    SciTech Connect

    Saha, Sushmita; Kirkham, Jennifer; Wood, David; Curran, Stephen; Yang, Xuebin

    2010-10-22

    Research highlights: {yields} This study has characterised three different cell types under conditions similar to those used for autologous chondrocyte implantation (ACI) for applications in cartilage repair/regeneration. {yields} Compared for the first time the chondrogenic potential of neonatal chondrocytes with human bone marrow stromal cells (HBMSCs) and adult chondrocytes. {yields} Demonstrated that adult chondrocytes hold greatest potential for use in ACI based on their higher proliferation rates, lower alkaline phosphatise activity and enhanced expression of chondrogenic genes. {yields} Demonstrated the need for chondroinduction as a necessary pre-requisite to efficient chondrogenesis in vitro and, by extrapolation, for cell based therapy (e.g. ACI or cartilage tissue engineering). -- Abstract: Cartilage tissue engineering is still a major clinical challenge with optimisation of a suitable source of cells for cartilage repair/regeneration not yet fully addressed. The aims of this study were to compare and contrast the differences in chondrogenic behaviour between human bone marrow stromal cells (HBMSCs), human neonatal and adult chondrocytes to further our understanding of chondroinduction relative to cell maturity and to identify factors that promote chondrogenesis and maintain functional homoeostasis. Cells were cultured in monolayer in either chondrogenic or basal medium, recapitulating procedures used in existing clinical procedures for cell-based therapies. Cell doubling time, morphology and alkaline phosphatase specific activity (ALPSA) were determined at different time points. Expression of chondrogenic markers (SOX9, ACAN and COL2A1) was compared via real time polymerase chain reaction. Amongst the three cell types studied, HBMSCs had the highest ALPSA in basal culture and lowest ALPSA in chondrogenic media. Neonatal chondrocytes were the most proliferative and adult chondrocytes had the lowest ALPSA in basal media. Gene expression analysis revealed

  9. Repopulation of laser-perforated chondroepiphyseal matrix with xenogeneic chondrocytes: An experimental model

    SciTech Connect

    Caruso, E.M.; Lewandrowski, K.U.; Ohlendorf, C.; Tomford, W.W.; Zaleske, D.J.

    1996-01-01

    Growth of chondrocytes into a xenogeneic chondroepiphyseal matrix was investigated in an in vitro experimental model by combining viable calf chondrocytes with chick epiphyseal matrix devoid of viable chondrocytes. The chondrocytes were harvested from the wrist joints of newborn calves and cultured for 2 days. The epiphyses were harvested from the distal femurs and the proximal tibias of fetal chicks after development was arrested at 17 days by freezing. The epiphyseal specimens were prepared in four ways. These included femoral and tibial epiphyses without holes and femoral and tibial epiphyses with holes made by a laser. These epiphyseal specimens were co-cultured with calf chondrocytes for various periods. After digestion of the epiphyseal matrix, viable chondrocytes were counted in suspension. Chondrocyte division in the matrix was assessed by [{sup 3}H]thymidine incorporation. The growth of calf chondrocytes into the xenogeneic chick matrix was evaluated by fluorescence microscopy on fresh thick epiphyseal sections. The percentage of viable chondrocytes in the xenogeneic epiphyseal matrix increased with culture time to a maximum at day 21. The addition of laser-drilled holes was found to extend a plateau of chondrocyte viability until day 29. A decrease in cell viability was detected at later observation points. This study demonstrates that xenogeneic matrix may serve as a morphogenetic scaffold for chondrocytic growth. 22 refs., 3 figs.

  10. Control of Grasp and Manipulation by Soft Fingers with 3-Dimensional Deformation

    NASA Astrophysics Data System (ADS)

    Nakashima, Akira; Shibata, Takeshi; Hayakawa, Yoshikazu

    In this paper, we consider control of grasp and manipulation of an object in a 3-dimensional space by a 3-fingered hand robot with soft finger tips. We firstly propose a 3-dimensional deformation model of a hemispherical soft finger tip and verify its relevance by experimental data. Second, we consider the contact kinematics and derive the dynamical equations of the fingers and the object where the 3-dimensional deformation is considered. For the system, we thirdly propose a method to regulate the object and the internal force with the information of the hand, the object and the deformation. A simulation result is presented to show the effectiveness of the control method.

  11. Construction of a functional silk-based biomaterial complex with immortalized chondrocytes in vivo.

    PubMed

    Ni, Yusu; Jiang, Yi; Wen, Jianchuan; Shao, Zhenzhong; Chen, Xin; Sun, Shan; Yu, Huiqian; Li, Wen

    2014-04-01

    To explore the feasibility of constructing a functional biomaterial complex with regenerated silk fibroin membrane and immortalized chondrocytes in vivo. Rat auricular chondrocytes (RACs) were transfected with the lentivirus vector pGC-FU-hTERT-3FLAG or pGC-FU-GFP-3FLAG, encoding the human telomerase reverse transcriptase (hTERT) or GFP gene. The effects of regenerated silk fibroin film on the adhesion, growth of immortalized chondrocytes and expression of collagen II in vitro were analyzed with immunofluorescent histochemistry. Immortalized RACs were transformed. Induction by nutrient medium promoted higher expression levels of collagen II in transformed chondrocytes. The regenerated silk fibroin film was not cytotoxic to immortalized chondrocytes and had no adverse influence on their adhesion. Collagen II expression was good in the immortalized chondrocytes in vivo. The construction of a silk-based biomaterial complex with immortalized chondrocytes may provide a feasible kind of functional biomaterial for the repair of cartilage defects in clinical applications.

  12. Generative surgery of cultured autologous auricular chondrocytes for nasal augmentation.

    PubMed

    Yanaga, Hiroko; Imai, Keisuke; Yanaga, Katsu

    2009-11-01

    Conventional treatment for nasal augmentation utilizes autologous grafts, allografts, or synthetic implants such as silicon implants. Silicon implants could protrude/expose or induce nasal bone resorption. Autologous grafts are usually associated with donor site morbidity and the volume of harvested tissue is limited. We had developed a new method for nasal augmentation using cultured autologous chondrocytes (CAC). The current report presents the results of a study using that method with a larger number of patients and an improved graft technique for the nasal tip. Approximately 1 cm2 of cartilage was harvested from the auricular concha and treated with collagenase, and then chondrocytes were obtained. In our multilayer culture system the chondrocytes formed immature cartilaginous tissues with a gelatinous chondroid matrix. They were injection-grafted into the subcutaneous pocket of the nasal dorsum. The chondrocytes with a gelatinous chondroid matrix change from a soft gel to hard neocartilage tissue within 2 to 3 weeks and then stabilize. The authors have used this procedure over a 6-year period on 75 cases: 58 secondary augmentation rhinoplasties following silicon implantation and 17 primary augmentation cases. The results have been satisfactory and long-lasting. Grafting of CAC is an optional method for nasal augmentation and could be used for a wide range of facial augmentation cases.

  13. Effect of thiram on avian growth plate chondrocytes in culture

    USDA-ARS?s Scientific Manuscript database

    Thiram (tetramethyl thiuram disulfide) is a general use pesticide. It causes tibial dyschondroplasia, a cartilage defect in poultry leading to growth plate deformation and lameness. The mechanism of its action on chondrocytes is not understood. Since proteins play significant role in development an...

  14. Polyhexanide and hydrogen peroxide inhibit proteoglycan synthesis of human chondrocytes.

    PubMed

    Röhner, Eric; Hoff, Paula; Winkler, Tobias; von Roth, Philipp; Seeger, Jörn Bengt; Perka, Carsten; Matziolis, Georg

    2011-03-01

    The use of local antiseptics is a common method in septic joint surgery. We tested polyhexanide and hydrogen peroxide, two of the most frequently used antiseptics with high efficacy and low toxicity. The purpose of this study was to evaluate the effects of both antiseptics on the extracellular cartilaginous matrix synthesis of human chondrocytes. Chondrocytes were isolated from donated human knee joints, embedded in alginate beads, and incubated for 10 and 30 minutes with polyhexanide (0.04%), hydrogen peroxide (3%), or phosphate-buffered saline (PBS) for control. Cartilaginous matrix production was quantified through light microscopic analysis of Alcian blue staining. Cell number and morphology were detected by histological analysis. Chondrocytes showed a decreased intensity of blue colouring after antiseptic treatment versus PBS. In contrast to that, neither the cell number per view field nor the cell morphology differed between the groups. Polyhexanide has more toxic potential than hydrogen peroxide. Based on the fact that the cell number and morphology was not altered by the substances at the examined concentrations, the lower intensity of Alcian blue staining of treated chondrocytes indicates a decreased cartilage-specific matrix synthesis by polyhexanide more than by hydrogen peroxide and control.

  15. Effects of concanavalin A on chondrocyte hypertrophy and matrix calcification.

    PubMed

    Yan, W; Pan, H; Ishida, H; Nakashima, K; Suzuki, F; Nishimura, M; Jikko, A; Oda, R; Kato, Y

    1997-03-21

    Resting chondrocytes do not usually undergo differentiation to the hypertrophic stage and calcification. However, incubating these cells with concanavalin A resulted in 10-100-fold increases in alkaline phosphatase activity, binding of 1,25(OH)2-vitamin D3, type X collagen synthesis, 45Ca incorporation into insoluble material, and calcium content. On the other hand, other lectins tested (including wheat germ agglutinin, lentil lectin, pea lectin, phytohemagglutinin-L, and phytohemagglutinin-E) marginally affected alkaline phosphatase activity, although they activate lymphocytes. Methylmannoside reversed the effect of concanavalin A on alkaline phosphatase within 48 h. Concanavalin A did not increase alkaline phosphatase activity in articular chondrocyte cultures. In resting chondrocyte cultures, succinyl concanavalin A was as potent as concanavalin A in increasing alkaline phosphatase activity, the incorporation of [35S]sulfate, D-[3H]glucosamine, and [3H]serine into proteoglycans, and the incorporation of [3H]serine into protein, although concanavalin A, but not succinyl concanavalin A, induced a rapid change in the shape of the cells from flat to spherical. These findings suggest that concanavalin A induces a switch from the resting, to the growth-plate stage, and that this action of concanavalin A is not secondary to changes in the cytoskeleton. Chondrocytes exposed to concanavalin A may be useful as a novel model of endochondral bone formation.

  16. Loading of Articular Cartilage Compromises Chondrocyte Respiratory Function

    PubMed Central

    Coleman, Mitchell C.; Ramakrishnan, Prem S.; Brouillette, Marc J.; Martin, James A.

    2015-01-01

    Objective Determine whether repeatedly overloading healthy cartilage disrupts mitochondrial function in a manner similar to that associated with osteoarthritis pathogenesis. Methods We exposed normal articular cartilage on bovine osteochondral explants to 1 day or 7 consecutive days of cyclic axial compression (0.25 or 1.0 MPa, 0.5 Hz, 3 hours) and evaluated effects on chondrocyte viability, ATP concentration, reactive oxygen species (ROS) production, indicators of oxidative stress, respiration, and mitochondrial membrane potential. Results Neither 0.25 nor 1.0 MPa cyclic compression caused extensive chondrocyte death, macroscopic tissue damage, or overt changes in stress-strain behavior. After one day of loading, differences in respiratory activities between the 0.25 and 1.0 MPa groups were minimal; after 7 loading days, however, respiratory activity and ATP levels were suppressed in the 1.0 MPa group relative to the 0.25 MPa group, an effect prevented with pretreatment with 10 mM N-acetylcysteine. These changes were accompanied by increased proton leakage and decreases in mitochondrial membrane potential as well as by increased ROS formation indicated by dihydroethidium staining and glutathione oxidation. Conclusion Repeated overloading leads to chondrocyte oxidant-dependent mitochondrial dysfunction. This mitochondrial dysfunction may contribute to destabilization of cartilage during various stages of OA in distinct ways by disrupting chondrocyte anabolic responses to mechanical stimuli. PMID:26473613

  17. Confocal microscopy indentation system for studying in situ chondrocyte mechanics.

    PubMed

    Han, Sang-Kuy; Colarusso, Pina; Herzog, Walter

    2009-10-01

    Chondrocytes synthesize extracellular matrix molecules, thus they are essential for the development, adaptation and maintenance of articular cartilage. Furthermore, it is well accepted that the biosynthetic activity of chondrocytes is influenced by the mechanical environment. Therefore, their response to mechanical stimuli has been studied extensively. Much of the knowledge in this area of research has been derived from testing of isolated cells, cartilage explants, and fixed cartilage specimens: systems that differ in important aspects from chondrocytes embedded in articular cartilage and observed during loading conditions. In this study, current model systems have been improved by working with the intact cartilage in real time. An indentation system was designed on a confocal microscope that allows for simultaneous loading and observation of chondrocytes in their native environment. Cell mechanics were then measured under precisely controlled loading conditions. The indentation system is based on a light transmissible cylindrical glass indentor of 0.17 mm thickness and 1.64 mm diameter that is aligned along the focal axis of the microscope and allows for real time observation of live cells in their native environment. The system can be used to study cell deformation and biological responses, such as calcium sparks, while applying prescribed loads on the cartilage surface. It can also provide novel information on the relationship between cell loading and cartilage adaptive/degenerative processes in the intact tissue.

  18. Stored human septal chondrocyte viability analyzed by confocal microscopy.

    PubMed

    Hicks, David L; Sage, August B; Schumacher, Barbara L; Jadin, Kyle D; Agustin, Ramses M; Sah, Robert L; Watson, Deborah

    2006-10-01

    To analyze the effects of prolonged storage time, at warm and cold temperatures, on the viability of human nasal septal chondrocytes and to understand the implications for tissue engineering of septal cartilage. Basic science. Septal cartilage was obtained from 10 patients and placed in bacteriostatic isotonic sodium chloride solution. Four specimens were kept at 23 degrees C, and 4 were kept at 4 degrees C. The viability of the chondrocytes within the cartilage was assessed using confocal laser scanning microscopy every 5 days. The 2 other specimens were assessed for viability on the day of harvest. Viability on the day of harvest was 96%, implying minimal cell death from surgical trauma. After 1 week, cell survival in all specimens was essentially unchanged from the day of harvest. At 23 degrees C, the majority (54%) of cells were alive after 20 days. At 4 degrees C, 70% of cells survived 1 month and 38% were alive at 2 months. Qualitatively, chondrocytes died in a topographically uniform distribution in warm specimens, whereas cold specimens displayed a more irregular pattern of cell death. Septal chondrocytes remain viable for prolonged periods when stored in simple bacteriostatic isotonic sodium chloride solution, and such survival is enhanced by cold storage.

  19. Cartilage homeoprotein 1, a homeoprotein selectively expressed in chondrocytes.

    PubMed

    Zhao, G Q; Zhou, X; Eberspaecher, H; Solursh, M; de Crombrugghe, B

    1993-09-15

    We identified a rat cDNA that encodes cartilage homeoprotein 1 (Cart-1). The deduced amino acid sequence of Cart-1 contains a paired-type homeodomain. Northern blot hybridization and RNase protection assay revealed that Cart-1 RNA was present at high levels in a well-differentiated rat chondrosarcoma tumor and in a cell line derived from this tumor. Cart-1 RNA was detected in primary mouse and rat chondrocytes but not in various fibroblasts including mouse 10T1/2 cells, NIH 3T3 cells, BALB 3T3 cells, and rat skin fibroblasts. It was also undetectable in mouse C2 myoblasts, S194 myeloma cells, and embryonic stem cells. Cart-1 RNA was present at a very low level in tested but was not detected in other soft tissues of 8-week-old rats. In situ hybridization of rat embryos between 14.5 and 16.5 days post coitum revealed relatively high levels of Cart-1 RNA in condensed prechondrocytic mesenchymal cells and in early chondrocytes of cartilage primordia. The levels of Cart-1 RNA were lower in mature chondrocytes. No hybridization was observed in brain, spinal cord, heart, spleen, gastrointestinal tract, liver, and muscle. We speculate that Cart-1 has a role in chondrocyte differentiation.

  20. Cartilage homeoprotein 1, a homeoprotein selectively expressed in chondrocytes.

    PubMed Central

    Zhao, G Q; Zhou, X; Eberspaecher, H; Solursh, M; de Crombrugghe, B

    1993-01-01

    We identified a rat cDNA that encodes cartilage homeoprotein 1 (Cart-1). The deduced amino acid sequence of Cart-1 contains a paired-type homeodomain. Northern blot hybridization and RNase protection assay revealed that Cart-1 RNA was present at high levels in a well-differentiated rat chondrosarcoma tumor and in a cell line derived from this tumor. Cart-1 RNA was detected in primary mouse and rat chondrocytes but not in various fibroblasts including mouse 10T1/2 cells, NIH 3T3 cells, BALB 3T3 cells, and rat skin fibroblasts. It was also undetectable in mouse C2 myoblasts, S194 myeloma cells, and embryonic stem cells. Cart-1 RNA was present at a very low level in tested but was not detected in other soft tissues of 8-week-old rats. In situ hybridization of rat embryos between 14.5 and 16.5 days post coitum revealed relatively high levels of Cart-1 RNA in condensed prechondrocytic mesenchymal cells and in early chondrocytes of cartilage primordia. The levels of Cart-1 RNA were lower in mature chondrocytes. No hybridization was observed in brain, spinal cord, heart, spleen, gastrointestinal tract, liver, and muscle. We speculate that Cart-1 has a role in chondrocyte differentiation. Images Fig. 1 Fig. 2 Fig. 3 PMID:7690966

  1. Insights on Molecular Mechanisms of Chondrocytes Death in Osteoarthritis

    PubMed Central

    Charlier, Edith; Relic, Biserka; Deroyer, Céline; Malaise, Olivier; Neuville, Sophie; Collée, Julie; Malaise, Michel G.; De Seny, Dominique

    2016-01-01

    Osteoarthritis (OA) is a joint pathology characterized by progressive cartilage degradation. Medical care is mainly based on alleviating pain symptoms. Compelling studies report the presence of empty lacunae and hypocellularity in cartilage with aging and OA progression, suggesting that chondrocyte cell death occurs and participates to OA development. However, the relative contribution of apoptosis per se in OA pathogenesis appears complex to evaluate. Indeed, depending on technical approaches, OA stages, cartilage layers, animal models, as well as in vivo or in vitro experiments, the percentage of apoptosis and cell death types can vary. Apoptosis, chondroptosis, necrosis, and autophagic cell death are described in this review. The question of cell death causality in OA progression is also addressed, as well as the molecular pathways leading to cell death in response to the following inducers: Fas, Interleukin-1β (IL-1β), Tumor Necrosis factor-α (TNF-α), leptin, nitric oxide (NO) donors, and mechanical stresses. Furthermore, the protective role of autophagy in chondrocytes is highlighted, as well as its decline during OA progression, enhancing chondrocyte cell death; the transition being mainly controlled by HIF-1α/HIF-2α imbalance. Finally, we have considered whether interfering in chondrocyte apoptosis or promoting autophagy could constitute therapeutic strategies to impede OA progression. PMID:27999417

  2. Influence of cell printing on biological characters of chondrocytes

    PubMed Central

    Qu, Miao; Gao, Xiaoyan; Hou, Yikang; Shen, Congcong; Xu, Yourong; Zhu, Ming; Wang, Hengjian; Xu, Haisong; Chai, Gang; Zhang, Yan

    2015-01-01

    Objective: To establish a two-dimensional biological printing technique of chondrocytes and compare the difference of related biological characters between printed chondrocytes and unprinted cells so as to control the cell transfer process and keep cell viability after printing. Methods: Primary chondrocytes were obtained from human mature and fetal cartilage tissues and then were regularly sub-cultured to harvest cells at passage 2 (P2), which were adjusted to the single cell suspension at a density of 1×106/mL. The experiment was divided into 2 groups: experimental group P2 chondrocytes were transferred by rapid prototype biological printer (driving voltage value 50 V, interval in x-axis 300 μm, interval in y-axis 1500 μm). Afterwards Live/Dead viability Kit and flow cytometry were respectively adopted to detect cell viability; CCK-8 Kit was adopted to detect cell proliferation viability; immunocytochemistry, immunofluorescence and RT-PCR was employed to identify related markers of chondrocytes; control group steps were the same as the printing group except that cell suspension received no printing. Results: Fluorescence microscopy and flow cytometry analyses showed that there was no significant difference between experimental group and control group in terms of cell viability. After 7-day in vitro culture, control group exhibited higher O.D values than experimental group from 2nd day to 7th day but there was no distinct difference between these two groups (P>0.05). Inverted microscope observation demonstrated that the morphology of these two groups had no significant difference either. Similarly, Immunocytochemistry, immunofluorescence and RT-PCR assays also showed that there was no significant difference in the protein and gene expression of type II collagen and aggrecan between these two groups (P>0.05). Conclusion Cell printing has no distinctly negative effect on cell vitality, proliferation and phenotype of chondrocytes. Biological printing technique may

  3. Influence of cell printing on biological characters of chondrocytes.

    PubMed

    Qu, Miao; Gao, Xiaoyan; Hou, Yikang; Shen, Congcong; Xu, Yourong; Zhu, Ming; Wang, Hengjian; Xu, Haisong; Chai, Gang; Zhang, Yan

    2015-01-01

    To establish a two-dimensional biological printing technique of chondrocytes and compare the difference of related biological characters between printed chondrocytes and unprinted cells so as to control the cell transfer process and keep cell viability after printing. Primary chondrocytes were obtained from human mature and fetal cartilage tissues and then were regularly sub-cultured to harvest cells at passage 2 (P2), which were adjusted to the single cell suspension at a density of 1×10(6)/mL. The experiment was divided into 2 groups: experimental group P2 chondrocytes were transferred by rapid prototype biological printer (driving voltage value 50 V, interval in x-axis 300 μm, interval in y-axis 1500 μm). Afterwards Live/Dead viability Kit and flow cytometry were respectively adopted to detect cell viability; CCK-8 Kit was adopted to detect cell proliferation viability; immunocytochemistry, immunofluorescence and RT-PCR was employed to identify related markers of chondrocytes; control group steps were the same as the printing group except that cell suspension received no printing. Fluorescence microscopy and flow cytometry analyses showed that there was no significant difference between experimental group and control group in terms of cell viability. After 7-day in vitro culture, control group exhibited higher O.D values than experimental group from 2nd day to 7th day but there was no distinct difference between these two groups (P>0.05). Inverted microscope observation demonstrated that the morphology of these two groups had no significant difference either. Similarly, Immunocytochemistry, immunofluorescence and RT-PCR assays also showed that there was no significant difference in the protein and gene expression of type II collagen and aggrecan between these two groups (P>0.05). Conclusion Cell printing has no distinctly negative effect on cell vitality, proliferation and phenotype of chondrocytes. Biological printing technique may provide a novel approach

  4. Rail Shear and Short Beam Shear Properties of Various 3-Dimensional (3-D) Woven Composites

    DTIC Science & Technology

    2016-01-01

    Woven Composites by Mark Pankow, Ashiq Quabili, Stephen Whittie, and Chian Yen Approved for public release; distribution...2016 US Army Research Laboratory Rail Shear and Short Beam Shear Properties of Various 3-Dimensional (3-D) Woven Composites by Mark...Properties of Various 3-Dimensional Woven Composites 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Mark Pankow

  5. Efficient, Low-Cost Nucleofection of Passaged Chondrocytes

    PubMed Central

    Parreno, Justin; Delve, Elizabeth; Andrejevic, Katarina; Paez-Parent, Sabrina; Wu, Po-han; Kandel, Rita

    2016-01-01

    Nucleofection of chondrocytes has been shown to be an adequate method of transfection. Using Amaxa’s nucleofection system, transfection efficiencies up to 89% were achievable for vector (pmaxGFP) and 98% for siRNA (siGLO) into passaged chondrocytes. However, such methods rely on costly commercial kits with proprietary reagents limiting its use in basic science labs and in clinical translation. Bovine-passaged chondrocytes were plated in serum reduced media conditionsand then nucleofected using various in laboratory-produced buffers. Cell attachment, confluency, viability, and transfection efficiency was assessed following nucleofection. For each parameter the buffers were scored and a final rank for each buffer was determined. Buffer denoted as 1M resulted in no significant difference for cell attachment, confluency, and viability as compared to non-nucleofected controls. Nucleofection in 1M buffer, in the absence of DNA vectors, resulted in increased col2, ki67, ccnd1 mRNA levels, and decreased col1 mRNA levels at 4 days of culture. Flow cytometry revealed that the transfection efficiency of 1M buffer was comparable to that obtained using the Amaxa commercial kit. siRNA designed against lamin A/C resulted in an average reduction of lamin A and C proteins to 19% and 8% of control levels, respectively. This study identifies a cost-effective, efficient method of nonviral nucleofection of bovine-passaged chondrocytes using known buffer formulations. Human-passaged chondrocytes could also be successfully nucleofected in 1M buffer. Thus this method should facilitate cost-efficient gene targeting of cells used for articular cartilage repair in a research setting. PMID:26958320

  6. Maturational differences in superficial and deep zone articular chondrocytes.

    PubMed

    Hidaka, Chisa; Cheng, Christina; Alexandre, Deborah; Bhargava, Madhu; Torzilli, Peter A

    2006-01-01

    To examine whether differences in chondrocytes from skeletally immature versus adult individuals are important in cartilage healing, repair, or tissue engineering, superficial zone chondrocytes (SZC, from within 100 microm of the articular surface) and deep zone chondrocytes (DZC, from 30%-45% of the deepest un-mineralized part of articular cartilage) were harvested from immature (1-4 months) and young adult (18-36 months) steers and compared. Cell size, matrix gene expression and protein levels, integrin levels, and chemotactic ability were measured in cells maintained in micromass culture for up to 7 days. Regardless of age, SZC were smaller, had a lower type II to type I collagen gene expression ratio, and higher gene expression of SZ proteins than their DZC counterparts. Regardless of zone, chondrocytes from immature steers had higher levels of Sox 9 and type II collagen gene expression. Over 7 days in culture, the SZC of immature steers had the highest rate of proliferation. Phenotypically, the SZC of immature and adult steers were more stable than their respective DZC. Cell surface alpha5 and alpha2 integrin subunit levels were higher in the SZC of immature than of adult steers, whereas beta1 integrin subunit levels were similar. Both immature and adult SZC were capable of chemotaxis in response to fetal bovine serum or basic fibroblast growth factor. Our data indicate that articular chondrocytes vary in the different zones of cartilage and with the age of the donor. These differences may be important for cartilage growth, tissue engineering, and/or repair.

  7. Efficient, Low-Cost Nucleofection of Passaged Chondrocytes.

    PubMed

    Parreno, Justin; Delve, Elizabeth; Andrejevic, Katarina; Paez-Parent, Sabrina; Wu, Po-Han; Kandel, Rita

    2016-01-01

    Nucleofection of chondrocytes has been shown to be an adequate method of transfection. Using Amaxa's nucleofection system, transfection efficiencies up to 89% were achievable for vector (pmaxGFP) and 98% for siRNA (siGLO) into passaged chondrocytes. However, such methods rely on costly commercial kits with proprietary reagents limiting its use in basic science labs and in clinical translation. Bovine-passaged chondrocytes were plated in serum reduced media conditionsand then nucleofected using various in laboratory-produced buffers. Cell attachment, confluency, viability, and transfection efficiency was assessed following nucleofection. For each parameter the buffers were scored and a final rank for each buffer was determined. Buffer denoted as 1M resulted in no significant difference for cell attachment, confluency, and viability as compared to non-nucleofected controls. Nucleofection in 1M buffer, in the absence of DNA vectors, resulted in increased col2, ki67, ccnd1 mRNA levels, and decreased col1 mRNA levels at 4 days of culture. Flow cytometry revealed that the transfection efficiency of 1M buffer was comparable to that obtained using the Amaxa commercial kit. siRNA designed against lamin A/C resulted in an average reduction of lamin A and C proteins to 19% and 8% of control levels, respectively. This study identifies a cost-effective, efficient method of nonviral nucleofection of bovine-passaged chondrocytes using known buffer formulations. Human-passaged chondrocytes could also be successfully nucleofected in 1M buffer. Thus this method should facilitate cost-efficient gene targeting of cells used for articular cartilage repair in a research setting.

  8. Characteristics of tissue-engineered cartilage from human auricular chondrocytes.

    PubMed

    Park, Stephen S; Jin, Hong Ryul; Chi, David H; Taylor, Ray S

    2004-05-01

    This study was done to define the mechanical and histological properties of tissue-engineered cartilage (TEC) derived from human chondrocytes and to compare these findings with those of native cartilage. Chondrocytes were obtained from 10 human auricular cartilages and seeded onto a biodegradable template of polyglycolic acid and poly L-lactic acid. Each template was shaped into a 1 cm x 2 cm rectangle. The templates were implanted in athymic mice for 8 weeks. Eight human auricular cartilages were used for comparison. Mechanical analysis with a tensile testing device provided values of ultimate tensile strength (UTS), stiffness, and resilience. Statistical analysis was performed with the Student's t-test. Histological assessment was done with hematoxylin-eosin staining along with other special stains. The TEC had UTS of 2.07 MPa, stiffness of 3.7 MPa, and resilience of 0.37 J/m3. The control specimens had UTS of 2.18 MPa, stiffness of 5.11 MPa, and resilience of 0.42 J/m3. No statistical difference was found between the experimental and control groups for each of the three parameters. Histological analysis showed mature cartilage with characteristic collagen, glycosaminoglycans, and elastin in the TEC. The neo-cartilage showed slightly smaller size and more irregular distribution of chondrocytes and unique fibrous capsule formation with peripheral infiltration of fibrous tissue. This study showed that the mechanical qualities of TEC from human chondrocytes are similar to those of native auricular cartilage. It suggests that the engineered cartilage from human chondrocytes may have sufficient strength and durability for clinical uses. The histological findings revealed some differences with neo-cartilage.

  9. RAGE, Receptor of Advanced Glycation Endoproducts, Negatively Regulates Chondrocytes Differentiation

    PubMed Central

    Kurosaka, Yuko; Nishimura, Haruka; Tanabe, Motoki; Takakura, Yuuki; Iwai, Keisuke; Waki, Takuya; Fujita, Takashi

    2014-01-01

    RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE) demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms. PMID:25275461

  10. Editorial Commentary: Chondrocytes Trump Ligaments! Partial Release of the Medial Collateral Ligament During Knee Arthroscopy Protects Chondrocytes.

    PubMed

    Leland, J Martin

    2016-10-01

    With knee arthroscopy being the most common orthopaedic procedure performed in the United States, it is crucial to be able to access the entire knee without iatrogenic injury. Frequently orthopaedic surgeons encounter tight medial compartments, creating difficulty in accessing the posterior horn of the medial meniscus without damaging the articular cartilage. Partial release of the medial collateral ligament during knee arthroscopy protects chondrocytes.

  11. Karyotyping of human chondrocytes in scaffold-assisted cartilage tissue engineering.

    PubMed

    Trimborn, Marc; Endres, Michaela; Bommer, Christiane; Janke, Una; Krüger, Jan-Philipp; Morawietz, Lars; Kreuz, Peter C; Kaps, Christian

    2012-04-01

    Scaffold-assisted autologous chondrocyte implantation (ACI) is an effective clinical procedure for cartilage repair. The aim of our study was to evaluate the chromosomal stability of human chondrocytes subjected to typical cell culture procedures needed for regenerative approaches in polymer-scaffold-assisted cartilage repair. Chondrocytes derived from post mortem donors and from donors scheduled for ACI were expanded, cryopreserved and re-arranged in polyglycolic acid (PGA)-fibrin scaffolds for tissue culture. Chondrocyte redifferentiation was analyzed by electron microscopy, histology and gene expression analysis. Karyotyping was performed using GTG banding and fluorescence in situ hybridization on a single cell basis. Chondrocytes showed de- and redifferentiation accompanied by the formation of extracellular matrix and induction of typical chondrocyte marker genes like type II collagen in PGA-fibrin scaffolds. Post mortem chondrocytes showed up to 1.7% structural and high numbers of numerical (up to 26.7%) chromosomal aberrations, while chondrocytes from living donors scheduled for ACI showed up to 1.8% structural and up to 1.3% numerical alterations. Cytogenetically, cell culture procedures and PGA-fibrin scaffolds did not significantly alter chromosomal integrity of the chondrocyte genome. Human chondrocytes derived from living donors subjected to regenerative medicine cell culture procedures like cell expansion, cryopreservation and culture in resorbable polymer-based scaffolds show normal chromosomal integrity and normal karyotypes. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  12. Human adipose-derived stem cells contribute to chondrogenesis in coculture with human articular chondrocytes.

    PubMed

    Hildner, Florian; Concaro, Sebastian; Peterbauer, Anja; Wolbank, Susanne; Danzer, Martin; Lindahl, Anders; Gatenholm, Paul; Redl, Heinz; van Griensven, Martijn

    2009-12-01

    Adipose tissue is easily available and contains high numbers of stem cells that are capable for chondrogenic differentiation. We hypothesize that a partial substitution of chondrocytes with autologous adipose-derived stem cells (ASC) might be a possible strategy to reduce the number of chondrocytes needed in matrix-associated autologous chondrocyte transplantation. To lay the ground, in vitro coculture experiments were performed using human chondrocytes and human ASC. Chondrocytes were obtained from donors undergoing matrix-associated autologous chondrocyte transplantation. ASC were isolated from liposuction material. Chondrocytes and ASC were seeded either in fibrin (Tisseel; Baxter, Vienna, Austria) or collagen matrix (Tissue Fleece; Baxter, Unterschleissheim, Germany). RNA for quantitative reverse transcriptase (RT)-polymerase chain reaction was isolated after 2 weeks of culture in chondrogenic medium, and after 4 weeks samples were processed for histology. Related to the number of chondrocytes used, coculture with ASC led to strong increase in collagen type IX mRNA expression, which is an indicator for long-term stability of cartilage. Moderate upregulation was shown for SOX9, aggrecan, melanoma inhibitory activity, cartilage link protein 1, and cartilage oligomeric matrix protein mRNA. However, expression of collagen I and collagen II indicates the synthesis of fibrous tissue, which might be due to the use of dedifferentiated chondrocytes. Tisseel provided slightly better chondrogenic conditions than Tissue Fleece. These data support the possibility to take advantage of ASC in cartilage regeneration in conjunction with autologous chondrocytes.

  13. Microfluidics‑based optimization of neuroleukin‑mediated regulation of articular chondrocyte proliferation.

    PubMed

    Tian, Kang; Zhong, Weiliang; Zhang, Yingqiu; Yin, Baosheng; Zhang, Weiguo; Liu, Han

    2016-01-01

    Due to the low proliferative and migratory capacities of chondrocytes, cartilage repair remains a challenging clinical problem. Current therapeutic strategies for cartilage repair result in unsatisfactory outcomes. Autologous chondrocyte implantation (ACI) is a cell based therapy that relies on the in vitro expansion of healthy chondrocytes from the patient, during which proliferation‑promoting factors are frequently used. Neuroleukin (NLK) is a multifunctional protein that possesses growth factor functions, and its expression has been associated with cartilage development and bone regeneration, however its direct role in chondrocyte proliferation remains to be fully elucidated. In the current study, the role of NLK in chondrocyte proliferation in vitro in addition to its potential to act as an exogenous factor during ACI was investigated. Furthermore, the concentration of NLK for in vitro chondrocyte culture was optimized using a microfluidic device. An NLK concentration of 12.85 ng/ml was observed to provide optimal conditions for the promotion of chondrocyte proliferation. Additionally, NLK stimulation resulted in an increase in type II collagen synthesis by chondrocytes, which is a cartilaginous secretion marker and associated with the phenotype of chondrocytes. Together these data suggest that NLK is able to promote cell proliferation and type II collagen synthesis during in vitro chondrocyte propagation, and thus may serve as an exogenous factor for ACI.

  14. Conditional expression of constitutively active estrogen receptor {alpha} in chondrocytes impairs longitudinal bone growth in mice

    SciTech Connect

    Ikeda, Kazuhiro; Tsukui, Tohru; Imazawa, Yukiko; Horie-Inoue, Kuniko; Inoue, Satoshi

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Conditional transgenic mice expressing constitutively active estrogen receptor {alpha} (caER{alpha}) in chondrocytes were developed. Black-Right-Pointing-Pointer Expression of caER{alpha} in chondrocytes impaired longitudinal bone growth in mice. Black-Right-Pointing-Pointer caER{alpha} affects chondrocyte proliferation and differentiation. Black-Right-Pointing-Pointer This mouse model is useful for understanding the physiological role of ER{alpha}in vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caER{alpha}{sup ColII}, expressing constitutively active mutant estrogen receptor (ER) {alpha} in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caER{alpha}{sup ColII} mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caER{alpha}{sup ColII} mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caER{alpha}{sup ColII} mice. These results suggest that ER{alpha} is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.

  15. Viability of chondrocytes seeded onto a collagen I/III membrane for matrix-induced autologous chondrocyte implantation.

    PubMed

    Hindle, Paul; Hall, Andrew C; Biant, Leela C

    2014-11-01

    Cell viability is crucial for effective cell-based cartilage repair. The aim of this study was to determine the effect of handling the membrane during matrix-induced autologous chondrocyte implantation surgery on the viability of implanted chondrocytes. Images were acquired under five conditions: (i) Pre-operative; (ii) Handled during surgery; (iii) Cut edge; (iv) Thumb pressure applied; (v) Heavily grasped with forceps. Live and dead cell stains were used. Images were obtained for cell counting and morphology. Mean cell density was 6.60 × 10(5) cells/cm(2) (5.74-7.11 × 10(5) ) in specimens that did not have significant trauma decreasing significantly in specimens that had been grasped with forceps (p < 0.001) or cut (p = 0.004). Cell viability on delivery grade membrane was 75.1%(72.4-77.8%). This dropped to 67.4%(64.1-69.7%) after handling (p = 0.002), 56.3%(51.5-61.6%) after being thumbed (p < 0.001) and 28.8%(24.7-31.2%) after crushing with forceps (p < 0.001). When cut with scissors there was a band of cell death approximately 275 µm in width where cell viability decreased to 13.7%(10.2-18.2%, p < 0.001). Higher magnification revealed cells without the typical rounded appearance of chondrocytes. We found that confocal laser-scanning microscope (CLSM) can be used to quantify and image the fine morphology of cells on a matrix-induced autologous chondrocyte implantation (MACI) membrane. Careful handling of the membrane is essential to minimise chondrocyte death during surgery. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  16. Application of 3-dimensional printing in hand surgery for production of a novel bone reduction clamp.

    PubMed

    Fuller, Sam M; Butz, Daniel R; Vevang, Curt B; Makhlouf, Mansour V

    2014-09-01

    Three-dimensional printing is being rapidly incorporated in the medical field to produce external prosthetics for improved cosmesis and fabricated molds to aid in presurgical planning. Biomedically engineered products from 3-dimensional printers are also utilized as implantable devices for knee arthroplasty, airway orthoses, and other surgical procedures. Although at first expensive and conceptually difficult to construct, 3-dimensional printing is now becoming more affordable and widely accessible. In hand surgery, like many other specialties, new or customized instruments would be desirable; however, the overall production cost restricts their development. We are presenting our step-by-step experience in creating a bone reduction clamp for finger fractures using 3-dimensional printing technology. Using free, downloadable software, a 3-dimensional model of a bone reduction clamp for hand fractures was created based on the senior author's (M.V.M.) specific design, previous experience, and preferences for fracture fixation. Once deemed satisfactory, the computer files were sent to a 3-dimensional printing company for the production of the prototypes. Multiple plastic prototypes were made and adjusted, affording a fast, low-cost working model of the proposed clamp. Once a workable design was obtained, a printing company produced the surgical clamp prototype directly from the 3-dimensional model represented in the computer files. This prototype was used in the operating room, meeting the expectations of the surgeon. Three-dimensional printing is affordable and offers the benefits of reducing production time and nurturing innovations in hand surgery. This article presents a step-by-step description of our design process using online software programs and 3-dimensional printing services. As medical technology advances, it is important that hand surgeons remain aware of available resources, are knowledgeable about how the process works, and are able to take advantage of

  17. Preoperative 3-dimensional Magnetic Resonance Imaging of Uterine Myoma and Endometrium Before Myomectomy.

    PubMed

    Kim, Young Jae; Kim, Kwang Gi; Lee, Sa Ra; Lee, Seung Hyun; Kang, Byung Chul

    2017-02-01

    Uterine myomas are the most common gynecologic benign tumor affecting women of childbearing age, and myomectomy is the main surgical option to preserve the uterus and fertility. During myomectomy for women with multiple myomas, it is advisable to identify and remove as many as possible to decrease the risk of future myomectomies. With deficient preoperative imaging, gynecologists are challenged to identify the location and size of myomas and the endometrium, which, in turn, can lead to uterine rupture during future pregnancies. Current conventional 2-dimensional imaging has limitations in identifying precise locations of multiple myomas and the endometrium. In our experience, we preferred to use 3-dimensional imaging to delineate the myomas, endometrium, or blood vessels, which we were able to successfully reconstruct by using the following imaging method. To achieve 3-dimensional imaging, we matched T2 turbo spin echo images to detect uterine myomas and endometria with T1 high-resolution isotropic volume excitation-post images used to detect blood vessels by using an algorithm based on the 3-dimensional region growing method. Then, we produced images of the uterine myomas, endometria, and blood vessels using a 3-dimensional surface rendering method and successfully reconstructed selective 3-dimensional imaging for uterine myomas, endometria, and adjacent blood vessels. A Web-based survey was sent to 66 gynecologists concerning imaging techniques used before myomectomy. Twenty-eight of 36 responding gynecologists answered that the 3-dimensional image produced in the current study is preferred to conventional 2-dimensional magnetic resonance imaging in identifying precise locations of uterine myomas and endometria. The proposed 3-dimensional magnetic resonance imaging method successfully reconstructed uterine myomas, endometria, and adjacent vessels. We propose that this will be a helpful adjunct to uterine myomectomy as a preoperative imaging technique in future

  18. Harpagoside suppresses IL-6 expression in primary human osteoarthritis chondrocytes.

    PubMed

    Haseeb, Abdul; Ansari, Mohammad Yunus; Haqqi, Tariq M

    2017-02-01

    There is growing evidence in support of the involvement of inflammatory response in the pathogenesis of osteoarthritis (OA). Harpagoside, one of the bioactive components of Harpagophytum procumbens (Hp), has been shown to possess anti-inflammatory properties. Here we used an in vitro model of inflammation in OA to investigate the potential of harpagoside to suppress the production of inflammatory cytokines/chemokines such as IL-6 and matrix degrading proteases. We further investigated the likely targets of harpagoside in primary human OA chondrocytes. OA chondrocytes were pre-treated with harpagoside before stimulation with IL-1β. mRNA expression profile of 92 cytokines/chemokines was determined using TaqMan Human Chemokine PCR Array. Expression levels of selected mRNAs were confirmed using TaqMan assays. Protein levels of IL-6 and MMP-13 were assayed by ELISA and immunoblotting. Total protein levels and phosphorylation of signaling proteins were determined by immunoblotting. Cellular localization of IL-6 and c-Fos was performed by immunofluorescence and confocal microscopy. DNA binding activity of c-FOS/AP-1 was determined by ELISA. Harpagoside significantly altered the global chemokine expression profile in IL-1β-stimulated OA chondrocytes. Expression of IL-6 was highly induced by IL-1β, which was significantly inhibited by pre-treatment of OA chondrocytes with harpagoside. Harpagoside did not inhibit the IL-1β-induced activation of NF-κB and C/EBPβ transcription factors but suppressed the IL-1β-triggered induction, phosphorylation, and DNA binding activity of c-FOS, one of the main components of AP-1 transcription factors. Further, harpagoside significantly inhibited the expression of MMP-13 in OA chondrocytes under pathological conditions. siRNA-mediated knockdown of IL-6 resulted in suppressed expression and secretion of MMP-13 directly linking the role of IL-6 with MMP-13 expression. Taken together, the present study suggests that harpagoside exerts a

  19. Chondrocyte intracellular calcium, cytoskeletal organization, and gene expression responses to dynamic osmotic loading.

    PubMed

    Chao, Pen-Hsiu Grace; West, Alan C; Hung, Clark T

    2006-10-01

    While chondrocytes in articular cartilage experience dynamic stimuli from joint loading activities, few studies have examined the effects of dynamic osmotic loading on their signaling and biosynthetic activities. We hypothesize that dynamic osmotic loading modulates chondrocyte signaling and gene expression differently than static osmotic loading. With the use of a novel microfluidic device developed in our laboratory, dynamic hypotonic loading (-200 mosM) was applied up to 0.1 Hz and chondrocyte calcium signaling, cytoskeleton organization, and gene expression responses were examined. Chondrocytes exhibited decreasing volume and calcium responses with increasing loading frequency. Phalloidin staining showed osmotic loading-induced changes to the actin cytoskeleton in chondrocytes. Real-time PCR analysis revealed a stimulatory effect of dynamic osmotic loading compared with static osmotic loading. These studies illustrate the utility of the microfluidic device in cell signaling investigations, and their potential role in helping to elucidate mechanisms that mediate chondrocyte mechanotransduction to dynamic stimuli.

  20. Axes of resistance for tooth movement: does the center of resistance exist in 3-dimensional space?

    PubMed

    Viecilli, Rodrigo F; Budiman, Amanda; Burstone, Charles J

    2013-02-01

    The center of resistance is considered the most important reference point for tooth movement. It is often stated that forces through this point will result in tooth translation. The purpose of this article is to report the results of numeric experiments testing the hypothesis that centers of resistance do not exist in space as 3-dimensional points, primarily because of the geometric asymmetry of the periodontal ligament. As an alternative theory, we propose that, for an arbitrary tooth, translation references can be determined by 2-dimensional projection intersections of 3-dimensional axes of resistance. Finite element analyses were conducted on a maxillary first molar model to determine the position of the axes of rotation generated by 3-dimensional couples. Translation tests were performed to compare tooth movement by using different combinations of axes of resistance as references. The couple-generated axes of rotation did not intersect in 3 dimensions; therefore, they do not determine a 3-dimensional center of resistance. Translation was obtained by using projection intersections of the 2 axes of resistance perpendicular to the force direction. Three-dimensional axes of resistance, or their 2-dimensional projection intersections, should be used to plan movement of an arbitrary tooth. Clinical approximations to a small 3-dimensional "center of resistance volume" might be adequate in nearly symmetric periodontal ligament cases. Copyright © 2013 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

  1. Dynamic formation of oriented patches in chondrocyte cell cultures.

    PubMed

    Grote, Marcus J; Palumberi, Viviana; Wagner, Barbara; Barbero, Andrea; Martin, Ivan

    2011-10-01

    Growth factors have a significant impact not only on the growth dynamics but also on the phenotype of chondrocytes (Barbero et al. in J. Cell. Phys. 204:830-838, 2005). In particular, as chondrocytes approach confluence, the cells tend to align and form coherent patches. Starting from a mathematical model for fibroblast populations at equilibrium (Mogilner et al. in Physica D 89:346-367, 1996), a dynamic continuum model with logistic growth is developed. Both linear stability analysis and numerical solutions of the time-dependent nonlinear integro-partial differential equation are used to identify the key parameters that lead to pattern formation in the model. The numerical results are compared quantitatively to experimental data by extracting statistical information on orientation, density and patch size through Gabor filters.

  2. Chondroprotective effects of taurine in primary cultures of human articular chondrocytes.

    PubMed

    Liu, Qin; Lu, Zhenhui; Wu, Huayu; Zheng, Li

    2015-03-01

    Articular cartilage is characterized by the lack of blood vessels and has a poor self-healing potential. Limited cell numbers and dedifferentiation of chondrocytes when expanded in vitro are the major obstacles of autologous chondrocyte implantation. Autologous chondrocyte implantation is a cell-based treatment that can be used as a second-line measure to regenerate chondral or osteochondral defects in younger, active patients. There is an urgent need to find an effective chondrogenic protection agent alleviating or inhibiting chondrocyte dedifferentiation. In this study, we explored the effect of taurine (2-aminoethane sulfonic acid) on proliferation and phenotype maintenance of human articular chondrocytes by analyzing the cell proliferation, morphology, viability, and expression of cartilage specific mRNAs and proteins. Primary chondrocytes were isolated from human articular cartilage tissues. Results showed that taurine effectively promoted chondrocyte growth and enhanced accumulation of glycosaminoglycans and collagens in the conditioned media of chondrocytes. Moreover, taurine exposure caused significant increases in the relative expression levels of mRNAs for cartilage specific markers, including aggrecan, collagen type II and SOX9. Aggrecan is a cartilage-specific proteoglycan, and SOX9 is a chondrogenic transcription factor. In contrast, the mRNA expression of collagen type I, a marker for chondrocyte dedifferentiation, was significantly decreased in cells treated with taurine, indicating that taurine inhibits the chondrocyte dedifferentiation. This study reveals that taurine is effective in proliferation promotion and phenotype maintenance of chondrocytes. Thus, taurine may be a useful pro-chondrogenic agent for autologous chondrocyte implantation in the treatment of cartilage repair.

  3. 13cRA regulates the differentiation of antler chondrocytes through targeting Runx3.

    PubMed

    Zhang, Hong-Liang; Cao, Hang; Yang, Zhan-Qing; Geng, Shuang; Wang, Kai; Yu, Hai-Fan; Guo, Bin; Yue, Zhan-Peng

    2017-03-01

    Although 13cRA is involved in the regulation of cellular proliferation and differentiation, its physiological roles in chondrocyte proliferation and differentiation still remain unknown. Here, we showed that 13cRA could induce the proliferation of sika deer antler chondrocytes and expression of Ccnd3 and Cdk6. Administration of 13cRA to antler chondrocytes resulted in an obvious increase in the expression of chondrocyte marker Col II and hypertrophic chondrocyte marker Col X. Silencing of Crabp2 expression by specific siRNA could prevent the 13cRA-induced up-regulation of Col X, whereas overexpression of Crabp2 showed the opposite effects. Further study found that Crabp2 mediated the regulation of 13cRA on the expression of Runx3 which was highly expressed in the antler cartilage and inhibited the differentiation of antler chondrocytes. Moreover, attenuation of Runx3 expression greatly raised 13cRA-induced chondrocyte differentiation. Simultaneously, 13cRA could stimulate the expression of Cyp26a1 and Cyp26b1 in the antler chondrocytes. Inhibition of Cyp26a1 and/or Cyp26b1 reinforced the effects of 13cRA on the expression of Col X and Runx3, while overexpression of Cyp26b1 rendered the antler chondrocytes hyposensitive to 13cRA. Collectively, 13cRA may play an important role in the differentiation of antler chondrocytes through targeting Runx3. Crabp2 enhances the effects of 13cRA on chondrocyte differentiation, while Cyp26a1 and Cyp26b1 weaken the sensitivity of antler chondrocytes to 13cRA.

  4. Human articular chondrocytes express functional leukotriene B4 receptors

    PubMed Central

    Hansen, Ann Kristin; Indrevik, Jill-Tove; Figenschau, Yngve; Martinez-Zubiaurre, Inigo; Sveinbjörnsson, Baldur

    2015-01-01

    Leukotriene B4 (LTB4) is a potent chemoattractant associated with the development of osteoarthritis (OA), while its receptors BLT1 and BLT2 have been found in synovium and subchondral bone. In this study, we have investigated whether these receptors are also expressed by human cartilage cells and their potential effects on cartilage cells. The expression of LTB4 receptors in native tissue and cultured cells was assessed by immunohistochemistry, immunocytochemistry, polymerase chain reaction (PCR) and electron microscopy. The functional significance of the LTB4 receptor expression was studied by Western blotting, using phospho-specific antibodies in the presence or absence of receptor antagonists. In further studies, the secretion of pro-inflammatory cytokines, growth factors and metalloproteinases by LTB4-stimulated chondrocytes was measured by multiplex protein assays. The effects of LTB4 in cartilage signature gene expression in cultured cells were assessed by quantitative PCR, whereas the LTB4-promoted matrix synthesis was determined using 3D pellet cultures. Both receptors were present in cultured chondrocytes, as was confirmed by immunolabelling and PCR. The relative quantification by PCR demonstrated a higher expression of the receptors in cells from healthy joints compared with OA cases. The stimulation of cultured chondrocytes with LTB4 resulted in a phosphorylation of downstream transcription factor Erk 1/2, which was reduced after blocking BLT1 signalling. No alteration in the secretion of cytokine and metalloproteinases was recorded after challenging cultured cells with LTB4; likewise, cartilage matrix gene expression and 3D tissue synthesis were unaffected. Chondrocytes express BLT1 and BLT2 receptors, and LTB4 activates the downstream Erk 1/2 pathway by engaging the high-affinity receptor BLT1. However, any putative role in cartilage biology could not be revealed, and remains to be clarified. PMID:25677035

  5. A practical way to prepare primer human chondrocyte culture.

    PubMed

    Isyar, Mehmet; Yilmaz, Ibrahim; Yasar Sirin, Duygu; Yalcin, Sercan; Guler, Olcay; Mahirogullari, Mahir

    2016-09-01

    Biological cartilage repair is one of the most important targets for orthopedic surgeons currently. For this purpose, it is mandatory to know how to prepare a chondrocyte culture. In this study, our purpose was to introduce a method enabling orthopedic surgeons to practice their knowledge and skills on molecular experimental setup at cellular level, based on our experiences from previous pilot studies. Thus, we believe it will encourage orthopedic surgeons.

  6. Engineering Superficial Zone Chondrocytes from Mesenchymal Stem Cells

    PubMed Central

    Coates, Emily E.

    2014-01-01

    Recent cartilage engineering efforts have focused on development of zonally organized tissue. However, there remains a need for protocols that differentiate progenitor populations into chondrocytes of zonal phenotype. Here, we evaluate the potential of coculture of bovine mesenchymal stem cells (MSCs) and zonal explants of bovine cartilage tissue to drive MSC differentiation to chondrocytes with the superficial zone phenotype. Two coculture systems were set up: one between alginate encapsulated MSCs and superficial zone cartilage explants, and one between MSCs and middle/deep zone cartilage explants. Chondrogenic and superficial zone markers were monitored over a 21-day differentiation period via gene and protein expression. A control conditioned media study was used to determine the impact of communication via soluble factors between cell populations during differentiation. At day 21, results show superficial zone explant coculture without transforming-growth factor β3 supplementation induces upregulation of chondrogenic gene expression markers SOX9 and type II collagen 3.4-fold and 11.4-fold, respectively, over standard chondrogenic control media. Further, coculture of MSCs and superficial zone explants can be used to upregulate mRNA expression of the superficial zone marker proteoglycan-4 in MSCs (1.75-fold over chondrogenic control at day 21), indicating the superficial zone chondrocyte phenotype. Gene expression data show middle/deep zone explant and MSC coculture did not induce the chondrogenesis observed in superficial zone explant coculture. Likewise, poor chondrogenesis was observed in all conditioned media groups. Results highlight the importance of superficial zone cartilage and cells in guiding stem cell fate and regulating differentiation of MSCs to chondrocytes of the superficial zone type. PMID:24279336

  7. Metabolic Effects of Avocado/Soy Unsaponifiables on Articular Chondrocytes

    PubMed Central

    Nardo, Joseph V.; Harlan, Robert; Chiou, Tiffany

    2008-01-01

    Avocado/soy unsaponifiable (ASU) components are reported to have a chondroprotective effect by virtue of anti-inflammatory and proanabolic effects on articular chondrocytes. The identity of the active component(s) remains unknown. In general, sterols, the major component of unsaponifiable plant material have been demonstrated to be anti-inflammatory in vitro and in animal models. These studies were designed to clarify whether the sterol content of ASU preparations were the primary contributors to biological activity in articular chondrocytes. ASU samples were analyzed by high pressure liquid chromatography (HPLC) and GC mass spectrometry. The sterol content was normalized between diverse samples prior to in vitro testing on bovine chondrocytes. Anabolic activity was monitored by uptake of 35-sulfate into proteoglycans and quantitation of labeled hydroxyproline and proline content after incubation with labeled proline. Anti-inflammatory activity was assayed by measuring reduction of interleukin-1 (IL-1)-induced synthesis of PGE2 and metalloproteases and release of label from tissue prelabeled with S-35.All ASU samples exerted a similar time-dependent up-regulation of 35-sulfate uptake in bovine cells reaching a maximum of greater than 100% after 72 h at sterol doses of 1–10 μg/ml. Non-collagenous protein (NCP) and collagen synthesis were similarly up-regulated. All ASU were equally effective in dose dependently inhibiting IL-1-induced MMP-3 activity (23–37%), labeled sulfate release (15–23%) and PGE2 synthesis (45–58%). Up-regulation of glycosaminoglycan and collagen synthesis and reduction of IL-1 effects in cartilage are consistent with chondroprotective activity. The similarity of activity of ASU from diverse sources when tested at equal sterol levels suggests sterols are important for biologic effects in articular chondrocytes. PMID:18604259

  8. The effects of fixed electrical charge on chondrocyte behavior.

    PubMed

    Dadsetan, Mahrokh; Pumberger, Matthias; Casper, Michelle E; Shogren, Kristin; Giuliani, Melissa; Ruesink, Terry; Hefferan, Theresa E; Currier, Bradford L; Yaszemski, Michael J

    2011-05-01

    In this study we have compared the effects of negative and positive fixed charges on chondrocyte behavior in vitro. Electrical charges have been incorporated into oligo(poly(ethylene glycol) fumarate) (OPF) using small charged monomers such as sodium methacrylate (SMA) and (2-(methacryloyloxy) ethyl)-trimethyl ammonium chloride (MAETAC) to produce negatively and positively charged hydrogels, respectively. The physical and electrical properties of the hydrogels were characterized by measuring and calculating the swelling ratio and zeta potential, respectively. Our results revealed that the properties of these OPF modified hydrogels varied according to the concentration of charged monomers. Zeta potential measurements demonstrated that the electrical properties of the OPF hydrogel surfaces changed on incorporation of SMA and MAETAC and that these changes in electrical properties were dose-dependent. Attenuated total reflectance Fourier transform infrared spectroscopy was used to determine the hydrogel surface composition. To assess the effects of surface properties on chondrocyte behavior primary chondrocytes isolated from rabbit ears were seeded as a monolayer on top of the hydrogels. We demonstrated that the cells remained viable over 7 days and began to proliferate while seeded on top of the hydrogels. Collagen type II staining was positive in all samples, however, the staining intensity was higher on negatively charged hydrogels. Similarly, glycosaminoglycan production was significantly higher on negatively charged hydrogels compared with a neutral hydrogel. Reverse transcriptase polymerase chain reaction showed up-regulation of collagen type II and down-regulation of collagen type I on the negatively charged hydrogels. These findings indicate that charge plays an important role in establishing an appropriate environment for chondrocytes and, hence, in the engineering of cartilage. Thus, further investigations into charged hydrogels for cartilage tissue

  9. Structural differences in epiphyseal and physeal hypertrophic chondrocytes

    PubMed Central

    Shapiro, Frederic; Flynn, Evelyn

    2015-01-01

    We have observed that epiphyseal and physeal hypertrophic chondrocytes in BALB/c mice show considerable differences of light microscopic and ultrastructural appearance, even when the cells are at the same stage of differentiation. In addition, cell structure maintenance improved with tissue preparation controlled for osmolarity and for membrane stabilization using 0.5% ruthenium hexammine trichloride (RHT) for both light microscopy (LM) and electron microscopy (EM) or 0.5% lanthanum nitrate for LM. Physeal hypertrophic chondrocytes showed a gradual increase in size closer to the metaphysis and a change in shape as cells elongated along the long axis. The nucleus remained central, with uniformly dispersed chromatin, and the rough endoplasmic reticulum (RER) was randomly dispersed throughout cytoplasm with little to no presence against the cell membrane. Even the lowermost cells showed thin elongated or dilated cisternae of RER and intact cell membranes. Epiphyseal chondrocytes remained circular to oval with no elongation. Nucleus and RER were positioned as a complete transcellular central nucleocytoplasmic column or as an incomplete bud with RER of the column/bud always continuous with RER peripherally against the intact cell membrane. RER was densely packed with parallel cisternae with adjacent cytoplasm empty of organelles but often filled with circular deposits of moderately electron-dense material consistent with fat. Optimal technique for LM involved fixation using glutaraldehyde (GA) 1.3%, paraformaldehyde (PFA) 1% and RHT 0.5% (mOsm 606) embedded in JB-4 plastic and stained with 0.5% toluidine blue. Optimal technique for EM used fixation with GA 1.3%, PFA 1%, RHT 0.5% and cacodylate buffer 0.03 M (mOsm 511) and post-fixation including 1% osmium tetroxide. These observations lead to the possibility that the same basic cell, the hypertrophic chondrocyte, has differing functional mechanisms at different regions of the developing bone. PMID:25987982

  10. Microenvironmental changes during differentiation of mesenchymal stem cells towards chondrocytes

    PubMed Central

    Djouad, Farida; Delorme, Bruno; Maurice, Marielle; Bony, Claire; Apparailly, Florence; Louis-Plence, Pascale; Canovas, François; Charbord, Pierre; Noël, Danièle; Jorgensen, Christian

    2007-01-01

    Chondrogenesis is a process involving stem-cell differentiation through the coordinated effects of growth/differentiation factors and extracellular matrix (ECM) components. Recently, mesenchymal stem cells (MSCs) were found within the cartilage, which constitutes a specific niche composed of ECM proteins with unique features. Therefore, we hypothesized that the induction of MSC differentiation towards chondrocytes might be induced and/or influenced by molecules from the microenvironment. Using microarray analysis, we previously identified genes that are regulated during MSC differentiation towards chondrocytes. In this study, we wanted to precisely assess the differential expression of genes associated with the microenvironment using a large-scale real-time PCR assay, according to the simultaneous detection of up to 384 mRNAs in one sample. Chondrogenesis of bone-marrow-derived human MSCs was induced by culture in micropellet for various periods of time. Total RNA was extracted and submitted to quantitative RT-PCR. We identified molecules already known to be involved in attachment and cell migration, including syndecans, glypicans, gelsolin, decorin, fibronectin, and type II, IX and XI collagens. Importantly, we detected the expression of molecules that were not previously associated with MSCs or chondrocytes, namely metalloproteases (MMP-7 and MMP-28), molecules of the connective tissue growth factor (CTGF); cef10/cyr61 and nov (CCN) family (CCN3 and CCN4), chemokines and their receptors chemokine CXC motif ligand (CXCL1), Fms-related tyrosine kinase 3 ligand (FlT3L), chemokine CC motif receptor (CCR3 and CCR4), molecules with A Disintegrin And Metalloproteinase domain (ADAM8, ADAM9, ADAM19, ADAM23, A Disintegrin And Metalloproteinase with thrombospondin type 1 motif ADAMTS-4 and ADAMTS-5), cadherins (4 and 13) and integrins (α4, α7 and β5). Our data suggest that crosstalk between ECM components of the microenvironment and MSCs within the cartilage is

  11. Hydrogen peroxide induces apoptosis via a mitochondrial pathway in chondrocytes

    NASA Astrophysics Data System (ADS)

    Zhuang, Cai-ping; Liang, Qian; Wang, Xiao-ping; Chen, Tong-sheng

    2012-03-01

    The degenerative joint disease such as osteoarthritis (OA) is closely associated with the death of chondrocytes in apoptosis fashion. Hydrogen peroxide (H2O2), higher expression following acute damage in OA patients, has been shown to be up-regulated during apoptosis in a bulk of experimental models. This study was aimed to explore the mechanism of H2O2-induced rabbit chondrocytes apoptosis. Articular cartilage was biopsied from the joints of 6 weeks old New Zealand rabbits. Cell Counting Kit (CCK-8) assay was used to assess the inhibitory effect of H2O2 on cell viability. H2O2 treatment induced a remarkable reduction of cell viability. We used flow cytometry to assess the form of cell death with Annexin-V/PI double staining, and found that H2O2 treatment induced apoptosis in a dose-and time-dependent manner. Exposure of chondrocytes to 1.5 mM of H2O2 for 2 h induced a burst apoptosis that can be alleviated by N-acetyl cysteine (NAC) pretreatment, an anti-oxidant amino-acid derivative. Loss of mitochondria membrane potential (▵Ψm) was evaluated using confocal microscopy imaging and flow cytometry (FCM). H2O2 treatment induced a marked reduction of ▵Ψm, and the abrupt disappearance of ▵Ψm occurred within 5 minutes. These results indicate that H2O2 induces a rapid apoptosis via a mitochondrial pathway in rabbit chondrocytes.

  12. MicroRNA-33 suppresses CCL2 expression in chondrocytes

    PubMed Central

    Wei, Meng; Xie, Qingyun; Zhu, Jun; Wang, Tao; Zhang, Fan; Cheng, Yue; Guo, Dongyang; Wang, Ying; Mo, Liweng; Wang, Shuai

    2016-01-01

    CCL2-mediated macrophage infiltration in articular tissues plays a pivotal role in the development of the osteoarthritis (OA). miRNAs regulate the onset and progression of diseases via controlling the expression of a series of genes. How the CCL2 gene was regulated by miRNAs was still not fully elucidated. In the present study, we demonstrated that the binding sites of miR-33 in the 3′UTR of CCL2 gene were conserved in human, mouse and rat species. By performing gain- or loss-of-function studies, we verified that miR-33 suppressed CCL2 expression in the mRNA and protein levels. We also found that miR-33 suppressed the CCL2 levels in the supernatant of cultured primary mouse chondrocytes. With reporter gene assay, we demonstrated that miR-33 targeted at AAUGCA in the 3′UTR of CCL2 gene. In transwell migration assays, we demonstrated that the conditional medium (CM) from miR-33 deficient chondrocytes potentiated the monocyte chemotaxis in a CCL2 dependent manner. Finally, we demonstrated that the level of miR-33 was decreased, whereas the CCL2 level was increased in the articular cartilage from the OA patients compared with the control group. In summary, we identified miR-33 as a novel suppressor of CCL2 in chondrocytes. The miR-33/CCL2 axis in chondrocytes regulates monocyte chemotaxis, providing a potential mechanism of macrophage infiltration in OA. PMID:27129293

  13. Fate of Meckel's cartilage chondrocytes in ocular culture

    SciTech Connect

    Richman, J.M.; Diewert, V.M.

    1988-09-01

    Modulation of the chondrocyte phenotype was observed in an organ culture system using Meckel's cartilage. First branchial arch cartilage was dissected from fetal rats of 16- and 17-day gestation. Perichondrium was mechanically removed, cartilage was split at the rostral process, and each half was grafted into the anterior chamber of an adult rat eye. The observed pattern of development in nonirradiated specimens was the following: hypertrophy of the rostral process and endochondral-type ossification, fibrous atrophy in the midsection, and mineralization of the malleus and incus. A change in matrix composition of the implanted cartilage was demonstrated with immunofluorescence staining for cartilage-specific proteoglycan (CSPG). After 15 days of culture, CSPG was found in the auricular process but not in the midsection or rostral process. In order to mark the implanted cells and follow their fate, cartilage was labeled in vitro with (3H)thymidine (3H)TdR). Immediately after labeling 20% of the chondrocytes contained (3H)TdR. After culturing for 5 days, 20% of the chondrocytes were still labeled and 10% of the osteogenic cells also contained radioactive label. The labeling index decreased in both cell types with increased duration of culture. Multinucleated clast-type cells did not contain label. Additional cartilages not labeled with (3H)TdR were exposed to between 20000 and 6000 rad of gamma irradiation before ocular implantation. Irradiated cartilage did not hypertrophy or form bone but a fibrous region developed in the midsection. Cells of the host animal were not induced to form bone around the irradiated cartilage. Our studies suggest that fully differentiated chondrocytes of Meckel's cartilage have the capacity to become osteocytes, osteoblasts, and fibroblasts.

  14. Microenvironmental changes during differentiation of mesenchymal stem cells towards chondrocytes.

    PubMed

    Djouad, Farida; Delorme, Bruno; Maurice, Marielle; Bony, Claire; Apparailly, Florence; Louis-Plence, Pascale; Canovas, François; Charbord, Pierre; Noël, Danièle; Jorgensen, Christian

    2007-01-01

    Chondrogenesis is a process involving stem-cell differentiation through the coordinated effects of growth/differentiation factors and extracellular matrix (ECM) components. Recently, mesenchymal stem cells (MSCs) were found within the cartilage, which constitutes a specific niche composed of ECM proteins with unique features. Therefore, we hypothesized that the induction of MSC differentiation towards chondrocytes might be induced and/or influenced by molecules from the microenvironment. Using microarray analysis, we previously identified genes that are regulated during MSC differentiation towards chondrocytes. In this study, we wanted to precisely assess the differential expression of genes associated with the microenvironment using a large-scale real-time PCR assay, according to the simultaneous detection of up to 384 mRNAs in one sample. Chondrogenesis of bone-marrow-derived human MSCs was induced by culture in micropellet for various periods of time. Total RNA was extracted and submitted to quantitative RT-PCR. We identified molecules already known to be involved in attachment and cell migration, including syndecans, glypicans, gelsolin, decorin, fibronectin, and type II, IX and XI collagens. Importantly, we detected the expression of molecules that were not previously associated with MSCs or chondrocytes, namely metalloproteases (MMP-7 and MMP-28), molecules of the connective tissue growth factor (CTGF); cef10/cyr61 and nov (CCN) family (CCN3 and CCN4), chemokines and their receptors chemokine CXC motif ligand (CXCL1), Fms-related tyrosine kinase 3 ligand (FlT3L), chemokine CC motif receptor (CCR3 and CCR4), molecules with A Disintegrin And Metalloproteinase domain (ADAM8, ADAM9, ADAM19, ADAM23, A Disintegrin And Metalloproteinase with thrombospondin type 1 motif ADAMTS-4 and ADAMTS-5), cadherins (4 and 13) and integrins (alpha4, alpha7 and beta5). Our data suggest that crosstalk between ECM components of the microenvironment and MSCs within the cartilage is

  15. The Role of the Membrane Potential in Chondrocyte Volume Regulation

    PubMed Central

    Lewis, Rebecca; Asplin, Katie E; Bruce, Gareth; Dart, Caroline; Mobasheri, Ali; Barrett-Jolley, Richard

    2011-01-01

    Many cell types have significant negative resting membrane potentials (RMPs) resulting from the activity of potassium-selective and chloride-selective ion channels. In excitable cells, such as neurones, rapid changes in membrane permeability underlie the generation of action potentials. Chondrocytes have less negative RMPs and the role of the RMP is not clear. Here we examine the basis of the chondrocyte RMP and possible physiological benefits. We demonstrate that maintenance of the chondrocyte RMP involves gadolinium-sensitive cation channels. Pharmacological inhibition of these channels causes the RMP to become more negative (100 µM gadolinium: ΔVm = −30 ± 4 mV). Analysis of the gadolinium-sensitive conductance reveals a high permeability to calcium ions (PCa/PNa ≈80) with little selectivity between monovalent ions; similar to that reported elsewhere for TRPV5. Detection of TRPV5 by PCR and immunohistochemistry and the sensitivity of the RMP to the TRPV5 inhibitor econazole (ΔVm = −18 ± 3 mV) suggests that the RMP may be, in part, controlled by TRPV5. We investigated the physiological advantage of the relatively positive RMP using a mathematical model in which membrane stretch activates potassium channels allowing potassium efflux to oppose osmotic water uptake. At very negative RMP potassium efflux is negligible, but at more positive RMP it is sufficient to limit volume increase. In support of our model, cells clamped at −80 mV and challenged with a reduced osmotic potential swelled approximately twice as much as cells at +10 mV. The positive RMP may be a protective adaptation that allows chondrocytes to respond to the dramatic osmotic changes, with minimal changes in cell volume. J. Cell. Physiol. 226: 2979–2986, 2011. © 2011 Wiley-Liss, Inc. PMID:21328349

  16. Application of 3-Dimensional Printing Technology to Kirschner Wire Fixation of Adolescent Condyle Fracture.

    PubMed

    Dong, Zhiwei; Li, Qihong; Bai, Shizhu; Zhang, Li

    2015-10-01

    Condyle fractures are common in children and are increasingly treated with open reduction. Three-dimensional printing has developed into an important method of assisting surgical treatment. This report describes the case of a 14-year-old patient treated for a right condyle fracture at the authors' hospital. Preoperatively, the authors designed a surgical guide using 3-dimensional printing and virtual surgery. The 3-dimensional surgical guide allowed accurate alignment of the fracture using Kirschner wire without additional dissection and tissue injury. Kirschner wire fixation augmented by 3-dimensional printing technology produced a good outcome in this adolescent condyle fracture. Copyright © 2015 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

  17. Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels.

    PubMed

    Rosenzweig, D H; Chicatun, F; Nazhat, S N; Quinn, T M

    2013-12-01

    Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However, standard culture techniques require cell passaging, leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes, grown either in CE culture or passaged twice on static silicone dishes (SS culture; comparable to standard methods), were seeded in dense collagen gels and cultured for 3 weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes, CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3 weeks in culture, correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition, yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3 weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties, making them more suitable than SS cultured cells for tissue engineering applications. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  18. Inflammatory synovial fluid microenvironment drives primary human chondrocytes to actively take part in inflammatory joint diseases.

    PubMed

    Röhner, Eric; Matziolis, Georg; Perka, Carsten; Füchtmeier, Bernd; Gaber, Timo; Burmester, Gerd-Rüdiger; Buttgereit, Frank; Hoff, Paula

    2012-06-01

    The role of human chondrocytes in the pathogenesis of cartilage degradation in rheumatic joint diseases has presently gained increasing interest. An active chondrocyte participation in local inflammation may play a role in the initiation and progression of inflammatory joint diseases and in a disruption of cartilage repair mechanisms resulting in cartilage degradation. In the present study, we hypothesized that inflammatory synovial fluid triggers human chondrocytes to actively take part in inflammatory processes in rheumatic joint diseases. Primary human chondrocytes were incubated in synovial fluids gained from patients with rheumatoid arthritis, psoriasis arthritis and reactive arthritis. The detection of vital cell numbers was determined by using Casy Cell Counter System. Apoptosis was measured by Annexin-V and 7AAD staining. Cytokine and chemokine secretion was determined by a multiplex suspension array. Detection of vital cells showed a highly significant decrease in chondrocyte numbers. Flow cytometry demonstrated a significant increase in apoptotic chondrocytes after the incubation. An active secretion of cytokines such as MCP-1 and MIF by chondrocytes was observed. The inflammatory synovial fluid microenvironment mediates apoptosis and cell death of chondrocytes. Moreover, in terms of cytokine secretion, it also induces an active participation of chondrocytes in ongoing inflammation.

  19. Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress.

    PubMed

    Lin, Pingdong; Weng, Xiaping; Liu, Fayuan; Ma, Yuhuan; Chen, Houhuang; Shao, Xiang; Zheng, Wenwei; Liu, Xianxiang; Ye, Hongzhi; Li, Xihai

    2015-12-01

    Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type Ⅱ collagen. The ER stress-mediated apoptosis of tunicamycin (TM)‑stimulated chondrocytes was detected using 4-phenylbutyric acid (4‑PBA). We found that 4‑PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4',6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM‑induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X‑box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP‑homologous protein (Chop), caspase‑9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase

  20. Topography-Guided Proliferation: Distinct Surface Microtopography Increases Proliferation of Chondrocytes In Vitro.

    PubMed

    Joergensen, Natasja Leth; Le, Dang Quang Svend; Andersen, Ole Zoffmann; Foss, Morten; Danielsen, Carl Christian; Foldager, Casper Bindzus; Lind, Martin; Lysdahl, Helle

    2015-11-01

    Chondrocyte-based cartilage repair techniques require control of articular chondrocyte expansion ex vivo. Articular chondrocytes have limited availability, and prolonged culturing to obtain a cell number sufficient for clinical use often results in phenotypic alterations and increased costs. In this study, we applied a screening library consisting of micrometer-sized topographical features, termed biosurface structure array (BSSA), to identify specific topographical microstructures affecting the proliferation of human chondrocytes in passage 1 (P1) or 2 (P2). The BSSA library comprised 10 patterns and 16 combinations of pillar size (X) and interpillar gap size (Y). Specific microstructures significantly increased the chondrocytes' proliferative responsiveness in term of patterns, X and Y for P2 compared with P1. The P1 and P2 chondrocytes responded independently to similar patterns after 4 days of culturing, whereas only chondrocytes at P2 responded to specific microstructures with Y = 1 μm and X = 2, 4 μm by a 2.3- and 4.4-fold increased proliferation, respectively. In conclusion, these findings indicate that specific surface topographies promote chondrocyte proliferation and may, indeed, be a tool to control the behavior of chondrocytes in vitro.

  1. Optimal 3-D culture of primary articular chondrocytes for use in the Rotating Wall Vessel Bioreactor

    PubMed Central

    Mellor, Liliana F.; Baker, Travis L.; Brown, Raquel J.; Catlin, Lindsey W.; Oxford, Julia Thom

    2014-01-01

    INTRODUCTION Reliable culturing methods for primary articular chondrocytes are essential to study the effects of loading and unloading on joint tissue at the cellular level. Due to the limited proliferation capacity of primary chondrocytes and their tendency to dedifferentiate in conventional culture conditions, long-term culturing conditions of primary chondrocytes can be challenging. The goal of this study was to develop a suspension culturing technique that not only would retain the cellular morphology but also maintain gene expression characteristics of primary articular chondrocytes. METHODS Three-dimensional culturing methods were compared and optimized for primary articular chondrocytes in the rotating wall vessel bioreactor, which changes the mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence. We performed gene expression analysis and morphological characterization of cells cultured in alginate beads, Cytopore-2 microcarriers, primary monolayer culture, and passaged monolayer cultures using reverse transcription-PCR and laser scanning confocal microscopy. RESULTS Primary chondrocytes grown on Cytopore-2 microcarriers maintained the phenotypical morphology and gene expression pattern observed in primary bovine articular chondrocytes, and retained these characteristics for up to 9 days. DISCUSSION Our results provide a novel and alternative culturing technique for primary chondrocytes suitable for studies that require suspension such as those using the rotating wall vessel bioreactor. In addition, we provide an alternative culturing technique for primary chondrocytes that can impact future mechanistic studies of osteoarthritis progression, treatments for cartilage damage and repair, and cartilage tissue engineering. PMID:25199120

  2. Elastic cartilage reconstruction by transplantation of cultured hyaline cartilage-derived chondrocytes.

    PubMed

    Mizuno, M; Takebe, T; Kobayashi, S; Kimura, S; Masutani, M; Lee, S; Jo, Y H; Lee, J I; Taniguchi, H

    2014-05-01

    Current surgical intervention of craniofacial defects caused by injuries or abnormalities uses reconstructive materials, such as autologous cartilage grafts. Transplantation of autologous tissues, however, places a significant invasiveness on patients, and many efforts have been made for establishing an alternative graft. Recently, we and others have shown the potential use of reconstructed elastic cartilage from ear-derived chondrocytes or progenitors with the unique elastic properties. Here, we examined the differentiation potential of canine joint cartilage-derived chondrocytes into elastic cartilage for expanding the cell sources, such as hyaline cartilage. Articular chondrocytes are isolated from canine joint, cultivated, and compared regarding characteristic differences with auricular chondrocytes, including proliferation rates, gene expression, extracellular matrix production, and cartilage reconstruction capability after transplantation. Canine articular chondrocytes proliferated less robustly than auricular chondrocytes, but there was no significant difference in the amount of sulfated glycosaminoglycan produced from redifferentiated chondrocytes. Furthermore, in vitro expanded and redifferentiated articular chondrocytes have been shown to reconstruct elastic cartilage on transplantation that has histologic characteristics distinct from hyaline cartilage. Taken together, cultured hyaline cartilage-derived chondrocytes are a possible cell source for elastic cartilage reconstruction. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

  3. Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress

    PubMed Central

    LIN, PINGDONG; WENG, XIAPING; LIU, FAYUAN; MA, YUHUAN; CHEN, HOUHUANG; SHAO, XIANG; ZHENG, WENWEI; LIU, XIANXIANG; YE, HONGZHI; LI, XIHAI

    2015-01-01

    Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type II collagen. The ER stress-mediated apoptosis of tunicamycin (TM)-stimulated chondrocytes was detected using 4-phenylbutyric acid (4-PBA). We found that 4-PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4′,6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM-induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X-box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), caspase-9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase-9, caspase-3

  4. Chondrocytes contain a growth factor that is localized in the nucleus and is associated with chromatin.

    PubMed Central

    Azizkhan, J C; Khagsbrun, M

    1980-01-01

    Bovine scapular and articular chondrocytes were isolated from fresh cartilage and disrupted by sonication. The disrupted cells had the ability to stimulate DNA synthesis and cell division in vitro in chondrocytes and in 3T3 cells. Subcellular fractions were prepared by two methods, enucleation with cytochalasin B and lysis of cells with Nonidet P-40. After enucleation of chondrocytes, karyoplasts and cytoplasts were collected, disrupted by sonication, and tested for their ability to stimulate DNA synthesis. Over 95% of the cellular growth factor activity was localized in the karyoplast. In addition, after lysis of chondrocytes in Nonidet P-40, over 95% of the growth factor activity was recovered in the nuclear fraction. Chondrocyte chromatin was prepared by low ionic strength detergent treatment of karyoplasts. All of the growth factor activity of the karyoplast was found to be associated with chromatin. The growth factor activity of chondrocytes, cytoplasts, karyoplasts, and chromatin was analyzed by gel filtration on Bio-Gel A-0.5 m equilibrated with 4 M guanidine . HCl and 5 mM dithiothreitol. Chondrocytes, chondrocyte karyoplasts, and chondrocyte chromatin had similar column elution profiles, with molecular weights in the range of 12,000-22,000. Images PMID:6930665

  5. Reduction of environmental temperature mitigates local anesthetic cytotoxicity in bovine articular chondrocytes.

    PubMed

    Onur, Tarik; Dang, Alexis

    2014-09-01

    The purpose of this study was to assess whether reducing environmental temperature will lead to increased chondrocyte viability following injury from a single-dose of local anesthetic treatment. Bovine articular chondrocytes from weight bearing portions of femoral condyles were harvested and cultured. 96-well plates were seeded with 15,000 chondrocytes per well. Chondrocytes were treated with one of the following conditions: ITS Media, 1x PBS, 2% lidocaine, 0.5% bupivacaine, or 0.5% ropivacaine. Each plate was then incubated at 37°C, 23°C, or 4°C for one hour and then returned to media at 37°C. Chondrocyte viability was assessed 24 hours after treatment. Chondrocyte viability is presented as a ratio of the fluorescence of the treatment group over the average of the media group at that temperature (ratio ± SEM). At 37°C, lidocaine (0.35 ± 0.04) and bupivacaine (0.30 ± 0.05) treated chondrocytes show low cell viability when compared to the media (1.00 ± 0.03) control group (p < 0.001). Lidocaine treated chondrocytes were significantly more viable at 23°C (0.84 ± 0.08) and 4°C (0.86±0.085) than at 37°C (p < 0.001). Bupivacaine treated chondrocytes were significantly more viable at 4°C (0.660 ± 0.073) than at 37°C or 23°C (0.330 ± 0.069) (p < 0.001 and p = 0.002 respectively). Reducing the temperature from 37°C to 23°C during treatment with lidocaine increases chondrocyte viability following injury. Chondrocytes treated with bupivacaine can be rescued by reducing the temperature to 4°C. Key pointsConfirm that local anesthetics, specifically bupivacaine and lidocaine, are toxic to chondrocytes in monolayerChondrocyte viability significantly improved for chondrocytes treated with bupivacaine when the environment was cooled to 23°C.Chondrocyte viability significantly improved for chondrocytes treated with bupivacaine or lidocaine when the environment was cooled to 4°CIt is the recommendation of the authors that physicians should be wary of the

  6. Engineering cartilage tissue by pellet coculture of chondrocytes and mesenchymal stromal cells.

    PubMed

    Wu, Ling; Post, Janine N; Karperien, Marcel

    2015-01-01

    Coculture of chondrocytes and mesenchymal stromal cells (MSCs) in pellets has been shown to be beneficial in engineering cartilage tissue in vitro. In these cultures trophic effects of MSCs increase the proliferation and matrix deposition of chondrocytes. Thus, large cartilage constructs can be made with a relatively small number of chondrocytes. In this chapter, we describe the methods for making coculture pellets of MSCs and chondrocytes. We also provide detailed protocols for analyzing coculture pellets with cell tracking, proliferation assays, species specific polymerase chain reactions (PCR), short tandem repeats analysis, and histological examination.

  7. Phenotypic diversity of neoplastic chondrocytes and extracellular matrix gene expression in cartilaginous neoplasms.

    PubMed Central

    Aigner, T.; Dertinger, S.; Vornehm, S. I.; Dudhia, J.; von der Mark, K.; Kirchner, T.

    1997-01-01

    Chondrocyte differentiation is characterized by distinct cellular phenotypes, which can be identified by specific extracellular matrix gene expression profiles. By applying in situ analysis on the mRNA and protein level in a series of benign and malignant human chondrogenic neoplasms, we were able to identify for the first time different phenotypes of neoplastic chondrocytes in vivo: 1) mature chondrocytes, which synthesized the characteristic cartilaginous extracellular tumor matrix, 2) cells resembling hypertrophic chondrocytes of the fetal growth plate, 3) cells resembling so-called dedifferentiated chondrocytes, and 4) well differentiated chondrocytic cells, which expressed type I collagen, indicating the presence of post-hypertrophic differentiated neoplastic chondrocytes. Chondrocytes exhibiting a range of phenotypes were found to be present in the same neoplasm. The different observed phenotypes, including the dedifferentiated phenotype, were in contrast to the anaplastic cells of high-grade chondrosarcomas. Comparison of expression data with tumor morphology revealed a relationship between the cellular phenotypes, the tumor matrix composition, and the matrix and cell morphology within the neoplasms. The distinctly different phenotypes of neoplastic chondrocytes are the basis of the characteristic high biochemical and morphological heterogeneity of chondroid neoplasms and shed light on their biological and clinical behavior. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9176404

  8. Cellular response of chondrocytes to magnesium alloys for orthopedic applications

    PubMed Central

    LIAO, YI; XU, QINGLI; ZHANG, JIAN; NIU, JIALING; YUAN, GUANGYIN; JIANG, YAO; HE, YAOHUA; WANG, XINLING

    2015-01-01

    In the present study, the effects of Mg-Nd-Zn-Zr (JDBM), brushite (CaHPO4·2H2O)-coated JDBM (C-JDBM), AZ31, WE43, pure magnesium (Mg) and Ti alloy (TC4) on rabbit chondrocytes were investigated in vitro. Adhesion experiments revealed the satisfactory morphology of chondrocytes on the surface of all samples. An indirect cytotoxicity test using MTT assay revealed that C-JDBM and TC4 exhibited results similar to those of the negative control, better than those obtained with JDBM, AZ31, WE43 and pure Mg (p<0.05). There were no statistically significant differences observed between the JDBM, AZ31, WE43 and pure Mg group (p>0.05). The results of indirect cell cytotoxicity and proliferation assays, as well as those of apoptosis assay, glycosaminoglycan (GAG) quantification, assessment of collagen II (Col II) levels and RT-qPCR revealed a similar a trend as was observed with MTT assay. These findings suggested that the JDBM alloy was highly biocompatible with chondrocytes in vitro, yielding results similar to those of AZ31, WE43 and pure Mg. Furthermore, CaHPO4·2H2O coating significantly improved the biocompatibility of this alloy. PMID:25975216

  9. Effects of Indigo Carmine on Human Chondrocytes In Vitro

    PubMed Central

    Zippelius, Timo; Hoburg, Arnd; Preininger, Bernd; Vörös, Pauline; Perka, Carsten; Matziolis, Georg; Röhner, Eric

    2013-01-01

    Joint infections following or accompanying superficious soft tissue infections are severe complication in orthopedic surgery. The use of intra-articular blue staining is a helpful method to visualize a fistula and to differentiate between superficial and intra-articular infections. Regarding this clinical implication data about the effects of indigo carmine, a frequently used blue staining substance, on cartilage is missing. The hypothesis of this study was that indigo carmine damages human chondrocytes in a time and concentration dependent manner. Human chondrocytes were isolated from donors with osteoarthritis who were treated with TKA. Cells were cultivated and treated with different concentrations of indigo carmine for 5 and 10 minutes. Morphologic damage was examined by light microscopy. Toxicity was quantified by counting vital cell number and lactate dehydrogenase (LDH) expression. Analysis by light microscopy showed defected cell structure and loss of cell number after treatment with 100% indigo carmine for 10 minutes. Treatment with 10% and 1% indigo carmine showed no significant cell defects and loss of cells. Counting vital cell number showed loss of vital cells after treatment with 100% and 10% indigo carmine for 10 minutes. LDH expression was significantly increased after treatment with 100% indigo carmine. Toxic effects were shown after treatment with indigo carmine. Therefore, it should be used in 1:100 dilution. This is both, sufficient for visualizing a fistula in a possible clinical application and could be protective for chondrocytes. PMID:23341851

  10. The effects of simulated microgravity on cultured chicken embryonic chondrocytes

    NASA Astrophysics Data System (ADS)

    Zhang, X.; Li, X. B.; Yang, S. Z.; Li, S. G.; Jiang, P. D.; Lin, Z. H.

    2003-10-01

    Using the cultured chicken embryonic chondrocytes as a model, the effects of simulated microgravity on the microtubular system of the cellular skeleton, extracellular matrix, alkaline phosphatase activity, intracellular free calcium concentration and mitochondrial ATP synthase activity with its oligomycin inhibition rate were studied with a clinostat. The microtubular content was measured by a flow cytometer. The decrease of microtubular content showed the impairment of the cellular skeleton system. Observation on the extracellualr matrix by the scanning electron microscopy showed that it decreased significantly after rotating, and the fibers in the extracellular matrix were more tiny and disorderly than that of the control group. It can be concluded that the simulated microgravity can affect the secreting and assembly of the extracellular matrix. In contrast to the control, there was a time course decrease in alkaline phosphatase activity of chondrocytes, a marker of matrix mineralization. Meanwhile a significant drop in the intracellular calcium concentration happened at the beginning of rotation. These results indicate that simulated microgravity can suppress matrix calcification of cultured chondrocytes, and intracellular free calcium may be involved in the regulation of matrix calcification as the second signal transmitter. No significant changes happened in the mitochondrial ATP synthase activity and its oligomycin inhibition rate. Perhaps the energy metabolism wasn't affected by the simulated microgravity. The possible mechanisms about them were discussed.

  11. Quantitative Proteomic Analysis of Rat Condylar Chondrocytes during Postnatal Development.

    PubMed

    Jiang, Li Ting; Xie, Yin Yin; Wei, Li; Zhou, Qi; Shen, Xing; Gao, Yi Ming; Jiang, Xin Quan

    To investigate differentially expressed proteins in rat mandibular condylar cartilage (MCC) chondrocytes caused by initial mastication for short postnatal periods. Four groups of protein samples were extracted from primary cultured rat MCC chondrocytes, harvested from eigthy postnatal SD rats aged 1,7,14 and 28 days, with twenty in each group. Total proteins were labelled with isobaric tags for relative and absolute quantification (iTRAQ) reagents. Two-dimensional nano-high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time-of-flight/ time-of-flight (MALDI-TOF/TOF) mass spectrometry analysis with iTRAQ technique were performed. All data were analysed by MASCOT software with the SWISSPROT protein database. Furthermore, bioinformatics and statistical analysis were performed to classify their cellular components, biological processes, molecular functions and metabolic pathway by the PANTHER database. In total, 137 differentially expressed proteins were identified during MCC growth and were assigned to one or more cellular components. According to the PANTHER analysis, a significant proportion of proteins are involved in the metabolic process, cellular process, biological regulation, developmental process and response to stimulus. The most extensive molecular function was 43% in catalytic activity. In addition, it was found that proteins in MCC chondrocytes change markedly on the growth stage of eruption of the teeth. This study provides an integrated perspective of molecular mechanisms regulating early normal postnatal growth and development of rat MCC at the protein level.

  12. Dosimetric Comparison Between 3-Dimensional Conformal and Robotic SBRT Treatment Plans for Accelerated Partial Breast Radiotherapy.

    PubMed

    Goggin, L M; Descovich, M; McGuinness, C; Shiao, S; Pouliot, J; Park, C

    2016-06-01

    Accelerated partial breast irradiation is an attractive alternative to conventional whole breast radiotherapy for selected patients. Recently, CyberKnife has emerged as a possible alternative to conventional techniques for accelerated partial breast irradiation. In this retrospective study, we present a dosimetric comparison between 3-dimensional conformal radiotherapy plans and CyberKnife plans using circular (Iris) and multi-leaf collimators. Nine patients who had undergone breast-conserving surgery followed by whole breast radiation were included in this retrospective study. The CyberKnife planning target volume (PTV) was defined as the lumpectomy cavity + 10 mm + 2 mm with prescription dose of 30 Gy in 5 fractions. Two sets of 3-dimensional conformal radiotherapy plans were created, one used the same definitions as described for CyberKnife and the second used the RTOG-0413 definition of the PTV: lumpectomy cavity + 15 mm + 10 mm with prescription dose of 38.5 Gy in 10 fractions. Using both PTV definitions allowed us to compare the dose delivery capabilities of each technology and to evaluate the advantage of CyberKnife tracking. For the dosimetric comparison using the same PTV margins, CyberKnife and 3-dimensional plans resulted in similar tumor coverage and dose to critical structures, with the exception of the lung V5%, which was significantly smaller for 3-dimensional conformal radiotherapy, 6.2% when compared to 39.4% for CyberKnife-Iris and 17.9% for CyberKnife-multi-leaf collimator. When the inability of 3-dimensional conformal radiotherapy to track motion is considered, the result increased to 25.6%. Both CyberKnife-Iris and CyberKnife-multi-leaf collimator plans demonstrated significantly lower average ipsilateral breast V50% (25.5% and 24.2%, respectively) than 3-dimensional conformal radiotherapy (56.2%). The CyberKnife plans were more conformal but less homogeneous than the 3-dimensional conformal radiotherapy plans. Approximately 50% shorter

  13. Comparison of 2-Dimensional and 3-Dimensional Metacarpal Fracture Plating Constructs Under Cyclic Loading.

    PubMed

    Tannenbaum, Eric P; Burns, Geoffrey T; Oak, Nikhil R; Lawton, Jeffrey N

    2017-03-01

    Metacarpal fractures are commonly treated by a variety of means including casting or open reduction internal fixation when unacceptable alignment is present following attempted closed reduction. Dorsal plating with either single-row 2-dimensional or double-row 3-dimensional plates has been proposed. This study's purpose was to determine if there are any differences in fixation construct stability under cyclic loading and subsequent load to failure between the lower profile 3-dimensional and the larger 2-dimensional plates in a metacarpal fracture gap sawbone model. Thirty metacarpal cortico-cancellous synthetic bones were cut with a 1.75-mm gap between the 2 fragments simulating mid-diaphyseal fracture comminution. Half of the metacarpals were plated with 2.0-mm locking 2-dimensional plates and half with 1.5-mm locking 3-dimensional plates. The plated metacarpals were mounted into a materials testing apparatus and cyclically loaded under cantilever bending for 2,000 cycles at 70 N, then 2,000 cycles at 120 N, and finally monotonically loaded to failure. Throughout testing, fracture gap sizes were measured, failure modes were recorded, and construct strength and stiffness values were calculated. All 3-dimensional constructs survived both cyclic loading conditions. Ten (67%) 2-dimensional constructs survived both loading conditions, whereas 5 (33%) failed the 120-N loading at 1377 ± 363 cycles. When loaded to failure, the 3-dimensional constructs failed at 265 N ± 21 N, whereas the 2-dimensional constructs surviving cyclic loading failed at 190 N ± 17 N. The shorter, thinner 3-dimensional metacarpal plates demonstrated increased resistance to failure in a cyclic loading model and increased load to failure compared with the relatively longer, thicker 2-dimensional metacarpal plates. The lower-profile 3-dimensional metacarpal plate fixation demonstrated greater stability for early postoperative resistance than the thicker 2-dimensional fixation, whereas the smaller

  14. Magnetic topologies of coronal mass ejection events: Effects of 3-dimensional reconnection

    SciTech Connect

    Gosling, J.T.

    1995-09-01

    New magnetic loops formed in the corona following coronal mass ejection, CME, liftoffs provide strong evidence that magnetic reconnection commonly occurs within the magnetic ``legs`` of the departing CMEs. Such reconnection is inherently 3-dimensional and naturally produces CMEs having magnetic flux rope topologies. Sustained reconnection behind CMEs can produce a mixture of open and disconnected field lines threading the CMES. In contrast to the results of 2-dimensional reconnection. the disconnected field lines are attached to the outer heliosphere at both ends. A variety of solar and solar wind observations are consistent with the concept of sustained 3-dimensional reconnection within the magnetic legs of CMEs close to the Sun.

  15. RNA Interference and BMP-2 Stimulation Allows Equine Chondrocytes Redifferentiation in 3D-Hypoxia Cell Culture Model: Application for Matrix-Induced Autologous Chondrocyte Implantation.

    PubMed

    Rakic, Rodolphe; Bourdon, Bastien; Hervieu, Magalie; Branly, Thomas; Legendre, Florence; Saulnier, Nathalie; Audigié, Fabrice; Maddens, Stéphane; Demoor, Magali; Galera, Philippe

    2017-08-24

    As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine.

  16. Matrix molecule influence on chondrocyte phenotype and proteoglycan 4 expression by alginate-embedded zonal chondrocytes and mesenchymal stem cells.

    PubMed

    Coates, Emily E; Riggin, Corinne N; Fisher, John P

    2012-12-01

    Articular cartilage resists load and provides frictionless movement at joint surfaces. The tissue is organized into the superficial, middle, deep, and calcified zones throughout its depth, each which serve distinct functions. Proteoglycan 4 (PRG4), found in the superficial zone, is a critical component of the joint's lubricating mechanisms. Maintenance of both the chondrocyte and zonal chondrocyte phenotype remain challenges for in vitro culture and tissue engineering. Here we investigate the expression of PRG4 mRNA and protein by primary bovine superficial zone chondrocytes, middle/deep zone chondrocytes, and mesenchymal stem cells encapsulated in alginate hydrogels with hyaluronic acid (HA) and chondroitin sulfate (CS) additives. Chondrogenic phenotype and differentiation markers are evaluated by mRNA expression, histochemical, and immunohistochemical staining. Results show middle/deep cells express no measurable PRG4 mRNA by day 7. In contrast, superficial zone cells express elevated PRG4 mRNA throughout culture time. This expression can be significantly enhanced up to 15-fold by addition of both HA and CS to scaffolds. Conversely, PRG4 mRNA expression is downregulated (up to 5-fold) by CS and HA in differentiating MSCs, possibly due to build up of entrapped protein. HA and CS demonstrate favorable effects on chondrogenesis by upregulating transcription factor Sox9 mRNA (up to 4.6-fold) and downregulating type I collagen mRNA (up to 18-fold). Results highlight the important relationship between matrix components and expression of critical lubricating proteins in an engineered cartilage scaffold. Copyright © 2012 Orthopaedic Research Society.

  17. The potential of human allogeneic juvenile chondrocytes for restoration of articular cartilage.

    PubMed

    Adkisson, H Davis; Martin, James A; Amendola, Richard L; Milliman, Curt; Mauch, Kelsey A; Katwal, Arbindra B; Seyedin, Mitchell; Amendola, Annuziato; Streeter, Philip R; Buckwalter, Joseph A

    2010-07-01

    Donor-site morbidity, limited numbers of cells, loss of phenotype during ex vivo expansion, and age-related decline in chondrogenic activity present critical obstacles to the use of autologous chondrocyte implantation for cartilage repair. Chondrocytes from juvenile cadaveric donors may represent an alternative to autologous cells. Hypothesis/ The authors hypothesized that juvenile chondrocyte would show stronger and more stable chondrogenic activity than adult cells in vitro and that juvenile cells pose little risk of immunologic incompatibility in adult hosts. Controlled laboratory study. Cartilage samples were from juvenile (<13 years old) and adult (>13 years old) donors. The chondrogenic activity of freshly isolated human articular chondrocytes and of expanded cells after monolayer culture was measured by proteoglycan assay, gene expression analysis, and histology. Lymphocyte proliferation assays were used to assess immunogenic activity. Proteoglycan content in neocartilage produced by juvenile chondrocytes was 100-fold higher than in neocartilage produced by adult cells. Collagen type II and type IX mRNA in fresh juvenile chondrocytes were 100- and 700-fold higher, respectively, than in adult chondrocytes. The distributions of collagens II and IX were similar in native juvenile cartilage and in neocartilage made by juvenile cells. Juvenile cells grew significantly faster in monolayer cultures than adult cells (P = .002) and proteoglycan levels produced in agarose culture was significantly higher in juvenile cells than in adult cells after multiple passages (P < .001). Juvenile chondrocytes did not stimulate lymphocyte proliferation. These results document a dramatic age-related decline in human chondrocyte chondrogenic potential and show that allogeneic juvenile chondrocytes do not stimulate an immunologic response in vivo. Juvenile human chondrocytes have greater potential to restore articular cartilage than adult cells, and may be transplanted without the

  18. THE POTENTIAL OF HUMAN ALLOGENEIC JUVENILE CHONDROCYTES FOR RESTORATION OF ARTICULAR CARTILAGE

    PubMed Central

    Adkisson, H. Davis; Martin, James A.; Amendola, Richard L.; Milliman, Curt; Mauch, Kelsey A.; Katwal, Arbindra B.; Seyedin, Mitchell; Amendola, Annuziato; Streeter, Philip R.; Buckwalter, Joseph A.

    2013-01-01

    Background Donor site morbidity, limited numbers of cells, loss of phenotype during ex vivo expansion, and age-related decline in chondrogenic activity present critical obstacles to the use of autologous chondrocyte implantation for cartilage repair. Chondrocytes from juvenile cadaveric donors may represent an alternative to autologous cells. Hypothesis/Purpose The authors hypothesized that juvenile chondrocyte would show stronger and more stable chondrogenic activity than adult cells in vitro and that juvenile cells pose little risk of immunologic incompatibility in adult hosts. Study Design Controlled laboratory study. Methods Cartilage samples were from juvenile (<13 years old) and adult (> 13 years old) donors. The chondrogenic activity of freshly isolated human articular chondrocytes and of expanded cells after monolayer culture was measured by proteoglycan assay, gene expression analysis, and histology. Lymphocyte proliferation assays were used to assess immunogenic activity. Results Proteoglycan content in neocartilage produced by juvenile chondrocytes was 100-fold higher than in neocartilage produced by adult cells. Collagen type II and type IX mRNAs in fresh juvenile chondrocytes were 100- and 700-fold higher, respectively, than in adult chondrocytes. The distributions of collagens II and IX were similar in native juvenile cartilage and in neocartilage made by juvenile cells. Juvenile cells grew significantly faster in monolayer cultures than adult cells (p = 0.002) and proteoglycan levels produced in agarose culture was significantly higher in juvenile cells than in adult cells after multiple passages (p < 0.001). Juvenile chondrocytes did not stimulate lymphocyte proliferation. Conclusions These results document a dramatic age related decline in human chondrocyte chondrogenic potential and show that allogeneic juvenile chondrocytes do not stimulate an immunologic response in vivo. Clinical Relevance Juvenile human chondrocytes have greater potential to

  19. Platelets promote cartilage repair and chondrocyte proliferation via ADP in a rodent model of osteoarthritis.

    PubMed

    Zhou, Qi; Xu, Chunhua; Cheng, Xingyao; Liu, Yangyang; Yue, Ming; Hu, Mengjiao; Luo, Dongjiao; Niu, Yuxi; Ouyang, Hongwei; Ji, Jiansong; Hu, Hu

    2016-01-01

    Osteoarthritis (OA) is the most common age-related degenerative joint disease and platelet-rich plasma (PRP) has been shown to be beneficial in OA. Therefore, in this study, we aimed to investigate the effects of platelets on chondrocytes and the underlying mechanisms. Anabolic and catabolic activity and the proliferation rate of chondrocytes were evaluated after co-culture with platelets. Chondrocyte gene expression was measured by real-time PCR. Chondrocyte protein expression and phosphorylation were measured by western blot. Chondrocytes treated with or without platelets were transplanted into a rat model of OA induced by intra-articular injection of monosodium iodoacetate and the repair of articular cartilage was evaluated macroscopically and histologically. Platelets significantly promoted the proliferation of chondrocytes, while mildly influencing anabolic and catabolic activity. Chondrocytes co-cultured with platelets showed significantly increased production of bone morphogenetic protein 7 (BMP7). The autocrine/paracrine effect of BMP7 was responsible for the increased proliferation of chondrocytes, via the ERK/CDK1/cyclin B1 signaling pathway. Transplantation of platelet-treated chondrocytes showed better cartilage repair in the OA model. Platelet-derived ADP was identified as the major mediator to promote the production of BMP7 and the proliferation of chondrocytes, through the ADP receptor P2Y1. Finally, direct injection of α,β-methyleneadenosine-5'-diphosphate into OA joints also enhanced cartilage repair. This study has identified that platelet-derived ADP, but not ATP, is the key mediator for platelet-promoted chondrocyte proliferation and cartilage repair in osteoarthritis. This finding may provide a key explanation for the therapeutic effect of platelets in OA and help shaping a strategy to improve OA therapy.

  20. Transcription Factor Erg Variants and Functional Diversification of Chondrocytes during Limb Long Bone Development

    PubMed Central

    Iwamoto, Masahiro; Higuchi, Yoshinobu; Koyama, Eiki; Enomoto-Iwamoto, Motomi; Kurisu, Kojiro; Yeh, Helena; Abrams, William R.; Rosenbloom, Joel; Pacifici, Maurizio

    2000-01-01

    During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27–amino acid segment located ∼80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes. PMID:10893254

  1. IGF1 regulates RUNX1 expression via IRS1/2: Implications for antler chondrocyte differentiation.

    PubMed

    Yang, Zhan-Qing; Zhang, Hong-Liang; Duan, Cui-Cui; Geng, Shuang; Wang, Kai; Yu, Hai-Fan; Yue, Zhan-Peng; Guo, Bin

    2017-03-19

    Although IGF1 is important for the proliferation and differentiation of chondrocytes, its underlying molecular mechanism is still unknown. Here we addressed the physiologic function of IGF1 in antler cartilage and explored the interplay of IGF1, IRS1/2 and RUNX1 in chondrocyte differentiation. The results showed that IGF1 was highly expressed in antler chondrocytes. Exogenous rIGF1 could increase the proliferation of chondrocytes and cell proportion in the S phase, whereas IGF1R inhibitor PQ401 abrogated the induction by rIGF1. Simultaneously, IGF1 could stimulate the expression of IHH which was a well-known marker for prehypertrophic chondrocytes. Further analysis evidenced that IGF1 regulated the expression of IRS1/2 whose silencing resulted in a rise of IHH mRNA levels, but the regulation was impeded by PQ401. Knockdown of IRS1 or IRS2 with specific siRNA could greatly enhance rIGF1-induced chondrocyte differentiation and reduce the expression of RUNX1. Extraneous rRUNX1 might rescue the effects of IRS1 or IRS2 siRNA on the differentiation. In antler chondrocytes, IGF1 played a role in modulating the expression of RUNX1 through IGF1R. Moreover, attenuation of RUNX1 expression advanced the differentiation elicited by rIGF1, while administration of rRUNX1 to chondrocytes treated with IGF1 siRNA or PQ401 reduced their differentiation. Additionally, siRNA-mediated downregulation of IRS1 or IRS2 in the chondrocytes impaired the interaction between IGF1 and RUNX1. Collectively, IGF1 could promote the proliferation and differentiation of antler chondrocytes. Furthermore, IRS1/2 might act downstream of IGF1 to regulate chondrocyte differentiation through targeting RUNX1.

  2. Autophagy protects end plate chondrocytes from intermittent cyclic mechanical tension induced calcification.

    PubMed

    Xu, Hong-guang; Yu, Yun-fei; Zheng, Quan; Zhang, Wei; Wang, Chuang-dong; Zhao, Xiao-yn; Tong, Wen-xue; Wang, Hong; Liu, Ping; Zhang, Xiao-ling

    2014-09-01

    Calcification of end plate chondrocytes is a major cause of intervertebral disc (IVD) degeneration. However, the underlying molecular mechanism of end plate chondrocyte calcification is still unclear. The aim of this study was to clarify whether autophagy in end plate chondrocytes could protect the calcification of end plate chondrocytes. Previous studies showed that intermittent cyclic mechanical tension (ICMT) contributes to the calcification of end plate chondrocytes in vitro. While autophagy serves as a cell survival mechanism, the relationship of autophagy and induced end plate chondrocyte calcification by mechanical tension in vitro is unknown. Thus, we investigated autophagy, the expression of the autophagy genes, Beclin-1 and LC3, and rat end plate chondrocyte calcification by ICMT. The viability of end plate chondrocytes was examined using the LIVE/DEAD viability/cytotoxicity kit. The reverse transcription-polymerase chain reaction and western blotting were used to detect the expression of Beclin-1; LC3; type I, II and X collagen; aggrecan; and Sox-9 genes. Immunofluorescent and fluorescent microscopy showed decreased autophagy in the 10- and 20-day groups loaded with ICMT. Additionally, Alizarin red and alkaline phosphatase staining detected the palpable calcification of end plate chondrocytes after ICMT treatment. We found that increased autophagy induced by short-term ICMT treatment was accompanied by an insignificant calcification of end plate chondrocytes. To the contrary, the suppressive autophagy inhibited by long-term ICMT was accompanied by a more significant calcification. The process of calcification induced by ICMT was partially resisted by increased autophagy activity induced by rapamycin, implicating that autophagy may prevent end plate chondrocyte calcification. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. 3-dimensional orthodontics visualization system with dental study models and orthopantomograms

    NASA Astrophysics Data System (ADS)

    Zhang, Hua; Ong, S. H.; Foong, K. W. C.; Dhar, T.

    2005-04-01

    The aim of this study is to develop a system that provides 3-dimensional visualization of orthodontic treatments. Dental plaster models and corresponding orthopantomogram (dental panoramic tomogram) are first digitized and fed into the system. A semi-auto segmentation technique is applied to the plaster models to detect the dental arches, tooth interstices and gum margins, which are used to extract individual crown models. 3-dimensional representation of roots, generated by deforming generic tooth models with orthopantomogram using radial basis functions, is attached to corresponding crowns to enable visualization of complete teeth. An optional algorithm to close the gaps between deformed roots and actual crowns by using multi-quadratic radial basis functions is also presented, which is capable of generating smooth mesh representation of complete 3-dimensional teeth. User interface is carefully designed to achieve a flexible system with as much user friendliness as possible. Manual calibration and correction is possible throughout the data processing steps to compensate occasional misbehaviors of automatic procedures. By allowing the users to move and re-arrange individual teeth (with their roots) on a full dentition, this orthodontic visualization system provides an easy and accurate way of simulation and planning of orthodontic treatment. Its capability of presenting 3-dimensional root information with only study models and orthopantomogram is especially useful for patients who do not undergo CT scanning, which is not a routine procedure in most orthodontic cases.

  4. 3-Dimensional and Interactive Istanbul University Virtual Laboratory Based on Active Learning Methods

    ERIC Educational Resources Information Center

    Ince, Elif; Kirbaslar, Fatma Gulay; Yolcu, Ergun; Aslan, Ayse Esra; Kayacan, Zeynep Cigdem; Alkan Olsson, Johanna; Akbasli, Ayse Ceylan; Aytekin, Mesut; Bauer, Thomas; Charalambis, Dimitris; Gunes, Zeliha Ozsoy; Kandemir, Ceyhan; Sari, Umit; Turkoglu, Suleyman; Yaman, Yavuz; Yolcu, Ozgu

    2014-01-01

    The purpose of this study is to develop a 3-dimensional interactive multi-user and multi-admin IUVIRLAB featuring active learning methods and techniques for university students and to introduce the Virtual Laboratory of Istanbul University and to show effects of IUVIRLAB on students' attitudes on communication skills and IUVIRLAB. Although there…

  5. Investigation of Measurement Condition for 3-Dimensional Spectroscopy by Scanning Transmission X-ray Microscopy

    NASA Astrophysics Data System (ADS)

    Ohigashi, T.; Inagaki, Y.; Ito, A.; Shinohara, K.; Kosugi, N.

    2017-06-01

    A sample cell for performing computed tomography (CT) was developed. The 3-dimensional (3D) structure of polystyrene spheres was observed and the fluctuation of reconstructed linear absorption coefficients (LAC) was 9.3%. To improve the quality of data in 3D spectro-microscopy, required measurement condition is discussed.

  6. 3-dimensional root phenotyping with a novel imaging and software platform

    USDA-ARS?s Scientific Manuscript database

    A novel imaging and software platform was developed for the high-throughput phenotyping of 3-dimensional root traits during seedling development. To demonstrate the platform’s capacity, plants of two rice (Oryza sativa) genotypes, Azucena and IR64, were grown in a transparent gellan gum system and ...

  7. Topological entropy and renormalization group flow in 3-dimensional spherical spaces

    NASA Astrophysics Data System (ADS)

    Asorey, M.; Beneventano, C. G.; Cavero-Peláez, I.; D'Ascanio, D.; Santangelo, E. M.

    2015-01-01

    We analyze the renormalization group (RG) flow of the temperature independent term of the entropy in the high temperature limit β/a ≪ 1 of a massive field theory in 3-dimensional spherical spaces, M 3, with constant curvature 6 /a 2. For masses lower than , this term can be identified with the free energy of the same theory on M 3 considered as a 3-dimensional Euclidean space-time. The non-extensive part of this free energy, S hol, is generated by the holonomy of the spatial metric connection. We show that for homogeneous spherical spaces the holonomy entropy S hol decreases monotonically when the RG scale flows to the infrared. At the conformal fixed points the values of the holonomy entropy do coincide with the genuine topological entropies recently introduced. The monotonic behavior of the RG flow leads to an inequality between the topological entropies of the conformal field theories connected by such flow, i.e. S {top/ UV } > S {top/ IR }. From a 3-dimensional viewpoint the same term arises in the 3-dimensional Euclidean effective action and has the same monotonic behavior under the RG group flow. We conjecture that such monotonic behavior is generic, which would give rise to a 3-dimensional generalization of the c-theorem, along the lines of the 2-dimensional c-theorem and the 4-dimensional a-theorem. The conjecture is related to recent formulations of the F -theorem. In particular, the holonomy entropy on lens spaces is directly related to the topological Rényi entanglement entropy on disks of 2-dimensional flat spaces.

  8. Heterotopic autologous chondrocyte transplantation--a realistic approach to support articular cartilage repair?

    PubMed

    El Sayed, Karym; Haisch, Andreas; John, Thilo; Marzahn, Ulrike; Lohan, Anke; Müller, Riccarda D; Kohl, Benjamin; Ertel, Wolfgang; Stoelzel, Katharina; Schulze-Tanzil, Gundula

    2010-12-01

    Injured articular cartilage is limited in its capacity to heal. Autologous chondrocyte transplantation (ACT) is a suitable technique for cartilage repair, but it requires articular cartilage biopsies for sufficient autologous chondrocyte expansion in vitro. Hence, ACT is restricted by donor-site morbidity and autologous articular chondrocytes availability. The use of nonarticular heterotopic chondrocytes such as auricular, nasoseptal, or costal chondrocytes for ACT might overcome these limitations: heterotopic sources show lesser donor-site morbidity and a comparable extracellular cartilage matrix synthesis profile to articular cartilage. However, heterotopic (h)ACT poses a challenge. Particular tissue characteristics of heterotopic cartilage, divergent culturing peculiarities of heterotopic chondrocytes, and the advantages and drawbacks related to these diverse cartilage sources were critically discussed. Finally, available in vitro and in vivo experimental (h)ACT approaches were summarized. The quality of the cartilage engineered using heterotopic chondrocytes remains partly controversy due to the divergent methodologies and culture conditions used. While some encouraging in vivo results using (h)ACT have been demonstrated, standardized culturing protocols are strongly required. However, whether heterotopic chondrocytes implanted into joint cartilage defects maintain their particular tissue properties or can be adapted via tissue engineering strategies to fulfill regular articular cartilage functions requires further studies.

  9. Chondrogenic capacity and alterations in hyaluronan synthesis of cultured human osteoarthritic chondrocytes.

    PubMed

    Ono, Yohei; Sakai, Tadahiro; Hiraiwa, Hideki; Hamada, Takashi; Omachi, Takaaki; Nakashima, Motoshige; Ishizuka, Shinya; Matsukawa, Tetsuya; Knudson, Warren; Knudson, Cheryl B; Ishiguro, Naoki

    2013-06-14

    During osteoarthritis there is a disruption and loss of the extracellular matrix of joint cartilage, composed primarily of type II collagen, aggrecan and hyaluronan. In young patients, autologous chondrocyte implantation can be used to repair cartilage defects. However, for more elderly patients with osteoarthritis, such a repair approach is contraindicated because the procedure requires a large expansion of autologous chondrocytes in vitro leading a rapid, perhaps irreversible, loss of the chondrocyte phenotype. This study investigates whether osteoarthritic chondrocytes obtained from older patients can be expanded in vitro and moreover, induced to re-activate their chondrocyte phenotype. A decrease in chondrocyte phenotype markers, collagen II, aggrecan and SOX9 mRNA was observed with successive expansion of cells in monolayer culture. However, chondrogenic induction in three-dimensional pellet culture successfully rescued the expression of all three marker genes to native levels, even with 4th passage cells-cells representing an approximate 625-fold expansion in cell number. This data supports the use of osteoarthritic cells for autologous implantation repair. In addition, another set of gene products were explored as useful markers of the chondrocyte phenotype. Differentiated primary chondrocytes exhibited a common pattern of hyaluronan synthase isoforms that changed upon cell expansion in vitro and, reverted back to the original pattern following pellet culture. Moreover, the change in isoform pattern correlated with changes in the molecular size of synthesized hyaluronan. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Modelling and Simulating the Adhesion and Detachment of Chondrocytes in Shear Flow

    NASA Astrophysics Data System (ADS)

    Hao, Jian; Pan, Tsorng-Whay; Rosenstrauch, Doreen

    Chondrocytes are typically studied in the environment where they normally reside such as the joints in hips, intervertebral disks or the ear. For example, in [SKE+99], the effect of seeding duration on the strength of chondrocyte adhesion to articulate cartilage has been studied in shear flow chamber since such adhesion may play an important role in the repair of articular defects by maintaining cells in positions where their biosynthetic products can contribute to the repair process. However, in this investigation, we focus mainly on the use of auricular chondrocytes in cardiovascular implants. They are abundant, easily and efficiently harvested by a minimally invasive technique. Auricular chondrocytes have ability to produce collagen type-II and other important extracellular matrix constituents; this allows them to adhere strongly to the artificial surfaces. They can be genetically engineered to act like endothelial cells so that the biocompatibility of cardiovascular prothesis can be improved. Actually in [SBBR+02], genetically engineered auricular chondrocytes can be used to line blood-contacting luminal surfaces of left ventricular assist device (LVAD) and a chondrocyte-lined LVAD has been planted into the tissue-donor calf and the results in vivo have proved the feasibility of using autologous auricular chondrocytes to improve the biocompatibility of the blood-biomaterial interface in LVADs and cardiovascular prothesis. Therefore, cultured chondrocytes may offer a more efficient and less invasive means of covering artificial surface with a viable and adherent cell layer.

  11. ADAM17 controls endochondral ossification by regulating terminal differentiation of chondrocytes.

    PubMed

    Hall, Katherine C; Hill, Daniel; Otero, Miguel; Plumb, Darren A; Froemel, Dara; Dragomir, Cecilia L; Maretzky, Thorsten; Boskey, Adele; Crawford, Howard C; Selleri, Licia; Goldring, Mary B; Blobel, Carl P

    2013-08-01

    Endochondral ossification is a highly regulated process that relies on properly orchestrated cell-cell interactions in the developing growth plate. This study is focused on understanding the role of a crucial regulator of cell-cell interactions, the membrane-anchored metalloproteinase ADAM17, in endochondral ossification. ADAM17 releases growth factors, cytokines, and other membrane proteins from cells and is essential for epidermal growth factor receptor (EGFR) signaling and for processing tumor necrosis factor alpha. Here, we report that mice lacking ADAM17 in chondrocytes (A17ΔCh) have a significantly expanded zone of hypertrophic chondrocytes in the growth plate and retarded growth of long bones. This abnormality is caused by an accumulation of the most terminally differentiated type of chondrocytes that produces a calcified matrix. Inactivation of ADAM17 in osteoclasts or endothelial cells does not affect the zone of hypertrophic chondrocytes, suggesting that the main role of ADAM17 in the growth plate is in chondrocytes. This notion is further supported by in vitro experiments showing enhanced hypertrophic differentiation of primary chondrocytes lacking Adam17. The enlarged zone of hypertrophic chondrocytes in A17ΔCh mice resembles that described in mice with mutant EGFR signaling or lack of its ligand transforming growth factor α (TGFα), suggesting that ADAM17 regulates terminal differentiation of chondrocytes during endochondral ossification by activating the TGFα/EGFR signaling axis.

  12. AP-1 family members act with Sox9 to promote chondrocyte hypertrophy.

    PubMed

    He, Xinjun; Ohba, Shinsuke; Hojo, Hironori; McMahon, Andrew P

    2016-08-15

    An analysis of Sox9 binding profiles in developing chondrocytes identified marked enrichment of an AP-1-like motif. Here, we have explored the functional interplay between Sox9 and AP-1 in mammalian chondrocyte development. Among AP-1 family members, Jun and Fosl2 were highly expressed within prehypertrophic and early hypertrophic chondrocytes. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) showed a striking overlap in Jun- and Sox9-bound regions throughout the chondrocyte genome, reflecting direct binding of each factor to the same enhancers and a potential for protein-protein interactions within AP-1- and Sox9-containing complexes. In vitro reporter analysis indicated that direct co-binding of Sox9 and AP-1 at target motifs promoted gene activity. By contrast, where only one factor can engage its DNA target, the presence of the other factor suppresses target activation consistent with protein-protein interactions attenuating transcription. Analysis of prehypertrophic chondrocyte removal of Sox9 confirmed the requirement of Sox9 for hypertrophic chondrocyte development, and in vitro and ex vivo analyses showed that AP-1 promotes chondrocyte hypertrophy. Sox9 and Jun co-bound and co-activated a Col10a1 enhancer in Sox9 and AP-1 motif-dependent manners consistent with their combined action promoting hypertrophic gene expression. Together, the data support a model in which AP-1 family members contribute to Sox9 action in the transition of chondrocytes to the hypertrophic program.

  13. Chondrocyte death in mechanically injured articular cartilage--the influence of extracellular calcium.

    PubMed

    Amin, Anish K; Huntley, James S; Bush, Peter G; Simpson, A Hamish R W; Hall, Andrew C

    2009-06-01

    Calcium is thought to be an important regulator of chondrocyte death associated with articular cartilage injury. Our objective was to determine the influence of extracellular calcium on chondrocyte death following mechanical injury. Using a surgically relevant model of sharp mechanical injury (with a scalpel) and confocal laser scanning microscopy (CLSM), in situ chondrocyte death was quantified within the full thickness of articular cartilage as a function of medium calcium concentration and time (2.5 h and 7 days). Exposure of articular cartilage to calcium-free media (approximately 0 mM) significantly reduced superficial zone chondrocyte death after mechanical injury compared with exposure to calcium-rich media (2-20 mM, ANOVA at 2.5 h, p = 0.002). In calcium-rich media, although the extent of chondrocyte death increased with increasing medium calcium concentration, cell death remained localized to the superficial zone of articular cartilage over 7 days (ANOVA, p < 0.05). However, in calcium-free media, there was an increase in chondrocyte death within deeper zones of articular cartilage over 7 days. The early (within hours) chondroprotective effect in calcium-free media suggests that the use of joint irrigation solutions without added calcium may decrease chondrocyte death from mechanical injury during articular surgery. The delayed (within days) increase in chondrocyte death in calcium-free media supports the use of calcium supplementation in media used during cartilage culture for tissue engineering or transplantation.

  14. Expansion of the number of human auricular chondrocytes: recycling of culture media containing floating cells.

    PubMed

    Kamil, S H; Rodrigruez, A; Vacanti, C A; Eavey, R D; Vacanti, M P

    2004-01-01

    To grow a complete human size auricle by utilizing the principles of tissue engineering, a large number of chondrocytes is required for initial implantation. The number of chondrocytes can be increased by repeated passaging or by incubation with different growth factors, both of which can promote dedifferentiation. New methods of chondrocyte expansion over a relatively brief time period for potential practical application are required. In this study auricular chondrocytes were obtained from patients and cultured in vitro. Two groups of cells were created. Group A chondrocyte number was increased by repeated passaging. Group B cells were grown from floating culture medium and their number was increased both by passaging and by repeated recycling of the culture medium. Chondrocytes from both groups were implanted in nude mice for 8 weeks to generate tissue-engineered cartilage. Flow cytometry studies performed on both groups confirmed the presence of two distinct populations of structures as the source of chondrocytes from the recycled medium. Repeated recycling of the culture medium demonstrates a promising method to increase the number of chondrocytes in vitro for clinical application.

  15. Atf4 regulates chondrocyte proliferation and differentiation during endochondral ossification by activating Ihh transcription

    PubMed Central

    Wang, Weiguang; Lian, Na; Li, Lingzhen; Moss, Heather E.; Wang, Weixi; Perrien, Daniel S.; Elefteriou, Florent; Yang, Xiangli

    2009-01-01

    Activating transcription factor 4 (Atf4) is a leucine-zipper-containing protein of the cAMP response element-binding protein (CREB) family. Ablation of Atf4 (Atf4−/−) in mice leads to severe skeletal defects, including delayed ossification and low bone mass, short stature and short limbs. Atf4 is expressed in proliferative and prehypertrophic growth plate chondrocytes, suggesting an autonomous function of Atf4 in chondrocytes during endochondral ossification. In Atf4−/− growth plate, the typical columnar structure of proliferative chondrocytes is disturbed. The proliferative zone is shortened, whereas the hypertrophic zone is transiently expanded. The expression of Indian hedgehog (Ihh) is markedly decreased, whereas the expression of other chondrocyte marker genes, such as type II collagen (Col2a1), PTH/PTHrP receptor (Pth1r) and type X collagen (Col10a1), is normal. Furthermore, forced expression of Atf4 in chondrocytes induces endogenous Ihh mRNA, and Atf4 directly binds to the Ihh promoter and activates its transcription. Supporting these findings, reactivation of Hh signaling pharmacologically in mouse limb explants corrects the Atf4−/− chondrocyte proliferation and short limb phenotypes. This study thus identifies Atf4 as a novel transcriptional activator of Ihh in chondrocytes that paces longitudinal bone growth by controlling growth plate chondrocyte proliferation and differentiation. PMID:19906842

  16. Curcumin Inhibits Chondrocyte Hypertrophy of Mesenchymal Stem Cells through IHH and Notch Signaling Pathways.

    PubMed

    Cao, Zhen; Dou, Ce; Dong, Shiwu

    2017-01-01

    Using tissue engineering technique to repair cartilage damage caused by osteoarthritis is a promising strategy. However, the regenerated tissue usually is fibrous cartilage, which has poor mechanical characteristics compared to hyaline cartilage. Chondrocyte hypertrophy plays an important role in this process. Thus, it is very important to find out a suitable way to maintain the phenotype of chondrocytes and inhibit chondrocyte hypertrophy. Curcumin deriving from turmeric was reported with anti-inflammatory and anti-tumor pharmacological effects. However, the role of curcumin in metabolism of chondrocytes, especially in the chondrocyte hypertrophy remains unclear. Mesenchymal stem cells (MSCs) are widely used in cartilage tissue engineering as seed cells. So we investigated the effect of curcumin on chondrogenesis and chondrocyte hypertrophy in MSCs through examination of cell viability, glycosaminoglycan synthesis and specific gene expression. We found curcumin had no effect on expression of chondrogenic markers including Sox9 and Col2a1 while hypertrophic markers including Runx2 and Col10a1 were down-regulated. Further exploration showed that curcumin inhibited chondrocyte hypertrophy through Indian hedgehog homolog (IHH) and Notch signalings. Our results indicated curcumin was a potential agent in modulating cartilage homeostasis and maintaining chondrocyte phenotype.

  17. Linking cell shape, elasticity and fate: in vitro re-differentiation of chondrocytes

    NASA Astrophysics Data System (ADS)

    Yuan, Xiaofei; Chim, Yahua; Yin, Huabing

    2014-02-01

    Autologous chondrocyte transplantation (ACT) has become a promising method for repairing large articular defects. However, dedifferentiation of chondrocytes during cell expansion remains a major limitation for ACT procedures. In this study, we explore the potential of confining cell shape for re-differentiation of dedifferentiated bovine chondrocytes. A novel culture system, combining 2D micropatterning with 3D matrix formation, was developed to control and maintain individual chondrocyte's shape. Both collagen II synthesis and the mechanical properties of cells were monitored during re-differentiation. We show that a spherical morphology without cell spreading plays a limited role in induction of re-differentiation. Instead, isolated, dedifferentiated chondrocytes partially regain chondrogenic properties if they have an appropriate cell shape and limited spreading.

  18. Maintaining the Phenotype Stability of Chondrocytes Derived from MSCs by C-Type Natriuretic Peptide

    PubMed Central

    Shi, Quan; Qian, Zhiyong; Liu, Donghua; Sun, Jie; Xu, Juan; Guo, Ximin

    2017-01-01

    Mesenchymal stem cells (MSCs) play a critical role in cartilage tissue engineering. However, MSCs-derived chondrocytes or cartilage tissues are not stable and easily lose the cellular and cartilage phenotype during long-term culture in vitro or implantation in vivo. As a result, chondrocytes phenotypic instability can contribute to accelerated ossification. Thus, it is a big challenge to maintain their correct phenotype for engineering hyaline cartilage. As one member of the natriuretic peptide family, C-type natriuretic peptide (CNP) is found to correlate with the development of the cartilage, affect the chondrocytes proliferation and differentiation. Besides, based on its biological effects on protection of extracellular matrix of cartilage and inhibition of mineralization, we hypothesize that CNP may contribute to the stability of chondrocyte phenotype of MSCs-derived chondrocytes. PMID:28337152

  19. Direct measurement of TRPV4 and PIEZO1 activity reveals multiple mechanotransduction pathways in chondrocytes

    PubMed Central

    Rocio Servin-Vences, M; Moroni, Mirko; Lewin, Gary R; Poole, Kate

    2017-01-01

    The joints of mammals are lined with cartilage, comprised of individual chondrocytes embedded in a specialized extracellular matrix. Chondrocytes experience a complex mechanical environment and respond to changing mechanical loads in order to maintain cartilage homeostasis. It has been proposed that mechanically gated ion channels are of functional importance in chondrocyte mechanotransduction; however, direct evidence of mechanical current activation in these cells has been lacking. We have used high-speed pressure clamp and elastomeric pillar arrays to apply distinct mechanical stimuli to primary murine chondrocytes, stretch of the membrane and deflection of cell-substrate contacts points, respectively. Both TRPV4 and PIEZO1 channels contribute to currents activated by stimuli applied at cell-substrate contacts but only PIEZO1 mediates stretch-activated currents. These data demonstrate that there are separate, but overlapping, mechanoelectrical transduction pathways in chondrocytes. DOI: http://dx.doi.org/10.7554/eLife.21074.001 PMID:28135189

  20. The Biological Effects of Sex Hormones on Rabbit Articular Chondrocytes from Different Genders

    PubMed Central

    Lin, Yen Ting

    2014-01-01

    The aim of this study was to investigate the biological effects of sex hormones (17β-estradiol and testosterone) on rabbit articular chondrocytes from different genders. We cultured primary rabbit articular chondrocytes from both genders with varying concentration of sex hormones. We evaluate cell proliferation and biochemical functions by MTT and GAG assay. The chondrocyte function and phenotypes were analyzed by mRNA level using RT-PCR. Immunocytochemical staining was also used to evaluate the generation of collagen-II. This study demonstrated that 17β-estradiol had greater positive regulation on the biological function and gene expressions of articular chondrocytes than testosterone, with the optimal concentrations of 10−6 and 10−7 M, particularly for female chondrocytes. PMID:24995337

  1. The biological effects of sex hormones on rabbit articular chondrocytes from different genders.

    PubMed

    Chang, Shwu Jen; Kuo, Shyh Ming; Lin, Yen Ting; Yang, Shan-Wei

    2014-01-01

    The aim of this study was to investigate the biological effects of sex hormones (17β-estradiol and testosterone) on rabbit articular chondrocytes from different genders. We cultured primary rabbit articular chondrocytes from both genders with varying concentration of sex hormones. We evaluate cell proliferation and biochemical functions by MTT and GAG assay. The chondrocyte function and phenotypes were analyzed by mRNA level using RT-PCR. Immunocytochemical staining was also used to evaluate the generation of collagen-II. This study demonstrated that 17β-estradiol had greater positive regulation on the biological function and gene expressions of articular chondrocytes than testosterone, with the optimal concentrations of 10(-6) and 10(-7) M, particularly for female chondrocytes.

  2. R-spondin 2 facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β-catenin signaling in endochondral ossification

    SciTech Connect

    Takegami, Yasuhiko; Ohkawara, Bisei; Ito, Mikako; Masuda, Akio; Nakashima, Hiroaki; Ishiguro, Naoki; Ohno, Kinji

    2016-04-22

    Endochondral ossification is a crucial process for longitudinal growth of bones. Differentiating chondrocytes in growth cartilage form four sequential zones of proliferation, alignment into column, hypertrophy, and substitution of chondrocytes with osteoblasts. Wnt/β-catenin signaling is essential for differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. R-spondin 2 (Rspo2), a member of R-spondin family, is an agonist for Wnt signaling, but its role in chondrocyte differentiation remains unknown. Here we report that growth cartilage of Rspo2-knockout mice shows a decreased amount of β-catenin and increased amounts collagen type II (CII) and Sox9 in the abnormally extended proliferating zone. In contrast, expression of collagen type X (CX) in the hypertrophic zone remains unchanged. Differentiating chondrogenic ATDC5 cells, mimicking proliferating chondrocytes, upregulate Rspo2 and its putative receptor, Lgr5, in parallel. Addition of recombinant human Rspo2 to differentiating ATDC5 cells decreases expressions of Col2a1, Sox9, and Acan, as well as production of proteoglycans. In contrast, lentivirus-mediated knockdown of Rspo2 has the opposite effect. The effect of Rspo2 on chondrogenic differentiation is mediated by Wnt/β-catenin signaling, and not by Wnt/PCP or Wnt/Ca{sup 2+} signaling. We propose that Rspo2 activates Wnt/β-catenin signaling to reduce Col2a1 and Sox9 and to facilitate differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. - Highlights: • Rspo2 is a secreted activator of Wnt, and its knockout shows extended proliferating chondrocytes in endochondral ossification. • In proliferating chondrocytes of Rspo2-knockout mice, Sox9 and collagen type 2 are increased and β-catenin is decreased. • Rspo2 and its receptor Lgr5, as well as Sox9 and collagen type 2, are expressed in differentiating ATDC5 chondrogenic cells. • In ATDC5 cells, Rspo2 decreases

  3. [Repair of chondral defects of the knee using a combination of autologous chondrocytes and osteochondral allograft--an animal model. Part I: in vitro culture of autologous chondrocytes].

    PubMed

    Bacenková, D; Rosocha, J; Svihla, R; Vasko, G; Bodnár, J

    2001-01-01

    In the study we used in vitro cultivated autologous chondrocytes in combination with osteochondral allografts for the treatment of local defects of articular cartilage on the animal model (rabbit). Chondrocytes for in vitro cultivation were harvested by biopsy of articular cartilage of rabbit. For the monolayer cultivation we used Nutrient mix F 12 (Gibco BRL) with addition of Lascorbic acid (50 micrograms/ml, Sigma) and insulin-trasferin-selenium (A 6.26 micrograms/ml, Gibco BRL), 20% of fectal serum (Gibco BRL) and antibiotic antimycotic solution (Gibco BRL). Cultivation of chondrocytes took place at 37 degrees in the atmosphere of 5% CO2. Multiplied chondrocytes re-suspended in fibrin glue in combination with two osteochondral allografts were used for the reparation of artificial defect of the rabbit cartilage. For the analysis of collagen type II in the cultivation medium we used the principle of salting out by 30% ammonium sulphate and subsequent pepsinization in an acid environment with a repeated salting out by means of 2M of NaCl. Precipitates were dissolved in 5.0 M of acetic acid and used for SDS PAGE and immunoblotting. As a detection system we used ECL (Amersham/Pharmacia Biotech). The final average number of chondrocytes multiplied by monolayer cultivation was 1.10(5). The presence of collagen of type II has proved the preservation of the original phenotype of chondrocytes during cultivation. Bioengineering use of cell and tissue cultivation provides new options of the treatment of defect of connective tissue. Transplantation of autologous chondrocytes in combination with osteochondral allografts is on the basis of our results obtained so far a promising therapy. The aim of our work was an ex vivo expansion of autologous chondrocytes for the purpose of cell transplantation.

  4. Autophagy Activation Protects from Mitochondrial Dysfunction in Human Chondrocytes

    PubMed Central

    de Figueroa, Paloma López; Lotz, Martin; Blanco, Francisco J.; Caramés, Beatriz

    2015-01-01

    Objective Autophagy, is a key pathway of cellular homeostasis for removing damaged macromolecules and organelles, including mitochondria. Recent studies indicate that autophagy activation is defective in aging and osteoarthritis (OA), contributing to the cell death and tissue damage. In addition, there is increasing evidence that mitochondrial dysfunction plays an important role in OA pathogenesis. The objective of this study is to determine whether activation of autophagy protects from mitochondrial dysfunction in human chondrocytes. Methods Human chondrocytes were treated with Oligomycin, an inhibitor of mitochondrial respiratory chain (MRC) complex V. Autophagy activation was analyzed by determination of LC3-II, a marker for autophagosome formation. To investigate whether autophagy protects from mitochondrial dysfunction, autophagy was induced by mammalian target of rapamycin complex 1 (mTORC1) selective inhibitor Rapamycin and the dual mTORC1 and mTORC2 inhibitor Torin 1. SiAtg5 was employed to evaluate the role of autophagy in mitochondrial dysfunction. Results Mitochondrial dysfunction was induced by treatment with Oligomycin, which significantly decreased mitochondrial membrane potential (Δψm). This was associated with increased ROS production and cell death. Autophagy activation, reflected by LC3-II, was decreased in a time dependent manner. To evaluate whether autophagy regulates mitochondrial function, chondrocytes were pre-treated with Rapamycin and Torin 1 before Oligomycin. Autophagy activation significantly protected against mitochondrial dysfunction. Conversely, genetic inhibition of autophagy induced significant mitochondrial function defects. Conclusion Our data highlight the role of autophagy as a critical protective mechanism against mitochondrial dysfunction. Pharmacological interventions that enhance autophagy may have chondroprotective activity in cartilage degenerative processes such as OA. PMID:25605458

  5. Increasing the Dose of Autologous Chondrocytes Improves Articular Cartilage Repair

    PubMed Central

    Guillén-García, Pedro; Rodríguez-Iñigo, Elena; Guillén-Vicente, Isabel; Caballero-Santos, Rosa; Guillén-Vicente, Marta; Abelow, Stephen; Giménez-Gallego, Guillermo

    2014-01-01

    Background: We hypothesized that implanting cells in a chondral defect at a density more similar to that of the intact cartilage could induce them to synthesize matrix with the features more similar to that of the uninjured one. Methods: We compared the implantation of different doses of chondrocytes: 1 million (n = 5), 5 million (n = 5), or 5 million mesenchymal cells (n = 5) in the femoral condyle of 15 sheep. Tissue generated by microfracture at the trochlea, and normal cartilage from a nearby region, processed as the tissues resulting from the implantation, were used as references. Histological and molecular (expression of type I and II collagens and aggrecan) studies were performed. Results: The features of the cartilage generated by implantation of mesenchymal cells and elicited by microfractures were similar and typical of a poor repair of the articular cartilage (presence of fibrocartilage, high expression of type I collagen and a low mRNA levels of type II collagen and aggrecan). Nevertheless, in the samples obtained from tissues generated by implantation of chondrocytes, hyaline-like cartilage, cell organization, low expression rates of type I collagen and high levels of mRNA corresponding to type II collagen and aggrecan were observed. These histological features, show less variability and are more similar to those of the normal cartilage used as control in the case of 5 million cells implantation than when 1 million cells were used. Conclusions: The implantation of autologous chondrocytes in type I/III collagen membranes at high density could be a promising tool to repair articular cartilage. PMID:26069691

  6. Effect of metal ions on calcifying growth plate cartilage chondrocytes.

    PubMed

    Litchfield, T M; Ishikawa, Y; Wu, L N; Wuthier, R E; Sauer, G R

    1998-04-01

    The effects of the trace metals zinc (Zn), manganese (Mn), and cadmium (Cd) on the metabolism of growth plate chondrocytes was examined using a mineralizing culture system. Supplementation of serum-free primary cultures of growth plate chondrocytes with 10-100 mu m Zn resulted in an increase in cell protein and greatly increased alkaline phosphatase (AP) activity; however, above 25 mu m Zn mineralization of the cultures was reduced. The effects of Zn on cellular protein and AP activity were enhanced by the addition of the albumin to the culture media. Removal of Zn from basal culture media resulted in recoverable reductions in cellular protein and AP activities. Cadmium was acutely toxic to chondrocyte cell cultures at concentrations above 5 mu m. Even at very low concentrations (0.25 mu m) Cd caused significant reductions in DNA, cellular protein, and matrix protein synthesis. In contrast, Cd had negligible effects on AP activity or culture mineralization. Manganese treatment (50 mu m) resulted in reduced levels of proteoglycan, cell protein, DNA synthesis, and collagen synthesis, although AP specific activity did not change. At 10 mu m, Mn significantly reduced mineralization but had only minor influence on other culture parameters. Both Zn (200 mu m) and Cd (0.1 mu m), but not Mn, induced the synthesis of metallothionein. The physiological and biochemical effects of specific metal ions is largely dependent on their physicochemical properties, especially their ligand affinities. Knowledge of these properties allows predictions to be made regarding whether the organic or the mineral phase are most likely to be affected in a mineralized tissue.

  7. IFT88 influences chondrocyte actin organization and biomechanics

    PubMed Central

    Wang, Z.; Wann, A.K.T.; Thompson, C.L.; Hassen, A.; Wang, W.; Knight, M.M.

    2016-01-01

    Summary Objectives Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. Methods The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88orpk). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. Results IFT88orpk cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88orpk cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88orpk cells. Following membrane blebbing, IFT88orpk cells exhibited slower reformation of the actin cortex. IFT88orpk cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. Conclusions This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. PMID:26493329

  8. Heterotopic and orthotopic autologous chondrocyte implantation using a minipig chondral defect model.

    PubMed

    Lohan, Anke; Marzahn, Ulrike; El Sayed, Karym; Bock, Christopher; Haisch, Andreas; Kohl, Benjamin; Stoelzel, Katharina; John, Thilo; Ertel, Wolfgang; Schulze-Tanzil, Gundula

    2013-10-01

    Implantation of non-articular (heterotopic) chondrocyte-based implants might be an alternative approach to articular cartilage repair. This strategy could be helpful in cases in which there are no or too few articular chondrocytes available. Therefore, this study was undertaken to compare joint cartilage defect healing in the minipig model after implantation of heterotopic auricular and orthotopic articular chondrocytes. Poly-glycolic acid (PGA) associated three-dimensional (3D) constructs were prepared culturing autologous minipig-derived articular and auricular chondrocytes for 7 days in a dynamic culture system. Chondrocyte PGA constructs were implanted into 8mm diameter and ∼1.1mm deep chondral defects within the medial and lateral condyles of the minipig knee joints. Empty defects served as controls for assessment of the intrinsic healing response. Defect healing was monitored 6 months post implantation using a macroscopic and microscopic score system and biomechanical analysis. Neo-cartilage formation could be observed in the PGA constructs seeded with articular and auricular chondrocytes in vivo. The defect healing did not significantly differ at the macroscopic and histological level in response to implantation of either autologous articular or auricular chondrocytes seeded constructs compared with the empty defects. Although the differences were not significant, the auricular chondrocytes-based implants led to a slightly inferior repair quality at the macroscopic level, but a histologically superior healing response when compared with the empty defect group. However, biomechanical analysis revealed a higher stiffness in repair tissues produced by auricular chondrocyte implantation compared with the other groups. Deduced from these results, articular chondrocytes represent the preferable cell source for implantation. Copyright © 2013 Elsevier GmbH. All rights reserved.

  9. Scaffold-assisted cartilage tissue engineering using infant chondrocytes from human hip cartilage.

    PubMed

    Kreuz, P C; Gentili, C; Samans, B; Martinelli, D; Krüger, J P; Mittelmeier, W; Endres, M; Cancedda, R; Kaps, C

    2013-12-01

    Studies about cartilage repair in the hip and infant chondrocytes are rare. The aim of our study was to evaluate the use of infant articular hip chondrocytes for tissue engineering of scaffold-assisted cartilage grafts. Hip cartilage was obtained from five human donors (age 1-10 years). Expanded chondrocytes were cultured in polyglycolic acid (PGA)-fibrin scaffolds. De- and re-differentiation of chondrocytes were assessed by histological staining and gene expression analysis of typical chondrocytic marker genes. In vivo, cartilage matrix formation was assessed by histology after subcutaneous transplantation of chondrocyte-seeded PGA-fibrin scaffolds in immunocompromised mice. The donor tissue was heterogenous showing differentiated articular cartilage and non-differentiated tissue and considerable expression of type I and II collagens. Gene expression analysis showed repression of typical chondrocyte and/or mesenchymal marker genes during cell expansion, while markers were re-induced when expanded cells were cultured in PGA-fibrin scaffolds. Cartilage formation after subcutaneous transplantation of chondrocyte loaded PGA-fibrin scaffolds in nude mice was variable, with grafts showing resorption and host cell infiltration or formation of hyaline cartilage rich in type II collagen. Addition of human platelet rich plasma (PRP) to cartilage grafts resulted robustly in formation of hyaline-like cartilage that showed type II collagen and regions with type X collagen. These results suggest that culture of expanded and/or de-differentiated infant hip cartilage cells in PGA-fibrin scaffolds initiates chondrocyte re-differentiation. The heterogenous donor tissue containing immature chondrocytes bears the risk of cartilage repair failure in vivo, which may be possibly overcome by the addition of PRP. Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  10. Salvianolic acid B regulates gene expression and promotes cell viability in chondrocytes.

    PubMed

    Yang, Xiaohong; Liu, Shaojie; Li, Siming; Wang, Pengzhen; Zhu, Weicong; Liang, Peihong; Tan, Jianrong; Cui, Shuliang

    2017-02-28

    Articular chondrocytes reside in lacunae distributed in cartilage responsible for the remodelling of the tissue with limited ability of damage repairing. The in vitro expanded chondrocytes enhanced by factors/agents to obtain large numbers of cells with strengthened phenotype are essential for successful repair of cartilage lesions by clinical cell implantation therapies. Because the salvianolic acid B (Sal B), a major hydrophilic therapeutic agent isolated from Salvia miltiorrhiza, has been widely used to treat diseases and able to stimulate activity of cells, this study examines the effects of Sal B on passaged chondrocytes. Chondrocytes were treated with various concentrations of Sal B in monolayer culture, their morphological properties and changes, and mitochondrial membrane potential were analysed using microscopic analyses, including cellular biochemical staining and confocal laser scanning microscopy. The proteins were quantified by BCA and Western blotting, and the transcription of genes was detected by qRT-PCR. The passaged chondrocytes treated with Sal B showed strengthened cellular synthesis and stabilized mitochondrial membrane potential with upregulated expression of the marker genes for chondrocyte phenotype, Col2-α1, Acan and Sox9, the key Wnt signalling molecule β-catenin and paracrine cytokine Cytl-1. The treatments using CYTL-1 protein significantly increased expression of Col2-α1 and Acan with no effect on Sox9, indicating the paracrine cytokine acts on chondrocytes independent of SOX9. Sal B has ultimately promoted cell growth and enhanced chondrocyte phenotype. The chondrocytes treated with pharmaceutical agent and cytokine in the formulated medium for generating large number of differentiated chondrocytes would facilitate the cell-based therapies for cartilage repair.

  11. Induction of cartilage integration by a chondrocyte/collagen-scaffold implant

    PubMed Central

    Pabbruwe, Moreica B.; Esfandiari, Ehsanollah; Kafienah, Wael; Tarlton, John F.; Hollander, Anthony P.

    2009-01-01

    The integration of implanted cartilage is a major challenge for the success of tissue engineering protocols. We hypothesize that in order for effective cartilage integration to take place, matrix-free chondrocytes must be induced to migrate between the two tissue surfaces. A chondrocyte/collagen-scaffold implant system was developed as a method of delivering dividing cells at the interface between two cartilage surfaces. Chondrocytes were isolated from bovine nasal septum and seeded onto both surfaces of a collagen membrane to create the chondrocyte/collagen-scaffold implant. A model of two cartilage discs and the chondrocyte/collagen-scaffold sandwiched in between was used to effect integration in vitro. The resulting tissue was analysed histologically and biomechanically. The cartilage–implant–cartilage sandwich appeared macroscopically as one continuous piece of tissue at the end of 40 day cultures. Histological analysis showed tissue continuum across the cartilage–scaffold interface. The integration was dependent on both cells and scaffold. Fluorescent labeling of implanted chondrocytes demonstrated that these cells invade the surrounding mature tissue and drive a remodelling of the extracellular matrix. Using cell-free scaffolds we also demonstrated that some chondrocytes migrated from the natural cartilage into the collagen scaffold. Quantification of integration levels using a histomorphometric repair index showed that the chondrocyte/collagen-scaffold implant achieved the highest repair index compared to controls, reflected functionally through increased tensile strength. In conclusion, cartilage integration can be achieved using a chondrocyte/collagen-scaffold implant that permits controlled delivery of chondrocytes to both host and graft mature cartilage tissues. This approach has the potential to be used therapeutically for implantation of engineered tissue. PMID:19539365

  12. Induction of cartilage integration by a chondrocyte/collagen-scaffold implant.

    PubMed

    Pabbruwe, Moreica B; Esfandiari, Ehsanollah; Kafienah, Wael; Tarlton, John F; Hollander, Anthony P

    2009-09-01

    The integration of implanted cartilage is a major challenge for the success of tissue engineering protocols. We hypothesize that in order for effective cartilage integration to take place, matrix-free chondrocytes must be induced to migrate between the two tissue surfaces. A chondrocyte/collagen-scaffold implant system was developed as a method of delivering dividing cells at the interface between two cartilage surfaces. Chondrocytes were isolated from bovine nasal septum and seeded onto both surfaces of a collagen membrane to create the chondrocyte/collagen-scaffold implant. A model of two cartilage discs and the chondrocyte/collagen-scaffold sandwiched in between was used to effect integration in vitro. The resulting tissue was analysed histologically and biomechanically. The cartilage-implant-cartilage sandwich appeared macroscopically as one continuous piece of tissue at the end of 40 day cultures. Histological analysis showed tissue continuum across the cartilage-scaffold interface. The integration was dependent on both cells and scaffold. Fluorescent labeling of implanted chondrocytes demonstrated that these cells invade the surrounding mature tissue and drive a remodelling of the extracellular matrix. Using cell-free scaffolds we also demonstrated that some chondrocytes migrated from the natural cartilage into the collagen scaffold. Quantification of integration levels using a histomorphometric repair index showed that the chondrocyte/collagen-scaffold implant achieved the highest repair index compared to controls, reflected functionally through increased tensile strength. In conclusion, cartilage integration can be achieved using a chondrocyte/collagen-scaffold implant that permits controlled delivery of chondrocytes to both host and graft mature cartilage tissues. This approach has the potential to be used therapeutically for implantation of engineered tissue.

  13. Parkin clearance of dysfunctional mitochondria regulates ROS levels and increases survival of human chondrocytes.

    PubMed

    Ansari, M Y; Khan, N M; Ahmad, I; Haqqi, T M

    2017-08-08

    Mitochondrial dysfunction, oxidative stress and chondrocyte death are important contributors to the development and pathogenesis of osteoarthritis (OA). In this study, we determined the expression and role of Parkin in the clearance of damaged/dysfunctional mitochondria, regulation of reactive oxygen species (ROS) levels and chondrocyte survival under pathological conditions. Human chondrocytes were from the unaffected area of knee OA cartilage (n = 12) and were stimulated with IL-1β to mimic pathological conditions. Mitochondrial membrane depolarization and ROS levels were determined using specific dyes and flow cytometry. Autophagy was determined by Western blotting for ATG5, Beclin1, immunofluorescence staining and confocal microscopy. Gene expression was determined by RT-qPCR. siRNA, wild-type and mutant Parkin plasmids were transfected using Amaxa system. Apoptosis was determined by PI staining of chondrocytes and TUNEL assay. IL-1β-stimulated OA chondrocytes showed high levels of ROS generation, mitochondrial membrane damage, accumulation of damaged mitochondria and higher incidence of apoptosis. IL-1β stimulation of chondrocytes with depleted Parkin expression resulted in sustained high levels of ROS, accumulation of damaged/dysfunctional mitochondria and enhanced apoptosis. Parkin translocation to depolarized/damaged mitochondria and recruitment of p62/SQSTM1 was required for the elimination of damaged/dysfunctional mitochondria in IL-1β-stimulated OA chondrocytes. Importantly we demonstrate that Parkin elimination of depolarized/damaged mitochondria required the Parkin ubiquitin ligase activity and resulted in reduced ROS levels and inhibition of apoptosis in OA chondrocytes under pathological conditions. Our data demonstrates that Parkin functions to eliminate depolarized/damaged mitochondria in chondrocytes which is necessary for mitochondrial quality control, regulation of ROS levels and chondrocyte survival under pathological conditions

  14. 3-dimensional versus conventional laparoscopy for benign hysterectomy: protocol for a randomized clinical trial.

    PubMed

    Hoffmann, Elise; Bennich, Gitte; Larsen, Christian Rifbjerg; Lindschou, Jannie; Jakobsen, Janus Christian; Lassen, Pernille Danneskiold

    2017-09-07

    Hysterectomy is one of the most common surgical procedures for women of reproductive age. Laparoscopy was introduced in the 1990es and is today one of the recommended routes of surgery. A recent observational study showed that operative time for hysterectomy was significantly lower for 3-dimensional compared to conventional laparoscopy. Complication rates were similar for the two groups. No other observational studies or randomized clinical trials have compared 3-dimensional to conventional laparoscopy in patients undergoing total hysterectomy for benign disease. The objective of the study is to determine if 3D laparoscopy gives better quality of life, less postoperative pain, less per- and postoperative complications, shorter operative time, or a shorter stay in hospital and a faster return to work or normal life, compared to conventional laparoscopy for benign hysterectomy. The design is a randomised multicentre clinical trial. Participants will be 400 women referred for laparoscopic hysterectomy for benign indications. Patients will be randomized to 3-dimensional or conventional laparoscopic hysterectomy. Operative procedures will follow the same principles and the same standard whether the surgeon's vision is 3-dimensional or conventional laparoscopy. Primary outcomes will be the impact of surgery on quality of life, assessed by the SF 36 questionnaire, and postoperative pain, assessed by a Visual Analogue scale for pain measurement. With a standard deviation of 12 points on SF 36 questionnaire, a risk of type I error of 3.3% and a risk of type II error of 10% a sample size of 190 patients in each arm of the trial is needed. Secondarily, we will investigate operative time, time to return to work, length of hospital stay, and - and postoperative complications. This trial will be the first randomized clinical trial investigating the potential clinical benefits and harms of 3-dimensional compared to conventional laparoscopy. The results may provide more evidence

  15. Efficacy of 3-Dimensional Endorectal Ultrasound for Staging Early Extraperitoneal Rectal Neoplasms.

    PubMed

    Pinto, Rodrigo Ambar; Corrêa Neto, Isaac José Felippe; Nahas, Sérgio Carlos; Rizkalah Nahas, Caio Sérgio; Sparapan Marques, Carlos Frederico; Ribeiro Junior, Ulysses; Kawaguti, Fábio Shiguehissa; Cecconello, Ivan

    2017-05-01

    Adequate oncologic staging of rectal neoplasia is important for treatment and prognostic evaluation of the disease. Diagnostic methods such as endorectal ultrasound can assess rectal wall invasion and lymph node involvement. The purpose of this study was to correlate findings of 3-dimensional endorectal ultrasound and pathologic diagnosis of extraperitoneal rectal tumors with regard to depth of rectal wall invasion, lymph node involvement, percentage of rectal circumference involvement, and tumor extension. Consecutive patients with extraperitoneal rectal tumors were prospectively assessed by 3-dimensional endorectal ultrasound blind to other staging methods and pathologic diagnosis. Patients who underwent endorectal ultrasound followed by surgery were included in the study. The study was conducted at a single academic institution. Sensitivity, specificity, positive and negative predictive values, area under curve, and κ coefficient between 3-dimensional endorectal ultrasound and pathologic diagnosis were determined. Intraclass correlation coefficient was calculated for tumor extension and percentage of rectal wall involvement. Forty-four patients (27 women; mean age = 63.5 years) were evaluated between September 2010 and June 2014. Most lesions were malignant (72.7%). For depth of submucosal invasion, 3-dimensional endorectal ultrasound showed sensitivity of 77.3%, specificity of 86.4%, positive predictive value of 85.0%, a negative predictive value of 79.2%, and an area under curve of 0.82. The weighted κ coefficient for depth of rectal wall invasion staging was 0.67, and there was no agreement between 3-dimensional endorectal ultrasound and pathologic diagnosis for lymph node involvement (κ = -0.164). Intraclass correlation coefficient for lesion extension and percentage of rectal circumference involvement were 0.45 and 0.66. A better correlation between 3-dimensional endorectal ultrasound and pathologic diagnosis was observed in tumors <5 cm and with <50

  16. FGFR1 signaling in hypertrophic chondrocytes is attenuated by the Ras-GAP neurofibromin during endochondral bone formation

    PubMed Central

    Karolak, Matthew R.; Yang, Xiangli; Elefteriou, Florent

    2015-01-01

    Aberrant fibroblast growth factor receptor 3 (FGFR3) signaling disrupts chondrocyte proliferation and growth plate size and architecture, leading to various chondrodysplasias or bone overgrowth. These observations suggest that the duration, intensity and cellular context of FGFR signaling during growth plate chondrocyte maturation require tight, regulated control for proper bone elongation. However, the machinery fine-tuning FGFR signaling in chondrocytes is incompletely defined. We report here that neurofibromin, a Ras-GAP encoded by Nf1, has an overlapping expression pattern with FGFR1 and FGFR3 in prehypertrophic chondrocytes, and with FGFR1 in hypertrophic chondrocytes during endochondral ossification. Based on previous evidence that neurofibromin inhibits Ras-ERK signaling in chondrocytes and phenotypic analogies between mice with constitutive FGFR1 activation and Nf1 deficiency in Col2a1-positive chondrocytes, we asked whether neurofibromin is required to control FGFR1-Ras-ERK signaling in maturing chondrocytes in vivo. Genetic Nf1 ablation in Fgfr1-deficient chondrocytes reactivated Ras-ERK1/2 signaling in hypertrophic chondrocytes and reversed the expansion of the hypertrophic zone observed in mice lacking Fgfr1 in Col2a1-positive chondrocytes. Histomorphometric and gene expression analyses suggested that neurofibromin, by inhibiting Rankl expression, attenuates pro-osteoclastogenic FGFR1 signaling in hypertrophic chondrocytes. We also provide evidence suggesting that neurofibromin in prehypertrophic chondrocytes, downstream of FGFRs and via an indirect mechanism, is required for normal extension and organization of proliferative columns. Collectively, this study indicates that FGFR signaling provides an important input into the Ras-Raf-MEK-ERK1/2 signaling axis in chondrocytes, and that this input is differentially regulated during chondrocyte maturation by a complex intracellular machinery, of which neurofibromin is a critical component. PMID:25616962

  17. Comparative potential of juvenile and adult human articular chondrocytes for cartilage tissue formation in three-dimensional biomimetic hydrogels.

    PubMed

    Smeriglio, Piera; Lai, Janice H; Dhulipala, Lakshmi; Behn, Anthony W; Goodman, Stuart B; Smith, Robert L; Maloney, William J; Yang, Fan; Bhutani, Nidhi

    2015-01-01

    Regeneration of human articular cartilage is inherently limited and extensive efforts have focused on engineering the cartilage tissue. Various cellular sources have been studied for cartilage tissue engineering including adult chondrocytes, and embryonic or adult stem cells. Juvenile chondrocytes (from donors below 13 years of age) have recently been reported to be a promising cell source for cartilage regeneration. Previous studies have compared the potential of adult and juvenile chondrocytes or adult and osteoarthritic (OA) chondrocytes. To comprehensively characterize the comparative potential of young, old, and diseased chondrocytes, here we examined cartilage formation by juvenile, adult, and OA chondrocytes in three-dimensional (3D) biomimetic hydrogels composed of poly(ethylene glycol) and chondroitin sulfate. All three human articular chondrocytes were encapsulated in the 3D biomimetic hydrogels and cultured for 3 or 6 weeks to allow maturation and extracellular matrix formation. Outcomes were analyzed using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. After 3 and 6 weeks, juvenile chondrocytes showed a greater upregulation of chondrogenic gene expression than adult chondrocytes, while OA chondrocytes showed a downregulation. Aggrecan and type II collagen deposition and glycosaminoglycan accumulation were high for juvenile and adult chondrocytes but not for OA chondrocytes. Similar trend was observed in the compressive moduli of the cartilage constructs generated by the three different chondrocytes. In conclusion, the juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogels. The 3D culture model described here may also provide a useful tool to further study the molecular differences among chondrocytes from different stages, which can help elucidate the mechanisms for age-related decline in the intrinsic capacity for cartilage repair.

  18. [Role of the type 3 sodium-dependent phosphate transporter in the calcification of growth plate chondrocytes].

    PubMed

    Sugita, Atsushi; Hayashibara, Tetsuyuki; Yoneda, Toshiyuki

    2006-10-01

    Phosphate is a second most abundant mineral next to calcium. The facts that hypophosphatemia is associated with the retardation of skeletal development and phosphate levels increase during endochondral ossification suggest that phosphate plays a role in cartilage differentiation. The type 3 sodium-dependent phosphate transporter (NPT3) expressed in growth plate chondrocytes transports extracellular phosphates into the cells. These phosphates are utilized for ATP synthesis, which in turn promotes apoptosis of growth plate chondrocytes through activation of the caspase signal pathways. Subsequently, matrix vesicles released from apoptotic chondrocytes accelerate calcification of chondrocytes. Our results suggest that phosphate plays a critical role in terminal differentiation of chondrocytes.

  19. Expression of type I collagen and tenascin C is regulated by actin polymerization through MRTF in dedifferentiated chondrocytes.

    PubMed

    Parreno, Justin; Raju, Sneha; Niaki, Mortah Nabavi; Andrejevic, Katarina; Jiang, Amy; Delve, Elizabeth; Kandel, Rita

    2014-10-16

    This study examined actin regulation of fibroblast matrix genes in dedifferentiated chondrocytes. We demonstrated that dedifferentiated chondrocytes exhibit increased actin polymerization, nuclear localization of myocardin related transcription factor (MRTF), increased type I collagen (col1) and tenascin C (Tnc) gene expression, and decreased Sox9 gene expression. Induction of actin depolymerization by latrunculin treatment or cell rounding, reduced MRTF nuclear localization, repressed col1 and Tnc expression, and increased Sox9 gene expression in dedifferentiated chondrocytes. Treatment of passaged chondrocytes with MRTF inhibitor repressed col1 and Tnc expression, but did not affect Sox9 expression. Our results show that actin polymerization regulates fibroblast matrix gene expression through MRTF in passaged chondrocytes.

  20. Construction of 3-Dimensional Printed Ultrasound Phantoms With Wall-less Vessels.

    PubMed

    Nikitichev, Daniil I; Barburas, Anamaria; McPherson, Kirstie; Mari, Jean-Martial; West, Simeon J; Desjardins, Adrien E

    2016-06-01

    Ultrasound phantoms are invaluable as training tools for vascular access procedures. We developed ultrasound phantoms with wall-less vessels using 3-dimensional printed chambers. Agar was used as a soft tissue-mimicking material, and the wall-less vessels were created with rods that were retracted after the agar was set. The chambers had integrated luer connectors to allow for fluid injections with clinical syringes. Several variations on this design are presented, which include branched and stenotic vessels. The results show that 3-dimensional printing can be well suited to the construction of wall-less ultrasound phantoms, with designs that can be readily customized and shared electronically. © 2016 by the American Institute of Ultrasound in Medicine.

  1. Intraoperative 3-Dimensional Computed Tomography and Navigation in Foot and Ankle Surgery.

    PubMed

    Chowdhary, Ashwin; Drittenbass, Lisca; Dubois-Ferrière, Victor; Stern, Richard; Assal, Mathieu

    2016-09-01

    Computer-assisted orthopedic surgery has developed dramatically during the past 2 decades. This article describes the use of intraoperative 3-dimensional computed tomography and navigation in foot and ankle surgery. Traditional imaging based on serial radiography or C-arm-based fluoroscopy does not provide simultaneous real-time 3-dimensional imaging, and thus leads to suboptimal visualization and guidance. Three-dimensional computed tomography allows for accurate intraoperative visualization of the position of bones and/or navigation implants. Such imaging and navigation helps to further reduce intraoperative complications, leads to improved surgical outcomes, and may become the gold standard in foot and ankle surgery. [Orthopedics.2016; 39(5):e1005-e1010.]. Copyright 2016, SLACK Incorporated.

  2. Hypoxia promotes redifferentiation and suppresses markers of hypertrophy and degeneration in both healthy and osteoarthritic chondrocytes

    PubMed Central

    2013-01-01

    Introduction Hypoxia is considered to be a positive influence on the healthy chondrocyte phenotype and cartilage matrix formation. However, hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of osteoarthritis (OA). Thus, we assessed whether healthy and OA chondrocytes have distinct responses to oxygen, particularly with regard to hypertrophy and degradation during redifferentiation. Methods Monolayer-expanded healthy and OA chondrocytes were redifferentiated for 14 days in pellet cultures under standard (20% oxygen) or hypoxic (2% oxygen) conditions. Cartilage matrix gene expression, matrix quality and quantity, degradative enzyme expression and HIF expression were measured. Results In hypoxia, both healthy and OA chondrocytes had higher human collagen type II, α1 gene (COL2A1), and aggrecan (ACAN) expression and sulfated glycosaminoglycan (sGAG) accumulation, concomitant with lower human collagen type X, α1 gene (COL10A1), and human collagen type I, α1 gene (COL1A1), expression and collagen I extracellular accumulation. OA chondrocytes had significantly lower sGAGs/DNA than healthy chondrocytes, but only in high oxygen conditions. Hypoxia also caused significantly greater sGAG retention and hyaluronic acid synthase 2 (HAS2) expression by OA chondrocytes. Both healthy and OA chondrocytes had significantly lower expression of matrix metalloproteinases (MMPs) MMP1, MMP2, MMP3 and MMP13 in hypoxia and less active MMP2 enzyme, consistent with lower MMP14 expression. However, aggrecanase (ADAMTS4 and ADAMTS5) expression was significantly lowered by hypoxia only in healthy cells, and COL10A1 and MMP13 remained significantly higher in OA chondrocytes than in healthy chondrocytes in hypoxic conditions. HIF-1α and HIF-2α had similar expression profiles in healthy and OA cells, increasing to maximal levels early in hypoxia and decreasing over time. Conclusions Hypoxic culture of human chondrocytes has long been acknowledged to result in increased

  3. DETECTORS AND EXPERIMENTAL METHODS: Decay vertex reconstruction and 3-dimensional lifetime determination at BESIII

    NASA Astrophysics Data System (ADS)

    Xu, Min; He, Kang-Lin; Zhang, Zi-Ping; Wang, Yi-Fang; Bian, Jian-Ming; Cao, Guo-Fu; Cao, Xue-Xiang; Chen, Shen-Jian; Deng, Zi-Yan; Fu, Cheng-Dong; Gao, Yuan-Ning; Han, Lei; Han, Shao-Qing; He, Miao; Hu, Ji-Feng; Hu, Xiao-Wei; Huang, Bin; Huang, Xing-Tao; Jia, Lu-Kui; Ji, Xiao-Bin; Li, Hai-Bo; Li, Wei-Dong; Liang, Yu-Tie; Liu, Chun-Xiu; Liu, Huai-Min; Liu, Ying; Liu, Yong; Luo, Tao; Lü, Qi-Wen; Ma, Qiu-Mei; Ma, Xiang; Mao, Ya-Jun; Mao, Ze-Pu; Mo, Xiao-Hu; Ning, Fei-Peng; Ping, Rong-Gang; Qiu, Jin-Fa; Song, Wen-Bo; Sun, Sheng-Sen; Sun, Xiao-Dong; Sun, Yong-Zhao; Tian, Hao-Lai; Wang, Ji-Ke; Wang, Liang-Liang; Wen, Shuo-Pin; Wu, Ling-Hui; Wu, Zhi; Xie, Yu-Guang; Yan, Jie; Yan, Liang; Yao, Jian; Yuan, Chang-Zheng; Yuan, Ye; Zhang, Chang-Chun; Zhang, Jian-Yong; Zhang, Lei; Zhang, Xue-Yao; Zhang, Yao; Zheng, Yang-Heng; Zhu, Yong-Sheng; Zou, Jia-Heng

    2009-06-01

    This paper focuses mainly on the vertex reconstruction of resonance particles with a relatively long lifetime such as K0S, Λ, as well as on lifetime measurements using a 3-dimensional fit. The kinematic constraints between the production and decay vertices and the decay vertex fitting algorithm based on the least squares method are both presented. Reconstruction efficiencies including experimental resolutions are discussed. The results and systematic errors are calculated based on a Monte Carlo simulation.

  4. Odontoid screw placement using Isocentric 3-dimensional C-arm fluoroscopy.

    PubMed

    Summers, Lori E; Kouri, Joshua G; Yang, Mu; Patrick Jacob, R

    2008-02-01

    We describe the use of isocentric 3-dimensional fluoroscopy to place odontoid screws in 9 patients. We wanted to show the benefits of using isocentric 3-dimensional fluroscopy in odontoid screw placement. Odontoid screw fixation for treatment of type II odontoid fractures has gained popularity since its introduction in the early 1980s. During the last several years, a multitude of new techniques have improved the ease of odontoid screw placement, including biplanar fluoroscopy, cannulated screw systems, and beveled bedside-fixed retractor systems. The use of isocentric C-arm fluoroscopy can improve the ease and facilitate placement of odontoid screws. Nine patients, ranging in ages from 30 to 89 years, presented with type II odontoid fractures. All fractures were either nondisplaced or minimally displaced (<4 mm) and occurred as a result of acute trauma. No patient had evidence of transverse atlantal ligament disruption. Isocentric 3-dimensional fluoroscopy, in conjunction with image-guided navigational software, was used to place 1 or 2 odontoid screws in each patient. Three-dimensional images were acquired intraoperatively, which were then reconstructed and uploaded to the navigational workstation. Screw trajectory was planned and performed with the use of tracked instruments. Successful screw placement, as judged by intraoperative computerized tomography, was attained in all 9 patients. Isocentric 3-dimensional fluoroscopy, in conjunction with an image-guided navigational software system, obviates the need for cumbersome biplanar fluoroscopy, allows for intraoperative image acquisition after surgical exposure, reduces intraoperative registration time, reduces both surgeon and patient radiation exposure, and allows immediate computerized tomographic imaging in the operating room to verify screw position.

  5. Computerized 3-dimensional localization of a video capsule in the abdominal cavity: validation by digital radiography.

    PubMed

    Marya, Neil; Karellas, Andrew; Foley, Anne; Roychowdhury, Abhijit; Cave, David

    2014-04-01

    Wireless video capsule endoscopy allows the noninvasive visualization of the small intestine. Currently, capsules do not provide localization information while traversing the GI tract. To report on the radiological validation of 3-dimensional localization software incorporated in a newly developed capsule. By using radiofrequency transmission, the software measures the strength of the capsule's signal to locate the position of the capsule. This study was performed at the University of Massachusetts Medical Center, Worcester, Mass. Thirty healthy volunteers consented to the experimental procedure. After ingestion of the capsule, subjects had 5 sets of anteroposterior and lateral radiographs taken every 30 minutes while the software calculated the position of the capsule. By using the radiographs, we calculated the location of the capsule in the abdominal cavity and compared the results with those generated by the software. Average error (and standard deviation) among the 3-dimensional coordinates was X, 2.00 cm (1.64); Y, 2.64 cm (2.39); and Z, 2.51 cm (1.83). The average total spatial error among all measurements was 13.26 cm(3) (22.72). There was a correlation between increased subject body mass index and the 3-dimensional software measurement error. This study was performed in healthy volunteers and needs further validation in patients with small intestinal disorders. The new 3-dimensional software provides localization of the capsule consistent with radiological observations. However, further validation of the software's clinical utility is required with a prospective clinical trial. Copyright © 2014 American Society for Gastrointestinal Endoscopy. Published by Mosby, Inc. All rights reserved.

  6. Energy Sources of the Dominant Frequency Dependent 3-dimensional Atmospheric Modes

    NASA Technical Reports Server (NTRS)

    Schubert, S.

    1985-01-01

    The energy sources and sinks associated with the zonally asymmetric winter mean flow are investigated as part of an on-going study of atmospheric variability. Distinctly different horizontal structures for the long, intermediate and short time scale atmospheric variations were noted. In previous observations, the 3-dimensional structure of the fluctuations is investigated and the relative roles of barotropic and baroclinic terms are assessed.

  7. Energy Sources of the Dominant Frequency Dependent 3-dimensional Atmospheric Modes

    NASA Technical Reports Server (NTRS)

    Schubert, S.

    1985-01-01

    The energy sources and sinks associated with the zonally asymmetric winter mean flow are investigated as part of an on-going study of atmospheric variability. Distinctly different horizontal structures for the long, intermediate and short time scale atmospheric variations were noted. In previous observations, the 3-dimensional structure of the fluctuations is investigated and the relative roles of barotropic and baroclinic terms are assessed.

  8. Reconstructing a 3-dimensional image of the results of antinuclear antibody testing by indirect immunofluorescence.

    PubMed

    Murai, Ryosei; Yamada, Koji; Tanaka, Maki; Kuribayashi, Kageaki; Kobayashi, Daisuke; Tsuji, Naoki; Watanabe, Naoki

    2013-01-31

    Indirect immunofluorescence anti-nuclear antibody testing (IIF-ANAT) is an essential screening tool in the diagnosis of various autoimmune disorders. ANA titer quantification and interpretation of immunofluorescence patterns are determined subjectively, which is problematic. First, we determined the examination conditions under which IIF-ANAT fluorescence intensities are quantified. Next, IIF-ANAT was performed using homogeneous, discrete speckled, and mixed serum samples. Images were obtained using Bio Zero BZ-8000, and 3-dimensional images were reconstructed using the BZ analyzer software. In the 2-dimensional analysis, homogeneous ANAs hid the discrete speckled pattern, resulting in a diagnosis of homogeneous immunofluorescence. However, 3-dimensional analysis of the same sample showed discrete speckled-type ANA in the homogeneous background. This study strengthened the current IIF-ANAT method by providing a new approach to quantify the fluorescence intensity and enhance the resolution of IIF-ANAT fluorescence patterns. Reconstructed 3-dimensional imaging of IIF-ANAT can be a powerful tool for routine laboratory examination.

  9. Fast Apriori-based Graph Mining Algorithm and application to 3-dimensional Structure Analysis

    NASA Astrophysics Data System (ADS)

    Nishimura, Yoshio; Washio, Takashi; Yoshida, Tetsuya; Motoda, Hiroshi; Inokuchi, Akihiro; Okada, Takashi

    Apriori-based Graph Mining (AGM) algorithm efficiently extracts all the subgraph patterns which frequently appear in graph structured data. The algorithm can deal with general graph structured data with multiple labels of vartices and edges, and is capable of analyzing the topological structure of graphs. In this paper, we propose a new method to analyze graph structured data for a 3-dimensional coordinate by AGM. In this method the distance between each vertex of a graph is calculated and added to the edge label so that AGM can handle 3-dimensional graph structured data. One problem in our approach is that the number of edge labels increases, which results in the increase of computational time to extract subgraph patterns. To alleviate this problem, we also propose a faster algorithm of AGM by adding an extra constraint to reduce the number of generated candidates for seeking frequent subgraphs. Chemical compounds with dopamine antagonist in MDDR database were analyzed by AGM to characterize their 3-dimensional chemical structure and correlation with physiological activity.

  10. Novel 3-dimensional analysis to evaluate temporomandibular joint space and shape.

    PubMed

    Ikeda, Renie; Oberoi, Snehlata; Wiley, David F; Woodhouse, Christian; Tallman, Melissa; Tun, Wint Wint; McNeill, Charles; Miller, Arthur J; Hatcher, David

    2016-03-01

    The purpose of this study was to present and validate a novel semiautomated method for 3-dimensional evaluation of the temporomandibular joint (TMJ) space and condylar and articular shapes using cone-beam computed tomographic data. The protocol for 3-dimensional analysis with the Checkpoint software (Stratovan, Davis, Calif) was established by analyzing cone-beam computed tomographic images of 14 TMJs representing a range of TMJ shape variations. Upon establishment of the novel method, analysis of 5 TMJs was further repeated by several investigators to assess the reliability of the analysis. Principal components analysis identified 3 key components that characterized how the condylar head shape varied among the 14 TMJs. Principal component analysis allowed determination of the minimum number of landmarks or patch density to define the shape variability in this sample. Average errors of landmark placement ranged from 1.15% to 3.65%, and none of the 121 landmarks showed significant average errors equal to or greater than 5%. Thus, the mean intraobserver difference was small and within the clinically accepted margin of error. Interobserver error was not significantly greater than intraobserver error, indicating that this is a reliable methodology. This novel semiautomatic method is a reliable tool for the 3-dimensional analysis of the TMJ including both the form and the space between the articular eminence and the condylar head. Copyright © 2016 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.

  11. The Preoperative Evaluation of Infective Endocarditis via 3-Dimensional Transesophageal Echocardiography.

    PubMed

    Yong, Matthew S; Saxena, Pankaj; Killu, Ammar M; Coffey, Sean; Burkhart, Harold M; Wan, Siu-Hin; Malouf, Joseph F

    2015-08-01

    Transesophageal echocardiography continues to have a central role in the diagnosis of infective endocarditis and its sequelae. Recent technological advances offer the option of 3-dimensional imaging in the evaluation of patients with infective endocarditis. We present an illustrative case and review the literature regarding the potential advantages and limitations of 3-dimensional transesophageal echocardiography in the diagnosis of complicated infective endocarditis. A 51-year-old man, an intravenous drug user who had undergone bioprosthetic aortic valve replacement 5 months earlier, presented with prosthetic valve endocarditis. Preoperative transesophageal echocardiography with 3D rendition revealed a large abscess involving the mitral aortic intervalvular fibrosa, together with a mycotic aneurysm that had ruptured into the left atrium, resulting in a left ventricle-to-left atrium fistula. Three-dimensional transesophageal echocardiography enabled superior preoperative anatomic delineation and surgical planning. We conclude that 3-dimensional transesophageal echocardiography can be a useful adjunct to traditional 2-dimensional transesophageal echocardiography as a tool in the diagnosis of infective endocarditis.

  12. Association between 3-dimensional mandibular morphology and condylar movement in subjects with mandibular asymmetry.

    PubMed

    Ikeda, Michiyo; Miyamoto, Jun J; Takada, Jun-Ichi; Moriyama, Keiji

    2017-02-01

    The purpose of this study was to evaluate the hypothesis that 3-dimensional mandibular morphology is correlated with condylar movement in patients with mandibular asymmetry. Subjects were classified into 2 groups (n = 25 each): mandibular asymmetry with a menton deviation greater than 4 mm and no mandibular asymmetry with a menton deviation less than 4 mm. Linear and volumetric measurements of 3-dimensional mandibular morphology were recorded using computed tomography. Mandibular functional movement was recorded by computerized axiography (CADIAX; Gamma Dental, Klosterneuburg, Austria), and condylar path length, sagittal condylar inclination, and transverse condylar inclination on protrusion were measured. We calculated side-to-side asymmetry (shifted side vs nonshifted side) in mandibular morphology and assessed condylar movement by using an asymmetry ratio (nonshifted side/shifted side). Significant differences in mandibular morphology and condylar movement were found between the 2 groups. In the group with menton deviation greater than 4 mm, significant correlations were found between the asymmetry ratio of mandibular morphology and condylar movement: ie, condylar path length and transverse condylar inclination. No significant correlations were found between any of these measurements in the group with menton deviation less than 4 mm. In support of our hypothesis, the results suggested that 3-dimensional mandibular morphologic asymmetry is associated with condylar movement in subjects with mandibular asymmetry. Copyright © 2017 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.

  13. The Neural Representation of 3-Dimensional Objects in Rodent Memory Circuits

    PubMed Central

    Burke, Sara N.; Barnes, Carol A.

    2014-01-01

    Three-dimensional objects are common stimuli that rodents and other animals encounter in the natural world that contribute to the associations that are the hallmark of explicit memory. Thus, the use of 3-dimensional objects for investigating the circuits that support associative and episodic memories has a long history. In rodents, the neural representation of these types of stimuli is a polymodal process and lesion data suggest that the perirhinal cortex, an area of the medial temporal lobe that receives afferent input from all sensory modalities, is particularly important for integrating sensory information across modalities to support object recognition. Not surprisingly, recent data from in vivo electrophysiological recordings have shown that principal cells within the perirhinal cortex are activated at locations of an environment that contain 3-dimensional objects. Interestingly, it appears that neural activity patterns related to object stimuli are ubiquitous across memory circuits and have now been observed in many medial temporal lobe structures as well as in the anterior cingulate cortex. This review summarizes behavioral and neurophysiological data that examine the representation of 3-dimensional objects across brain regions that are involved in memory. PMID:25205370

  14. The effects of monosodium urate monohydrate crystals on chondrocyte viability and function: implications for development of cartilage damage in gout.

    PubMed

    Chhana, Ashika; Callon, Karen E; Pool, Bregina; Naot, Dorit; Gamble, Gregory D; Dray, Michael; Pitto, Rocco; Bentley, Jarome; McQueen, Fiona M; Cornish, Jillian; Dalbeth, Nicola

    2013-12-01

    Cartilage damage is frequently observed in advanced destructive gout. The aim of our study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on chondrocyte viability and function. The alamarBlue assay and flow cytometry were used to assess the viability of primary human chondrocytes and cartilage explants following culture with MSU crystals. The number of dead chondrocytes in cartilage explants cultured with MSU crystals was quantified. Real-time PCR was used to determine changes in the relative mRNA expression levels of chondrocytic genes. The histological appearance of cartilage in joints affected by gout was also examined. MSU crystals rapidly reduced primary human chondrocyte and cartilage explant viability in a dose-dependent manner (p < 0.01 for both). Cartilage explants cultured with MSU crystals had a greater percentage of dead chondrocytes at the articular surface compared to untreated cartilage (p = 0.004). Relative mRNA expression of type II collagen and the cartilage matrix proteins aggrecan and versican was decreased in chondrocytes following culture with MSU crystals (p < 0.05 for all). However, expression of the degradative enzymes ADAMTS4 and ADAMTS5 was increased (p < 0.05 for both). In joints affected by gout, normal cartilage architecture was lost, with empty chondrocyte lacunae observed. MSU crystals have profound inhibitory effects on chondrocyte viability and function. Interactions between MSU crystals and chondrocytes may contribute to cartilage damage in gout through reduction of chondrocyte viability and promotion of a catabolic state.

  15. Antisense Reduction of Mutant COMP Reduces Growth Plate Chondrocyte Pathology.

    PubMed

    Posey, Karen L; Coustry, Francoise; Veerisetty, Alka C; Hossain, Mohammad; Gattis, Danielle; Booten, Sheri; Alcorn, Joseph L; Seth, Punit P; Hecht, Jacqueline T

    2017-03-01

    Mutations in cartilage oligomeric matrix protein cause pseudoachondroplasia, a severe disproportionate short stature disorder. Mutant cartilage oligomeric matrix protein produces massive intracellular retention of cartilage oligomeric matrix protein, stimulating ER and oxidative stresses and inflammation, culminating in post-natal loss of growth plate chondrocytes, which compromises linear bone growth. Treatments for pseudoachondroplasia are limited because cartilage is relatively avascular and considered inaccessible. Here we report successful delivery and treatment using antisense oligonucleotide technology in our transgenic pseudoachondroplasia mouse model. We demonstrate delivery of human cartilage oligomeric matrix protein-specific antisense oligonucleotides to cartilage and reduction of cartilage oligomeric matrix protein expression, which largely alleviates pseudoachondroplasia growth plate chondrocyte pathology. One antisense oligonucleotide reduced steady-state levels of cartilage oligomeric matrix protein mRNA and dampened intracellular retention of mutant cartilage oligomeric matrix protein, leading to a reduction of inflammatory markers and cell death and partial restoration of proliferation. This novel and exciting work demonstrates that antisense-based therapy is a viable approach for treating pseudoachondroplasia and other human cartilage disorders. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  16. Characterization of collagenous matrix assembly in a chondrocyte model system

    PubMed Central

    Yingst, Sorcha; Bloxham, Kaci; Warner, Lisa R.; Brown, Raquel J.; Cole, Jennifer; Kenoyer, Linda; Knowlton, William B.; Oxford, Julia Thom

    2010-01-01

    Collagen is a major component of the newly synthesized pericellular microenvironment of chondrocytes. Collagen types II, IX, and XI are synthesized and assembled into higher ordered complexes by a mechanism in which type XI collagen plays a role in nucleation of new fibrils, and in limiting fibril diameter. This study utilizes a cell line derived from the Swarm rat chondrosarcoma that allows the accumulation and assembly of pericellular matrix. Immunofluorescence and atomic force microscopy were used to assess early intermediates of fibril formation. Results indicate that this cell line synthesizes and secretes chondrocyte-specific pericellular matrix molecules including types II, IX, and XI collagen and is suitable for the study of newly synthesized collagen matrix under the experimental conditions used. AFM data indicate that small fibrils or assemblies of microfibrils are detectable and may represent precursors of the ~20 nm thin fibrils reported in cartilage. Treatment with hyaluronidase indicates that the dimensions of the small fibrils may be dependent upon the presence of hyaluronan within the matrix. This study provides information on the composition and organization of the newly synthesized extracellular matrix that plays a role in establishing the material properties and performance of biological materials such as cartilage. PMID:18496861

  17. AUTOLOGOUS CHONDROCYTE TRANSPLANTATION-SERIES OF 3 CASES

    PubMed Central

    Gobbi, Riccardo Gomes; Demange, Marco Kawamura; Barreto, Ronald Bispo; Pécora, José Ricardo; Rezende, Múrcia Uchõa de; Filho, Tarcisio E.P Barros; Lombello, Christiane Bertachini

    2015-01-01

    Hyaline cartilage covers joint surfaces and plays an important role in reducing friction and mechanical loading on synovial joints such as the knee. This tissue is not supplied with blood vessels, nerves or lymphatic circulation, which may be one of the reasons why joint cartilage has such poor capacity for healing. Chondral lesions that reach the subchondral bone (osteochondral lesions) do not heal and may progress to arthrosis with the passage of time. In young patients, treatment of chondral defects of the knee is still a challenge, especially in lesions larger than 4 cm. One option for treating these patients is autologous chondrocyte transplantation/implantation. Because this treatment does not violate the subchondral bone and repairs the defect with tissue similar to hyaline cartilage, it has the theoretical advantage of being more biological, and mechanically superior, compared with other techniques. In this paper, we describe our experience with autologous chondrocyte transplantation/implantation at the Institute of Orthopedics and Traumatology, Hospital das Clínicas, University of Sâo Paulo, through a report on three cases. PMID:27022579

  18. Osmotic loading of in situ chondrocytes in their native environment.

    PubMed

    Korhonen, Rami K; Han, Sang-Kuy; Herzog, Walter

    2010-09-01

    Changes in the osmotic environment cause changes in volume of isolated cells and cells in tissue explants, and the osmotic environment becomes hypotonic in cartilage diseases such as osteoarthritis (OA). However, it is not known how cells respond to a hypotonic osmotic challenge when situated in the fully intact articular cartilage. A confocal laser scanning microscope was used to image chondrocytes of intact rabbit patellae in an isotonic (300 mOsm) and hypotonic (172 mOsm) immersion medium. Cell volumes were calculated before and 5, 15, 60, 120 and 240 minutes after the change in saline concentration. Local tissue strains and swelling of the entire tissue were estimated from the relative movements of cells and displacements of single cells, respectively. Cell volumes increased rapidly (< or = 5 minutes, p<0.05) by approximately 22%, after which they remained constant for an hour (p>0.05). However, two and four hours post the hypotonic challenge, cell volumes were statistically greater (p<0.05) than those at all earlier time points, and swelling of the entire tissue continued throughout the four hour loading period. The results of our study suggest that osmotic loading induced volume changes of in situ chondrocytes in their native environment occur quickly and continue for hours. Understanding the behaviour of cells in their native environment provides novel insigth into the cell mechanics in ostearthritic joints and so may help understand the onset and progression of this disease.

  19. Loss of the Mammalian DREAM Complex Deregulates Chondrocyte Proliferation

    PubMed Central

    Forristal, Chantal; Henley, Shauna A.; MacDonald, James I.; Bush, Jason R.; Ort, Carley; Passos, Daniel T.; Talluri, Srikanth; Ishak, Charles A.; Thwaites, Michael J.; Norley, Chris J.; Litovchick, Larisa; DeCaprio, James A.; DiMattia, Gabriel; Holdsworth, David W.; Beier, Frank

    2014-01-01

    Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes. PMID:24710275

  20. Loss of the mammalian DREAM complex deregulates chondrocyte proliferation.

    PubMed

    Forristal, Chantal; Henley, Shauna A; MacDonald, James I; Bush, Jason R; Ort, Carley; Passos, Daniel T; Talluri, Srikanth; Ishak, Charles A; Thwaites, Michael J; Norley, Chris J; Litovchick, Larisa; DeCaprio, James A; DiMattia, Gabriel; Holdsworth, David W; Beier, Frank; Dick, Frederick A

    2014-06-01

    Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  1. Generation and characterization of osteochondral grafts with human nasal chondrocytes.

    PubMed

    Barandun, Marina; Iselin, Lukas Daniel; Santini, Francesco; Pansini, Michele; Scotti, Celeste; Baumhoer, Daniel; Bieri, Oliver; Studler, Ueli; Wirz, Dieter; Haug, Martin; Jakob, Marcel; Schaefer, Dirk Johannes; Martin, Ivan; Barbero, Andrea

    2015-08-01

    We investigated whether nasal chondrocytes (NC) can be used to generate composite constructs with properties necessary for the repair of osteochondral (OC) lesions, namely maturation, integration and capacity to recover from inflammatory burst. OC grafts were fabricated by combining engineered cartilage tissues (generated by culturing NC or articular chondrocytes - AC - onto Chondro-Gide® matrices) with devitalized spongiosa cylinders (Tutobone®). OC tissues were then exposed to IL-1β for three days and cultured for additional 2 weeks in the absence of IL-1β. Cartilage maturation extent was assessed (immune) histologically, biochemically and by delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) while cartilage/bone integration was assessed using a peel-off mechanical test. The use of NC as compared to AC allowed for more efficient cartilage matrix accumulation and superior integration of the cartilage/bone layers. dGEMRIC and biochemical analyzes of the OC constructs showed a reduced glycosaminoglycan (GAG) contents upon IL-1β administration. Cartilaginous matrix contents and integration forces returned to baseline up on withdrawal of IL-1β. By having a cartilage layer well developed and strongly integrated to the subchondral layer, OC tissues generated with NC may successfully engraft in an inflammatory post-surgery joint environment. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  2. The Wnt/β-Catenin Pathway Interacts Differentially with PTHrP Signaling to Control Chondrocyte Hypertrophy and Final Maturation

    PubMed Central

    Guo, Xizhi; Mak, Kinglun Kingston; Taketo, Makoto M.; Yang, Yingzi

    2009-01-01

    Sequential proliferation, hypertrophy and maturation of chondrocytes are required for proper endochondral bone development and tightly regulated by cell signaling. The canonical Wnt signaling pathway acts through β-catenin to promote chondrocyte hypertrophy whereas PTHrP signaling inhibits it by holding chondrocytes in proliferating states. Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/β-catenin and PTHrP signaling. Wnt/β-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate PTHrP expression. In addition, Wnt/β-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways. Furthermore, Wnt/β-catenin signaling also controls final maturation of hypertrophic chondrocytes, but such regulation is PTHrP signaling-independent. PMID:19557172

  3. The Wnt/beta-catenin pathway interacts differentially with PTHrP signaling to control chondrocyte hypertrophy and final maturation.

    PubMed

    Guo, Xizhi; Mak, Kinglun Kingston; Taketo, Makoto M; Yang, Yingzi

    2009-06-26

    Sequential proliferation, hypertrophy and maturation of chondrocytes are required for proper endochondral bone development and tightly regulated by cell signaling. The canonical Wnt signaling pathway acts through beta-catenin to promote chondrocyte hypertrophy whereas PTHrP signaling inhibits it by holding chondrocytes in proliferating states. Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/beta-catenin and PTHrP signaling. Wnt/beta-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate PTHrP expression. In addition, Wnt/beta-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways. Furthermore, Wnt/beta-catenin signaling also controls final maturation of hypertrophic chondrocytes, but such regulation is PTHrP signaling-independent.

  4. 3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

    PubMed Central

    Walter, Teagan J.; Sparks, Erin E.; Huppert, Stacey S.

    2012-01-01

    In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks. A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6. Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing

  5. Crucial Role of Elovl6 in Chondrocyte Growth and Differentiation during Growth Plate Development in Mice

    PubMed Central

    Kikuchi, Manami; Matsuzaka, Takashi; Ishii, Kiyoaki; Nakagawa, Yoshimi; Takayanagi, Misa; Yamada, Nobuhiro; Shimano, Hitoshi

    2016-01-01

    ELOVL family member 6, elongation of very long chain fatty acids (Elovl6) is a microsomal enzyme, which regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 has been shown to be associated with various pathophysiologies including insulin resistance, atherosclerosis, and non-alcoholic steatohepatitis. To investigate a potential role of Elovl6 during bone development, we here examined a skeletal phenotype of Elovl6 knockout (Elovl6-/-) mice. The Elovl6-/- skeleton was smaller than that of controls, but exhibited no obvious patterning defects. Histological analysis revealed a reduced length of proliferating and an elongated length of hypertrophic chondrocyte layer, and decreased trabecular bone in Elovl6-/- mice compared with controls. These results were presumably due to a modest decrease in chondrocyte proliferation and accelerated differentiation of cells of the chondrocyte lineage. Consistent with the increased length of the hypertrophic chondrocyte layer in Elovl6-/- mice, Collagen10α1 was identified as one of the most affected genes by ablation of Elovl6 in chondrocytes. Furthermore, this elevated expression of Collagen10α1 of Elovl6-null chondrocytes was likely associated with increased levels of Foxa2/a3 and Mef2c mRNA expression. Relative increases in protein levels of nuclear Foxa2 and cytoplasmic histone deacethylase 4/5/7 were also observed in Elovl6 knockdown cells of the chondrocyte lineage. Collectively, our data suggest that Elovl6 plays a critical role for proper development of embryonic growth plate. PMID:27467521

  6. Pediatric auricular chondrocytes gene expression analysis in monolayer culture and engineered elastic cartilage.

    PubMed

    Ruszymah, B H I; Lokman, B S; Asma, A; Munirah, S; Chua, Kienhui; Mazlyzam, A L; Isa, M R; Fuzina, N H; Aminuddin, B S

    2007-08-01

    This study was aimed at regenerating autologous elastic cartilage for future use in pediatric ear reconstruction surgery. Specific attentions were to characterize pediatric auricular chondrocyte growth in a combination culture medium and to assess the possibility of elastic cartilage regeneration using human fibrin. Laboratory experiment using human pediatric auricular chondrocytes. Pediatric auricular chondrocytes growth kinetics and quantitative gene expression profile in three different types of media were compared in primary culture and subsequent three passages. Large-scale culture-expanded chondrocytes from the combination medium were then mixed with human fibrin for the formation of elastic cartilage via tissue engineering technique. The equal mixture of Ham's F12 and Dulbecco's Modified Eagle Medium (FD) promoted the best chondrocyte growth at every passage compared to the individual media. Chondrocytes differentiation index; ratio of type II to type I collagen gene expression level, aggrecan and elastin expression gradually decreased while passaging but they were then restored in engineered tissues after implantation. The engineered cartilage was glistening white in color and firm in consistency. Histological evaluation, immunohistochemistry analysis and quantitative gene expression assessment demonstrated that the engineered cartilage resemble the features of native elastic cartilage. Pediatric auricular chondrocytes proliferate better in the combination medium (FD) and the utilization of human fibrin as a biomaterial hold promises for the regeneration of an autologous elastic cartilage for future application in ear reconstructive surgery.

  7. Enhanced chondrocyte densities on carbon nanotube composites: the combined role of nanosurface roughness and electrical stimulation.

    PubMed

    Khang, Dongwoo; Park, Grace E; Webster, Thomas J

    2008-07-01

    Simultaneous incorporation of intrinsic nanosurface roughness and external electrical stimulation may maximize the regeneration of articular cartilage tissue more than on nanosmooth, electrically nonstimulated biomaterials. Here, we report enhanced functions of chondrocytes (cartilage synthesizing cells) on electrically and nonelectrically stimulated highly dispersed carbon nanotubes (CNT) in polycarbonate urethane (PCU) compared to, respectively, stimulated pure PCU. Specifically, compared to conventional longitudinal (or vertical) electrical stimulation of chondrocytes on conducting surfaces which require high voltage, we developed a lateral electrical stimulation across CNT/PCU composite films of low voltage that enhanced chondrocyte functions. Chondrocyte adhesion and long-term cell densities (up to 2 days) were enhanced (more than 50%) on CNT/PCU composites compared to PCU alone without electrical stimulation. This study further explained why by measuring greater amounts of initial fibronectin adsorption (a key protein that mediates chondrocyte adhesion) on CNT/PCU composites which were more hydrophilic (than pure PCU) due to greater nanometer roughness. Importantly, the same trend was observed and was even significantly enhanced when chondrocytes were subjected to electrical stimulation (more than 200%) compared to nonstimulated CNT/PCU. For this reason, this study provided direct evidence of the positive role that conductive CNT/PCU films can play in promoting functions of chondrocytes for cartilage regeneration.

  8. IL-36α: a novel cytokine involved in the catabolic and inflammatory response in chondrocytes

    PubMed Central

    Conde, Javier; Scotece, Morena; Abella, Vanessa; Lois, Ana; López, Verónica; García-Caballero, Tomás; Pino, Jesús; Gómez-Reino, Juan Jesús; Gómez, Rodolfo; Lago, Francisca; Gualillo, Oreste

    2015-01-01

    Recent studies confer to IL-36α pro-inflammatory properties. However, little is known about the expression and function of IL-36α in cartilage. This study sought to analyze the expression of IL-36α in healthy and OA cartilage. Next, we determined the effects of recombinant IL-36α on catabolism and inflammation in chondrocytes. For completeness, part of the signaling pathway elicited by IL-36α was also explored. IL-36α expression was evaluated by immunohistochemistry and RT-qPCR. Expression of MMP-13, NOS2 and COX-2 was also determined in OA articular chondrocytes treated with recombinant IL-36α. IκB-α and P-p38 was explored by western blot. We observed a low constitutive expression of IL-36α in healthy human chondrocytes. However, OA chondrocytes likely expressed more IL-36α than healthy chondrocytes. In addition, immune cells infiltrated into the joint and PBMCs express higher levels of IL-36α in comparison to chondrocytes. OA chondrocytes, treated with IL-36α, showed significant increase in the expression of MMP-13, NOS2 and COX-2. Finally, IL-36α stimulated cells showed NFκB and p38 MAPK activated pathways. IL-36α acts as a pro-inflammatory cytokine at cartilage level, by increasing the expression of markers of inflammation and cartilage catabolism. Like other members of IL-1 family, IL-36α acts through the activation of NFκB and p38 MAPK pathway. PMID:26560022

  9. Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia.

    PubMed

    Zhou, Libin; Ding, Ruiying; Li, Baowei; Han, Haolun; Wang, Hongnan; Wang, Gang; Xu, Bingxin; Zhai, Suoqiang; Wu, Wei

    2015-01-01

    The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering.

  10. Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia

    PubMed Central

    Zhou, Libin; Ding, Ruiying; Li, Baowei; Han, Haolun; Wang, Hongnan; Wang, Gang; Xu, Bingxin; Zhai, Suoqiang; Wu, Wei

    2015-01-01

    The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering. PMID:25755694

  11. Functional Properties of Chondrocytes and Articular Cartilage using Optical Imaging to Scanning Probe Microscopy.

    PubMed

    Xia, Yang; Darling, Eric M; Herzog, Walter

    2017-10-03

    Mature chondrocytes in adult articular cartilage vary in number, size, and shape, depending on their depth in the tissue, location in the joint, and source species. Chondrocytes are the primary structural, functional, and metabolic unit in articular cartilage, the loss of which will induce fatigue to the extracellular matrix (ECM), eventually leading to failure of the cartilage and impairment of the joint as a whole. This brief review focuses on the functional and biomechanical studies of chondrocytes and articular cartilage, using microscopic imaging from optical microscopies to scanning probe microscopy. Three topics are covered in this review, including the functional studies of chondrons by optical imaging (unpolarized and polarized light and infrared light, two-photon excitation microscopy), the probing of chondrocytes and cartilage directly using microscale measurement techniques, and different imaging approaches that can measure chondrocyte mechanics and chondrocyte biological signaling under in situ and in vivo environments. Technical advancement in chondrocyte research during recent years has enabled new ways to study the biomechanical and functional properties of these cells and cartilage. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  12. Effects of osmotic challenges on membrane potential in human articular chondrocytes from healthy and osteoarthritic cartilage.

    PubMed

    Sánchez, Julio C; López-Zapata, Diego F

    2010-01-01

    Changes in external osmolarity arise from variations in mechanical loads on joints and may affect the homeostasis of chondrocytes, which are the only cell type responsible for matrix turnover. Accordingly, variations in membrane potential may affect cartilage production. The present study assessed the effects of variations in external osmolarity on membrane potential and the possible mechanisms responsible for this response. Membrane potential was measured by the patch clamp whole-cell technique using human articular chondrocytes freshly isolated from healthy and osteoarthritic cartilage. The membrane potential was -39±4 mV in articular human chondrocytes from healthy cartilage and -26±4 mV in those from osteoarthritic cartilage. Increasing the osmolarity produced a reversible hyperpolarization mediated by K+ efflux through BKCa channels in both groups of chondrocytes, but the response in osteoarthritic cells was significantly reduced; no other K+ pathways were involved in this effect. Alternatively, decreasing the osmolarity elicited depolarization in healthy chondrocytes but did not produce any response in chondrocytes from osteoarthritic cartilage. The depolarization was dependent on Na+ influx through Gd3+-sensitive stretch-activated cation channels and was independent of external Ca2+. The differential responses observed in chondrocytes from osteoarthritic cartilage suggest that disregulation on the responses to external osmolarity may be involved in the process that leads to the alterations in the cartilage structure observed in osteoarthritis.

  13. Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and causes dwarfism with osteopenia

    PubMed Central

    Horiki, Mitsuru; Imamura, Takeshi; Okamoto, Mina; Hayashi, Makoto; Murai, Junko; Myoui, Akira; Ochi, Takahiro; Miyazono, Kohei; Yoshikawa, Hideki; Tsumaki, Noriyuki

    2004-01-01

    Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo. PMID:15123739

  14. Mitogen-activated protein kinase p38 mediates regulation of chondrocyte differentiation by parathyroid hormone.

    PubMed

    Zhen, X; Wei, L; Wu, Q; Zhang, Y; Chen, Q

    2001-02-16

    Parathyroid hormone (PTH) and its related peptide regulate endochondral ossification by inhibiting chondrocyte differentiation toward hypertrophy. However, the intracellular pathway for transducing PTH/PTH-related peptide signals in chondrocytes remains unclear. Here, we show that this pathway is mediated by mitogen-activated protein kinase (MAPK) p38. Incubation of hypertrophic chondrocytes with PTH (1-34) induces an inhibition of p38 kinase activity in a time- and dose-dependent manner. Inhibition of protein kinase C prevents PTH-induced p38 MAPK inhibition, whereas inhibition of protein kinase A has no effect. Thus, protein kinase C, but not protein kinase A, is required for the inhibition of p38 MAPK by PTH. Treatment of hypertrophic chondrocytes by PTH or by p38 MAPK inhibitor SB203580 up-regulates Bcl-2, suggesting that Bcl-2 lies downstream of p38 MAPK in the PTH signaling pathway. Inhibition of p38 MAPK in hypertrophic chondrocytes by either PTH, SB303580, or both together leads to a decrease of hypertrophic marker type X collagen mRNA and an increase of the expression of prehypertrophic marker cartilage matrix protein. Therefore, inhibition of p38 converts a hypertrophic cell phenotype to a prehypertrophic one, thereby preventing precocious chondrocyte hypertrophy. Taken together, these data suggest a major role for p38 MAPK in transmitting PTH signals to regulate chondrocyte differentiation.

  15. Regulation of Articular Chondrocyte Proliferation and Differentiation by Indian Hedgehog and Parathyroid Hormone-related Protein

    PubMed Central

    Chen, Xuesong; Macica, Carolyn; Nasiri, Ali; Broadus, Arthur E.

    2008-01-01

    Objective The chondrocytes of the epiphyseal growth zone are regulated by the Indian hedgehog (Ihh)-parathyroid hormone-related protein (PTHrP) axis. In weight-bearing joints, this growth zone comes to be subdivided by the secondary ossification center into distinct articular and growth cartilage structures. Here, we explored the cells of origin, localization, regulation of expression, and putative functions of Ihh and PTHrP in articular cartilage in the mouse. Methods We assessed Ihh and PTHrP expression in an allelic PTHrP-lacZ knockin mouse and several versions of PTHrP-null mice. Selected joints were unloaded surgically to examine load-induction of PTHrP and Ihh. Results The embryonic growth zone appears to serve as the source of PTHrP-expressing proliferative chondrocytes that populate both the forming articular cartilage and growth plate structures. In articular cartilage, these cells take the form of articular chondrocytes in the mid-zone. In PTHrP-knockout mice, mineralizing chondrocytes encroach upon developing articular cartilage but appear to be prevented from mineralizing the joint space by Ihh-driven surface chondrocyte proliferation. In growing and adult mice, PTHrP expression in articular chondrocytes is load-induced, and unloading is associated with rapid changes in PTHrP expression and articular chondrocyte differentiation. Conclusion We conclude that the PTHrP-Ihh axis participates in the maintenance of articular cartilage. Dysregulation of this system might contribute to the pathogenesis of arthritis. PMID:19035497

  16. Chondrocyte differentiation for auricular cartilage reconstruction using a chitosan based hydrogel.

    PubMed

    García-López, J; Garciadiego-Cázares, D; Melgarejo-Ramírez, Y; Sánchez-Sánchez, R; Solís-Arrieta, L; García-Carvajal, Z; Sánchez-Betancourt, J I; Ibarra, C; Luna-Bárcenas, G; Velasquillo, C

    2015-12-01

    Tissue engineering with the use of biodegradable and biocompatible scaffolds is an interesting option for ear repair. Chitosan-Polyvinyl alcohol-Epichlorohydrine hydrogel (CS-PVA-ECH) is biocompatible and displays appropriate mechanical properties to be used as a scaffold. The present work, studies the potential of CS-PVA-ECH scaffolds seeded with chondrocytes to develop elastic cartilage engineered-neotissues. Chondrocytes isolated from rabbit and swine elastic cartilage were independently cultured onto CS-PVA-ECH scaffolds for 20 days to form the appropriate constructs. Then, in vitro cell viability and morphology were evaluated by calcein AM and EthD-1 assays and Scanning Electron Microscopy (SEM) respectively, and the constructs were implanted in nu/nu mice for four months, in order to evaluate the neotissue formation. Histological analysis of the formed neotissues was performed by Safranin O, Toluidine blue (GAG's), Verhoeff-Van Gieson (elastic fibers), Masson's trichrome (collagen) and Von Kossa (Calcium salts) stains and SEM. Results indicate appropriate cell viability, seeded with rabbit or swine chondrocyte constructs; nevertheless, upon implantation the constructs developed neotissues with different characteristics depending on the animal species from which the seeded chondrocytes came from. Neotissues developed from swine chondrocytes were similar to auricular cartilage, while neotissues from rabbit chondrocytes were similar to hyaline cartilage and eventually they differentiate to bone. This result suggests that neotissue characteristics may be influenced by the animal species source of the chondrocytes isolated.

  17. In vitro effect of a synthesized sulfonamido-based gallate on articular chondrocyte metabolism.

    PubMed

    Lin, Xiao; Zheng, Li; Liu, Qin; Liu, Buming; Jiang, Bingli; Peng, Xiaoyu; Lin, Cuiwu

    2014-06-01

    Autologous chondrocyte implantation (ACI) is a promising strategy for cartilage repair and reconstitution. However, limited cell numbers and the dedifferentiation of chondrocytes present major difficulties to the success of ACI therapy. Therefore, it is important to find effective pro-chondrogenic agents that restore these defects to ensure a successful therapy. In this study, we synthesized a sulfonamido-based gallate, namely N-[4-(4,6-dimethyl-pyrimidin-2-ylsulfamoyl)-phenyl]-3,4,5-trihydroxy-benzamide (EJTC), and investigated its effects on rabbit articular chondrocytes through an examination of its specific effects on cell proliferation, morphology, viability, GAG synthesis, and cartilage-specific gene expression. The results show that EJTC can effectively promote chondrocyte growth and enhance the secretion and synthesis of cartilage ECM by upregulating the expression levels of the aggrecan, collagen II, and Sox9 genes. The expression of the collagen I gene was effectively downregulated, which indicates that EJTC inhibits chondrocytes dedifferentiation. Chondrocyte hypertrophy, which may lead to chondrocyte ossification, was also undetectable in the EJTC-treated groups. The recommended dose of EJTC ranges from 3.125 μg/mL to 7.8125 μg/mL, and the most profound response was observed with 7.8125 μg/mL. This study may provide a basis for the development of a novel agent for the treatment of articular cartilage defects.

  18. Encapsulation of chondrocytes in high-stiffness agarose microenvironments for in vitro modeling of osteoarthritis mechanotransduction.

    PubMed

    Jutila, Aaron A; Zignego, Donald L; Schell, William J; June, Ronald K

    2015-05-01

    In articular cartilage, chondrocytes reside within a gel-like pericellular matrix (PCM). This matrix provides a mechanical link through which joint loads are transmitted to chondrocytes. The stiffness of the PCM decreases in the most common degenerative joint disease, osteoarthritis. To develop a system for modeling the stiffness of both the healthy and osteoarthritic PCM, we determined the concentration-stiffness relationships for agarose. We extended these results to encapsulate chondrocytes in agarose of physiological stiffness. Finally, we assessed the relevance of stiffness for chondrocyte mechanotransduction by examining the biological response to mechanical loading for cells encapsulated in low- and high-stiffness gels. We achieved agarose equilibrium stiffness values as large as 51.3 kPa. At 4.0% agarose, we found equilibrium moduli of 34.3 ± 1.65 kPa, and at 4.5% agarose, we found equilibrium moduli of 35.7 ± 0.95 kPa. Cyclical tests found complex moduli of ~100-300 kPa. Viability was >96% for all studies. We observed distinct metabolomic responses in >500 functional small molecules describing changes in cell physiology, between primary human chondrocytes encapsulated in 2.0 and 4.5% agarose indicating that the gel stiffness affects cellular mechanotransduction. These data demonstrate both the feasibility of modeling the chondrocyte pericellular matrix stiffness and the importance of the physiological pericellular stiffness for understanding chondrocyte mechanotransduction.

  19. Chondrocyte viability in fresh and frozen large human osteochondral allografts: effect of cryoprotective agents.

    PubMed

    Judas, F; Rosa, S; Teixeira, L; Lopes, C; Ferreira Mendes, A

    2007-10-01

    Chondrocyte survival is a major goal for the effective storage and clinical performance of human osteochondral allografts. The majority of animal and human cryopreservation studies conducted so far have been performed in small osteochondral cylinders. Using human tibial plateaus as a model for large osteochondral pieces, this work sought to evaluate the cryoprotective efficiency of glycerol and dimethylsulfoxide (DMSO), and to identify cryopreservation conditions suitable for use in tissue banks. Human tibial plateaus harvested from 7 cadaveric tissue donors were incubated in the presence or absence of cryoprotective agents (CPA): 10% or 15% glycerol and 10% DMSO in a Ham F-12 nutrient mixture. Chondrocyte viability was assessed immediately after thawing, using the MTT reduction assay and a fluorescence microscopic method. The tibial plateaus frozen in the absence of CPA showed a significant decrease in chondrocyte viability. The use of CPA significantly increased chondrocyte viability compared with cartilage frozen without CPA (nearly 50% versus 80% living chondrocytes with 10% glycerol versus 10% DMSO, respectively) relative to that in fresh cartilage. In this regard, 10% DMSO was slightly more effective than either 10% or 15% glycerol, eliciting the recovery of approximately 15% relative to the living chondrocyte content in fresh cartilage. In all conditions, fluorescence microscopic studies showed that surviving chondrocytes were restricted to the superficial cartilage layer. Human tibial plateaus seemed to be a good experimental model to establish cryopreservation methods applicable to large human osteochondral pieces in tissue banks.

  20. Chondrogenic capability of osteoarthritic chondrocytes from the trapeziometacarpal and hip joints.

    PubMed

    Lovati, Arianna B; Colombini, Alessandra; Recordati, Camilla; Ceriani, Cristina; Zagra, Luigi; Berzero, Gianfranco; Moretti, Matteo

    2016-03-01

    Osteoarthritis is the most common degenerative disease of joints like the hip and the trapeziometacarpal joint (rhizarthrosis). In this in vitro study, we compared the chondrogenesis of chondrocytes derived from the trapezium and the femoral head cartilage of osteoarthritic patients to have a deeper insight on trapezium chondrocyte behavior as autologous cell source for the repair of cartilage lesions in rhizarthrosis. Chondrocytes collected from trapezium and femoral head articular cartilage were cultured in pellets and analyzed for chondrogenic differentiation, cell proliferation, glycosaminoglycan production, gene expression of chondrogenic and fibrous markers, histological and immunohistochemical analyses. Our results showed a higher cartilaginous matrix deposition and a lower fibrocartilaginous phenotype of the femoral chondrocytes with respect to the trapezium chondrocytes assessed by a higher absolute glycosaminoglycan and type II collagen production, thus demonstrating a superior chondrogenic potential of the femoral with respect to the trapezium chondrocytes. The differences in chondrogenic potential between trapezium and femoral head chondrocytes confirmed a lower regenerative capability in the trapezium than in the femoral head cartilage due to the different environment and loading acting on these joints that affects the metabolism of the resident cells. This could represent a limitation to apply the cell therapy for rhizoarthrosis.

  1. Disparate Response of Articular- and Auricular-derived Chondrocytes to Oxygen Tension

    PubMed Central

    Kean, Thomas J.; Mera, Hisashi; Whitney, G. Adam; MacKay, Danielle L.; Awadallah, Amad; Fernandes, Russell J.; Dennis, James E.

    2016-01-01

    Purpose/Aim To determine the effect of reduced (5%) oxygen tension on chondrogenesis of auricular-derived chondrocytes. Currently, many cell and tissue culture experiments are performed at 20% oxygen with 5% carbon dioxide. Few cells in the body are subjected to this supra-physiological oxygen tension. Chondrocytes and their mesenchymal progenitors are widely reported to have greater chondrogenic expression when cultured at low, more physiological, oxygen tension (1–7%). Although generally accepted, there is still some controversy, and different culture methods, species, and outcome metrics cloud the field. These results are, however, articular chondrocyte biased and have not been reported for auricular-derived chondrocytes. Materials and Methods Auricular and articular chondrocytes were isolated from skeletally mature New Zealand White rabbits, expanded in culture and differentiated in high density cultures with serum free chondrogenic media. Cartilage tissue derived from aggregate cultures or from the tissue engineered sheets were assessed for biomechanical, glycosaminoglycan, collagen, collagen cross-links, and lysyl oxidase activity and expression. Results Our studies show increased proliferation rates for both auricular and articular chondrocytes at low (5%) O2 versus standard (20%) O2. In our scaffold free chondrogenic cultures, low O2 was found to increase articular chondrocyte accumulation of glycosaminoglycan, but not cross-linked type II collagen, or total collagen. Conversely, auricular chondrocytes accumulated less glycosaminoglycan, cross-linked type II collagen and total collagen under low oxygen tension. Conclusions This study highlights the dramatic difference in response to low O2 of chondrocytes isolated from different anatomical sites. Low O2 is beneficial for articular-derived chondrogenesis but detrimental for auricular-derived chondrogenesis. PMID:27128439

  2. Regulation of PTHrP expression by cyclic mechanical strain in postnatal growth plate chondrocytes.

    PubMed

    Xu, Tao; Yang, Kaixiang; You, Hongbo; Chen, Anmin; Wang, Jiang; Xu, Kai; Gong, Chen; Shao, Jingfan; Ma, Zhongxi; Guo, Fengjing; Qi, Jun

    2013-10-01

    Mechanical loading has been widely considered to be a crucial regulatory factor for growth plate development, but the exact mechanisms of this regulation are still not completely understood. In the growth plate, parathyroid hormone-related protein (PTHrP) regulates chondrocyte differentiation and longitudinal growth. Cyclic mechanical strain has been demonstrated to influence growth plate chondrocyte differentiation and metabolism, whereas the relationship between cyclic mechanical strain and PTHrP expression is not clear. The objective of this study was to investigate whether short-term cyclic tensile strain regulates PTHrP expression in postnatal growth plate chondrocytes in vitro and to explore whether the organization of cytoskeletal F-actin microfilaments is involved in this process. To this end, we obtained growth plate chondrocytes from 2-week-old Sprague-Dawley rats and sorted prehypertrophic and hypertrophic chondrocytes using immunomagnetic beads coated with anti-CD200 antibody. The sorted chondrocytes were subjected to cyclic tensile strain of varying magnitude and duration at a frequency of 0.5 Hz. We found that cyclic strain regulates PTHrP expression in a magnitude- and time-dependent manner. Incubation of chondrocytes with cytochalasin D, an actin microfilament-disrupting reagent, blocked the induction of PTHrP expression in response to strain. The results suggest that short-term cyclic tensile strain induces PTHrP expression in postnatal growth plate prehypertrophic and hypertrophic chondrocytes and that PTHrP expression by these chondrocytes may subsequently affect growth plate development. The results also support the idea that the organization of cytoskeletal F-actin microfilaments plays an important role in mechanotransduction.

  3. Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products.

    PubMed

    Cecil, Denise L; Johnson, Kristen; Rediske, John; Lotz, Martin; Schmidt, Ann Marie; Terkeltaub, Robert

    2005-12-15

    The multiligand receptor for advanced glycation end products (RAGE) mediates certain chronic vascular and neurologic degenerative diseases accompanied by low-grade inflammation. RAGE ligands include S100/calgranulins, a class of low-molecular-mass, calcium-binding polypeptides, several of which are chondrocyte expressed. Here, we tested the hypothesis that S100A11 and RAGE signaling modulate osteoarthritis (OA) pathogenesis by regulating a shift in chondrocyte differentiation to hypertrophy. We analyzed human cartilages and cultured human articular chondrocytes, and used recombinant human S100A11, soluble RAGE, and previously characterized RAGE-specific blocking Abs. Normal human knee cartilages demonstrated constitutive RAGE and S100A11 expression, and RAGE and S100A11 expression were up-regulated in OA cartilages studied by immunohistochemistry. CXCL8 and TNF-alpha induced S100A11 expression and release in cultured chondrocytes. Moreover, S100A11 induced cell size increase and expression of type X collagen consistent with chondrocyte hypertrophy in vitro. CXCL8-induced, IL-8-induced, and TNF-alpha-induced but not retinoic acid-induced chondrocyte hypertrophy were suppressed by treatment with soluble RAGE or RAGE-specific blocking Abs. Last, via transfection of dominant-negative RAGE and dominant-negative MAPK kinase 3, we demonstrated that S100A11-induced chondrocyte type X collagen expression was dependent on RAGE-mediated p38 MAPK pathway activation. We conclude that up-regulated chondrocyte expression of the RAGE ligand S100A11 in OA cartilage, and RAGE signaling through the p38 MAPK pathway, promote inflammation-associated chondrocyte hypertrophy. RAGE signaling thereby has the potential to contribute to the progression of OA.

  4. Transamidation by transglutaminase 2 transforms S100A11 calgranulin into a procatabolic cytokine for chondrocytes.

    PubMed

    Cecil, Denise L; Terkeltaub, Robert

    2008-06-15

    In osteoarthritis (OA), low-grade joint inflammation promotes altered chondrocyte differentiation and cartilage catabolism. S100/calgranulins share conserved calcium-binding EF-hand domains, associate noncovalently as homodimers and heterodimers, and are secreted and bind receptor for advanced glycation end products (RAGE). Chondrocyte RAGE expression and S100A11 release are stimulated by IL-1beta in vitro and increase in OA cartilage in situ. Exogenous S100A11 stimulates chondrocyte hypertrophic differentiation. Moreover, S100A11 is covalently cross-linked by transamidation catalyzed by transglutaminase 2 (TG2), itself an inflammation-regulated and redox stress-inducible mediator of chondrocyte hypertrophic differentiation. In this study, we researched mouse femoral head articular cartilage explants and knee chondrocytes, and a soluble recombinant double point mutant (K3R/Q102N) of S100A11 TG2 transamidation substrate sites. Both TG2 and RAGE knockout cartilage explants retained IL-1beta responsiveness. The K3R/Q102N mutant of S100A11 retained the capacity to bind to RAGE and chondrocytes but lost the capacity to signal via the p38 MAPK pathway or induce chondrocyte hypertrophy and glycosaminoglycans release. S100A11 failed to induce hypertrophy, glycosaminoglycan release, and appearance of the aggrecanase neoepitope NITEGE in both RAGE and TG2 knockout cartilages. We conclude that transamidation by TG2 transforms S100A11 into a covalently bonded homodimer that acquires the capacity to signal through the p38 MAPK pathway, accelerate chondrocyte hypertrophy and matrix catabolism, and thereby couple inflammation with chondrocyte activation to potentially promote OA progression.

  5. Disparate response of articular- and auricular-derived chondrocytes to oxygen tension.

    PubMed

    Kean, Thomas J; Mera, Hisashi; Whitney, G Adam; MacKay, Danielle L; Awadallah, Amad; Fernandes, Russell J; Dennis, James E

    2016-07-01

    To determine the effect of reduced (5%) oxygen tension on chondrogenesis of auricular-derived chondrocytes. Currently, many cell and tissue culture experiments are performed at 20% oxygen with 5% carbon dioxide. Few cells in the body are subjected to this supra-physiological oxygen tension. Chondrocytes and their mesenchymal progenitors are widely reported to have greater chondrogenic expression when cultured at low, more physiological, oxygen tension (1-7%). Although generally accepted, there is still some controversy, and different culture methods, species, and outcome metrics cloud the field. These results are, however, articular chondrocyte biased and have not been reported for auricular-derived chondrocytes. Auricular and articular chondrocytes were isolated from skeletally mature New Zealand White rabbits, expanded in culture and differentiated in high density cultures with serum-free chondrogenic media. Cartilage tissue derived from aggregate cultures or from the tissue engineered sheets were assessed for biomechanical, glycosaminoglycan, collagen, collagen cross-links, and lysyl oxidase activity and expression. Our studies show increased proliferation rates for both auricular and articular chondrocytes at low (5%) O2 versus standard (20%) O2. In our scaffold-free chondrogenic cultures, low O2 was found to increase articular chondrocyte accumulation of glycosaminoglycan, but not cross-linked type II collagen, or total collagen. Conversely, auricular chondrocytes accumulated less glycosaminoglycan, cross-linked type II collagen and total collagen under low oxygen tension. This study highlights the dramatic difference in response to low O2 of chondrocytes isolated from different anatomical sites. Low O2 is beneficial for articular-derived chondrogenesis but detrimental for auricular-derived chondrogenesis.

  6. Alpha B-Crystallin Protects Rat Articular Chondrocytes against Casein Kinase II Inhibition-Induced Apoptosis.

    PubMed

    Lee, Sung Won; Rho, Jee Hyun; Lee, Sang Yeob; Yoo, Seung Hee; Kim, Hye Young; Chung, Won Tae; Yoo, Young Hyun

    2016-01-01

    Although alpha (α)B-crystallin is expressed in articular chondrocytes, little is known about its role in these cells. Protein kinase casein kinase 2 (CK2) inhibition induces articular chondrocyte death. The present study examines whether αB-crystallin exerts anti-apoptotic activity in articular chondrocytes. Primary rat articular chondrocytes were isolated from knee joint slices. Cells were treated with CK2 inhibitors with or without αB-crystallin siRNA. To examine whether the silencing of αB-crystallin sensitizes rat articular chondrocytes to CK2 inhibition-induced apoptosis, we assessed apoptosis by performing viability assays, mitochondrial membrane potential measurements, flow cytometry, nuclear morphology observations, and western blot analysis. To investigate the mechanism by which αB-crystallin modulates the extent of CK2 inhibition-mediated chondrocyte death, we utilized confocal microscopy to observe the subcellular location of αB-crystallin and its phosphorylated forms and performed a co-immunoprecipitation assay to observe the interaction between αB-crystallin and CK2. Immunochemistry was employed to examine αB-crystallin expression in cartilage obtained from rats with experimentally induced osteoarthritis (OA). Our results demonstrated that silencing of αB-crystallin sensitized rat articular chondrocytes to CK2 inhibitor-induced apoptosis. Furthermore, CK2 inhibition modulated the expression and subcellular localization of αB-crystallin and its phosphorylated forms and dissociated αB-crystallin from CK2. The population of rat articular chondrocytes expressing αB-crystallin and its phosphorylated forms was reduced in an experimentally induced rat model of OA. In summary, αB-crystallin protects rat articular chondrocytes against CK2 inhibition-induced apoptosis. αB-crystallin may represent a suitable target for pharmacological interventions to prevent OA.

  7. Parathyroid hormone 1-34 reduces dexamethasone-induced terminal differentiation in human articular chondrocytes.

    PubMed

    Chang, Ling-Hua; Wu, Shun-Cheng; Chen, Chung-Hwan; Wang, Gwo-Jaw; Chang, Je-Ken; Ho, Mei-Ling

    2016-08-10

    Intra-articular injection of dexamethasone (Dex) is occasionally used to relieve pain and inflammation in osteoarthritis (OA) patients. Dex induces terminal differentiation of chondrogenic mesenchymal stem cells in vitro and causes impaired longitudinal skeletal growth in vivo. Parathyroid hormone 1-34 (PTH 1-34) has been shown to reverse terminal differentiation of osteoarthritic articular chondrocytes. We hypothesized that Dex induces terminal differentiation of articular chondrocytes and that this effect can be mitigated by PTH 1-34 treatment. We tested the effect of Dex on terminal differentiation in human articular chondrocytes and further tested if PTH 1-34 reverses the effects. We found that Dex treatment downregulated chondrogenic-induced expressions of SOX-9, collagen type IIa1 (Col2a1), and aggrecan and reduced synthesis of cartilaginous matrix (Col2a1 and sulfated glycosaminoglycan) synthesis. Dex treatment upregulated chondrocyte hypertrophic markers of collagen type X and alkaline phosphatase at mRNA and protein levels, and it increased the cell size of articular chondrocytes and induced cell death. These results indicated that Dex induces terminal differentiation of articular chondrocytes. To test whether PTH 1-34 treatment reverses Dex-induced terminal differentiation of articular chondrocytes, PTH 1-34 was co-administered with Dex. Results showed that PTH 1-34 treatment reversed both changes of chondrogenic and hypertrophic markers in chondrocytes induced by Dex. PTH 1-34 also decreased Dex-induced cell death. PTH 1-34 treatment reduces Dex-induced terminal differentiation and apoptosis of articular chondrocytes, and PTH 1-34 treatment may protect articular cartilage from further damage when received Dex administration.

  8. Chondrocyte survival in osteochondral transplant cylinders depends on the harvesting technique.

    PubMed

    Hafke, Benedikt; Petri, Maximilian; Suero, Eduardo; Neunaber, Claudia; Kwisda, Sebastian; Krettek, Christian; Jagodzinski, Michael; Omar, Mohamed

    2016-07-01

    In autologous osteochondral transplantation, the edges of the harvested plug are particularly susceptible to mechanical or thermal damage to the chondrocytes. We hypothesised that the applied harvesting device has an impact on chondrocyte vitality. Both knees of five blackhead sheep (ten knees) underwent open osteochondral plug harvesting with three different circular harvesting devices (osteoarticular transfer system harvester [OATS; diameter 8 mm; Arthrex, Munich, Germany], diamond cutter [DC; diameter 8.35 mm; Karl Storz, Tuttlingen, Germany] and hollow reamer with cutting crown [HRCC; diameter 7 mm; Dannoritzer, Tuttlingen, Germany]) from distinctly assigned anatomical sites of the knee joint. The rotary cutters (DC and HRCC) were either used with (+) or without cooling (-). Surgical cuts of the cartilage with a scalpel blade were chosen as control method. After cryotomy cutting, chondrocyte vitality was assessed using fluorescence microscopy and a Live/Dead assay. There were distinct patterns of chondrocyte vitality, with reproducible accumulations of dead chondrocytes along the harvesting edge. No statistical difference in chondrocyte survivorship was seen between the OATS technique and the control method, or between the HRCC+ technique and the control method (P > 0.05). The DC+, HRCC- and DC- techniques yielded significantly lower chondrocyte survival rates compared with the control method (P < 0.05). Chondrocyte survival in osteochondral cylinders depends on the applied harvesting technique. The use of rotary cutters without cooling yielded worst results, while the traditional OATS punch and rotary cutters with cooling achieved comparable rates of chondrocyte vitality.

  9. Benzamil sensitive ion channels contribute to volume regulation in canine chondrocytes

    PubMed Central

    Lewis, R; Feetham, CH; Gentles, L; Penny, J; Tregilgas, L; Tohami, W; Mobasheri, A; Barrett-Jolley, R

    2013-01-01

    Background and Purpose Chondrocytes exist within cartilage and serve to maintain the extracellular matrix. It has been postulated that osteoarthritic (OA) chondrocytes lose the ability to regulate their volume, affecting extracellular matrix production. In previous studies, we identified expression of epithelial sodium channels (ENaC) in human chondrocytes, but their function remained unknown. Although ENaC typically has Na+ transport roles, it is also involved in the cell volume regulation of rat hepatocytes. ENaC is a member of the degenerin (Deg) family, and ENaC/Deg-like channels have a low conductance and high sensitivity to benzamil. In this study, we investigated whether canine chondrocytes express functional ENaC/Deg-like ion channels and, if so, what their function may be. Experimental Approach Canine chondrocytes were harvested from dogs killed for unassociated welfare reasons. We used immunohistochemistry and patch-clamp electrophysiology to investigate ENaC expression and video microscopy to analyse the effects of pharmacological inhibition of ENaC/Deg on cell volume regulation. Key Results Immunofluorescence showed that canine chondrocytes expressed ENaC protein. Single-channel recordings demonstrated expression of a benzamil-sensitive Na+ conductance (9 pS), and whole-cell experiments show this to be approximately 1.5 nS per cell with high selectivity for Na+. Benzamil hyperpolarized chondrocytes by approximately 8 mV with a pD2 8.4. Chondrocyte regulatory volume decrease (RVI) was inhibited by benzamil (pD2 7.5) but persisted when extracellular Na+ ions were replaced by Li+. Conclusion and Implications Our data suggest that benzamil inhibits RVI by reducing the influx of Na+ ions through ENaC/Deg-like ion channels and present ENaC/Deg as a possible target for pharmacological modulation of chondrocyte volume. PMID:22928819

  10. Alpha B-Crystallin Protects Rat Articular Chondrocytes against Casein Kinase II Inhibition-Induced Apoptosis

    PubMed Central

    Rho, Jee Hyun; Lee, Sang Yeob; Yoo, Seung Hee; Kim, Hye Young; Chung, Won Tae; Yoo, Young Hyun

    2016-01-01

    Although alpha (α)B-crystallin is expressed in articular chondrocytes, little is known about its role in these cells. Protein kinase casein kinase 2 (CK2) inhibition induces articular chondrocyte death. The present study examines whether αB-crystallin exerts anti-apoptotic activity in articular chondrocytes. Primary rat articular chondrocytes were isolated from knee joint slices. Cells were treated with CK2 inhibitors with or without αB-crystallin siRNA. To examine whether the silencing of αB-crystallin sensitizes rat articular chondrocytes to CK2 inhibition-induced apoptosis, we assessed apoptosis by performing viability assays, mitochondrial membrane potential measurements, flow cytometry, nuclear morphology observations, and western blot analysis. To investigate the mechanism by which αB-crystallin modulates the extent of CK2 inhibition-mediated chondrocyte death, we utilized confocal microscopy to observe the subcellular location of αB-crystallin and its phosphorylated forms and performed a co-immunoprecipitation assay to observe the interaction between αB-crystallin and CK2. Immunochemistry was employed to examine αB-crystallin expression in cartilage obtained from rats with experimentally induced osteoarthritis (OA). Our results demonstrated that silencing of αB-crystallin sensitized rat articular chondrocytes to CK2 inhibitor-induced apoptosis. Furthermore, CK2 inhibition modulated the expression and subcellular localization of αB-crystallin and its phosphorylated forms and dissociated αB-crystallin from CK2. The population of rat articular chondrocytes expressing αB-crystallin and its phosphorylated forms was reduced in an experimentally induced rat model of OA. In summary, αB-crystallin protects rat articular chondrocytes against CK2 inhibition-induced apoptosis. αB-crystallin may represent a suitable target for pharmacological interventions to prevent OA. PMID:27851782

  11. Enhanced apoptotic and reduced protective response in chondrocytes following endoplasmic reticulum stress in osteoarthritic cartilage

    PubMed Central

    Takada, Koji; Hirose, Jun; Senba, Kei; Yamabe, Soichiro; Oike, Yuichi; Gotoh, Tomomi; Mizuta, Hiroshi

    2011-01-01

    Endoplasmic reticulum (ER) stress has been shown to participate in many disease pathologies. Although recent reports have demonstrated that ER stress in chondrocytes is present in human osteoarthritis (OA), its role in the pathology of cartilage degeneration, such as chondrocyte apoptosis, remains unclear. In the present study, we investigated the expression of phosphorylated PERK (pPERK), ubiquitin (Ub), GRP78, CHOP, phosphorylated JNK (pJNK) and cleaved caspase-3 (C-CASP3) and the mRNA splicing of XBP1 (XBP1 splicing) in human OA cartilage by immunohistochemistry and RT-PCR. Additionally, human chondrocytes were treated with several concentrations of tunicamycin, an ER stress inducer, to assess the impact of ER stress on the mRNA expression of CHOP, XBP1 splicing and apoptosis, as determined by real-time PCR, RT-PCR and ELISA analyses respectively. In human OA cartilage, the number of chondrocytes expressing pPERK, Ub, CHOP and pJNK positively correlated with cartilage degeneration and the number of C-CASP3-positive chondrocytes. XBP1 splicing and GRP78 expression in severe OA containing the greatest number of C-CASP3-positive chondrocytes were similar to the levels in mild OA, however, XBP1 splicing was higher in moderate OA than in mild and severe OA. Tunicamycin dose dependently increased CHOP expression and apoptosis of cultured chondrocytes. Although tunicamycin upregulated XBP1 splicing in cultured chondrocytes, its impact on XBP1 splicing was weakened at higher concentrations. In conclusion, the present results indicate that ER stress may contribute to chondrocyte apoptosis along with OA progression, which was closely associated with an enhanced apoptotic response and a reduced protective response by the cells. PMID:21294793

  12. Generation of Immortalized Equine Chondrocytes With Inducible Sox9 Expression Allows Control of Hypertrophic Differentiation.

    PubMed

    Gurusinghe, Saliya; Hilbert, Bryan; Trope, Gareth; Wang, Lexin; Bandara, Nadeeka; Strappe, Padraig

    2016-10-27

    Immortalization of chondrocytes enables long term in vitro culture; however, the chondrogenic capacity of transformed cells varies, thus highlighting the need to develop a proliferative and tuneable chondrocyte cell line where hypertrophic differentiation can be controlled. In this study the SV40 large T antigen and human telomerase reverse transcriptase were employed to immortalize pooled equine chondrocytes through lentiviral vector mediated transduction either singly or on combination. Transformed chondrocytes proliferated stably over multiple passages, but resulted in significantly lower expression of chondrocyte specific collagen II mRNA (P < 0.0001) and up regulation of the hypertrophic marker collagen X (P < 0.0001) in three dimensional cultures. A Col2a1 promoter driven GFP reporter was constructed for real time monitoring of chondrogenic differentiation and a significant increase in promoter activation was observed in cultures treated with the growth factor TGFβ-3 (P < 0.05). To recapitulate the native articular chondrocyte phenotype we further transduced large T antigen immortalized chondrocytes with lentiviral vectors allowing either constitutive or doxycycline inducible expression of Sox9. In 3D cultures, the Sox9 over-expressing chondrocytes secreted significantly higher levels of extracellular matrix polysaccharide glycosaminoglycan (P < 0.05), while up-regulating collagen II and Aggrecan mRNA (P < 0.05) in both expression systems with a similar patterns observed with imunohistochemical staining. High levels of collagen X mRNA and protein were maintained with constitutive sox9 reflecting hypetrophic differentiation but significantly lower expression could be achieved with inducible Sox9. In conclusion, immortalization of equine chondrocytes results in stable proliferation but a reduction of chondrogenic potential whilst modulation of sox9 expression enabled control of hypertrophic characteristics. J. Cell. Biochem. 9999: 1

  13. Cartilage tissue engineering of nasal septal chondrocyte-macroaggregates in human demineralized bone matrix.

    PubMed

    Liese, Juliane; Marzahn, Ulrike; El Sayed, Karym; Pruss, Axel; Haisch, Andreas; Stoelzel, Katharina

    2013-06-01

    Tissue Engineering is an important method for generating cartilage tissue with isolated autologous cells and the support of biomaterials. In contrast to various gel-like biomaterials, human demineralized bone matrix (DBM) guarantees some biomechanical stability for an application in biomechanically loaded regions. The present study combined for the first time the method of seeding chondrocyte-macroaggregates in DBM for the purpose of cartilage tissue engineering. After isolating human nasal chondrocytes and creating a three-dimensional macroaggregate arrangement, the DBM was cultivated in vitro with the macroaggregates. The interaction of the cells within the DBM was analyzed with respect to cell differentiation and the inhibitory effects of chondrocyte proliferation. In contrast to chondrocyte-macroaggregates in the cell-DBM constructs, morphologically modified cells expressing type I collagen dominated. The redifferentiation of chondrocytes, characterized by the expression of type II collagen, was only found in low amounts in the cell-DBM constructs. Furthermore, caspase 3, a marker for apoptosis, was detected in the chondrocyte-DBM constructs. In another experimental setting, the vitality of chondrocytes as related to culture time and the amount of DBM was analyzed with the BrdU assay. Higher amounts of DBM tended to result in significantly higher proliferation rates of the cells within the first 48 h. After 96 h, the vitality decreased in a dose-dependent fashion. In conclusion, this study provides the proof of concept of chondrocyte-macroaggregates with DBM as an interesting method for the tissue engineering of cartilage. The as-yet insufficient redifferentiation of the chondrocytes and the sporadic initiation of apoptosis will require further investigations.

  14. Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation

    SciTech Connect

    Liu, Qi; Wan, Qilong; Yang, Rongtao; Zhou, Haihua; Li, Zubing

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Different PTH administration exerts different effects on condylar chondrocyte. Black-Right-Pointing-Pointer Intermittent PTH administration suppresses condylar chondrocyte proliferation. Black-Right-Pointing-Pointer Continuous PTH administration maintains condylar chondrocyte proliferating. Black-Right-Pointing-Pointer Intermittent PTH administration enhances condylar chondrocyte differentiation. -- Abstract: Endochondral ossification is a complex process involving chondrogenesis and osteogenesis regulated by many hormones and growth factors. Parathyroid hormone (PTH), one of the key hormones regulating bone metabolism, promotes osteoblast differentiation and osteogenesis by intermittent administration, whereas continuous PTH administration inhibits bone formation. However, the effects of PTH on chondrocyte proliferation and differentiation are still unclear. In this study, intermittent PTH administration presented enhanced effects on condylar chondrocyte differentiation and bone formation, as demonstrated by increased mineral nodule formation and alkaline phosphatase (ALP) activity, up-regulated runt-related transcription factor 2 (RUNX2), ALP, collagen type X (COL10a1), collagen type I (COL1a1), osteocalcin (OCN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2) and osterix (OSX) mRNA and/or protein expression. On the contrary, continuous PTH administration promoted condylar chondrocyte proliferation and suppressed its differentiation, as demonstrated by up-regulated collagen type II (COL2a1) mRNA expression, reduced mineral nodule formation and down-regulated expression of the mRNAs and/or proteins mentioned above. Our data suggest that PTH can regulate condylar chondrocyte proliferation and differentiation, depending on the type of PTH administration. These results provide new insight into the effects of PTH on condylar chondrocytes and new evidence for using local PTH administration to cure mandibular

  15. Precipitant induced porosity augmentation of polystyrene preserves the chondrogenicity of human chondrocytes.

    PubMed

    Joergensen, Natasja L; Foldager, Casper B; Le, Dang Q S; Lind, Martin; Lysdahl, Helle

    2016-12-01

    Cells constantly sense and receive chemical and physical signals from neighboring cells, interstitial fluid, and extracellular matrix, which they integrate and translate into intracellular responses. Thus, the nature of the surface on which cells are cultured in vitro plays an important role for cell adhesion, proliferation, and differentiation. Autologs chondrocyte implantation is considered the treatment of choice for larger cartilage defects in the knee. To obtain a sufficient number of chondrocytes for implantation multiple passaging is often needed, which raises concerns about the changes in the chondrogenic phenotype. In the present study, we analyzed the effect at cellular and molecular level of precipitant induced porosity augmentation (PIPA) of polystyrene surfaces on proliferation and differentiation of human chondrocytes. Human chondrocytes were isolated from healthy patients undergoing anterior cruciate ligament reconstruction and cultured on PIPA modified polystyrene surfaces. Microscopical analysis revealed topographically arranged porosity with micron pores and nanometer pits. Chondrocytes cultured on PIPA surfaces revealed no difference in cell viability and proliferation, but gene- and protein expressions of collagen type II were pronounced in the first passage of chondrocytes when compared to chondrocytes cultured on control surfaces. Additionally, an analysis of 40 kinases revealed that chondrocytes expanded on PIPA caused upregulated PI3K/mTOR pathway activation and inhibition of mTORC1 resulted in reduced sGAG synthesis. These findings indicate that PIPA modified polystyrene preserved the chondrogenicity of expanded human chondrocytes at gene and protein levels, which clinically may be attractive for the next generation of cell-culture surfaces for ex vivo cell growth. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3073-3081, 2016. © 2016 Wiley Periodicals, Inc.

  16. Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes.

    PubMed

    Taylor, Drew W; Ahmed, Nazish; Hayes, Anthony J; Ferguson, Peter; Gross, Allan E; Caterson, Bruce; Kandel, Rita A

    2012-08-01

    To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks.

  17. Growth and differentiation of primary chick embryonic chondrocytes on agar plates.

    PubMed

    Yasumoto, S; Kondo, S; Kato, Y

    1980-06-01

    Freshly dissociated chondrocytes from chick embryonic sterna grew on the surface of solid substratum made of agar or agarose. The chondrocytes retained their differentiated traits such as morphology including intracellular organella and matrix forming ability. Extracellular matrix deposited around the cells exhibited strong metachromasia by staining with toluidine blue. Fibrous network was formed in the extracellular matrix. These characteristics are quite similar to those of the differentiated cartilage tissue. The agar plate seems useful in culturing the differentiated chondrocyte either in mass culture or clonal culture.

  18. Interaction of electromagnetic fields with chondrocytes in gel culture. Final report, February-August 1989

    SciTech Connect

    Grodzinsky, A.J.; Gluzband, Y.A.; Buschmann, M.D.

    1990-02-01

    The research accomplished during this project period focused on control experiments designed to establish whether cartilage cells from normal cartilage will continue to synthesize and accumulate normal extracellular matrix in agarose gel culture. This information is essential to properly design experiments to qualify changes in chondrocyte biosynthesis due to applied electromagnetic fields. The results suggest that both normal chondrocytes and swarm rat chondrosarcoma cells in agarose culture can continue to synthesize matrix macromolecules at a rate similar to or slightly higher than that in normal cartilage; also, that chondrocytes in agarose can successfully mediate assembly and accumulation of normal, mechanically functional extracellular matrix.

  19. Treatment of growth arrest by transfer of cultured chondrocytes into physeal defects.

    PubMed

    Lee, E H; Chen, F; Chan, J; Bose, K

    1998-01-01

    Chondrocytes were cultured from cartilage harvested from the iliac apophysis and knee joints of New Zealand White (NZW) rabbits. An experimental model for growth arrest was created by excising the medial half of the proximal growth plate of the tibia of 6-week-old NZW rabbits. The cultured chondrocytes were embedded in agarose and transferred into the growth-plate defect after excision of the physis. Transfer also was performed after excision of the bony bridge in established growth arrest. In both cases, growth arrest with angular deformation of the tibia was prevented. Histologic studies confirmed the viability of the chondrocytes in the new host physis.

  20. Interplay between cytoskeletal polymerization and the chondrogenic phenotype in chondrocytes passaged in monolayer culture.

    PubMed

    Parreno, Justin; Nabavi Niaki, Mortah; Andrejevic, Katarina; Jiang, Amy; Wu, Po-Han; Kandel, Rita A

    2017-02-01

    Tubulin and actin exist as monomeric units that polymerize to form either microtubules or filamentous actin. As the polymerization status (monomeric/polymeric ratio) of tubulin and/or actin have been shown to be important in regulating gene expression and phenotype in non-chondrocyte cells, the objective of this study was to examine the role of cytoskeletal polymerization on the chondrocyte phenotype. We hypothesized that actin and/or tubulin polymerization status modulates the chondrocyte phenotype during monolayer culture as well as in 3D culture during redifferentiation. To test this hypothesis, articular chondrocytes were grown and passaged in 2D monolayer culture. Cell phenotype was investigated by assessing cell morphology (area and circularity), actin/tubulin content, organization and polymerization status, as well as by determination of proliferation, fibroblast and cartilage matrix gene expression with passage number. Bovine chondrocytes became larger, more elongated, and had significantly (P < 0.05) increased gene expression of proliferation-associated molecules (cyclin D1 and ki67), as well as significantly (P < 0.05) decreased cartilage matrix (type II collagen and aggrecan) and increased fibroblast-like matrix, type I collagen (COL1), gene expression by passage 2 (P2). Although tubulin polymerization status was not significantly (P > 0.05) modulated, actin polymerization was increased in bovine P2 cells. Actin depolymerization, but not tubulin depolymerization, promoted the chondrocyte phenotype by inducing cell rounding, increasing aggrecan and reducing COL1 expression. Knockdown of actin depolymerization factor, cofilin, in these cells induced further P2 cell actin polymerization and increased COL1 gene expression. To confirm that actin status regulated COL1 gene expression in human P2 chondrocytes, human P2 chondrocytes were exposed to cytochalasin D. Cytochalasin D decreased COL1 gene expression in human passaged chondrocytes. Furthermore

  1. Hexosaminidase and alkaline phosphatase activities in articular chondrocytes and relationship to cell culture conditions.

    PubMed

    Mokonjimobe, E; Hecquet, C; Robic, D; Bourbouze, R; Adolphe, M

    1992-04-15

    Hexosaminidase and alkaline phosphatase activities in rabbit articular chondrocytes have been studied under different cell culture conditions. Chondrocytes were cultured in monolayer primary culture, monolayer subcultured to the fifth passage (in vitro aging) and cultured within a collagen gel; enzymatically released cartilage cells were used as control. Under these conditions, the two enzymes behave quite differently in relationship to alteration of the chondrocyte phenotype in culture. Increased lysosomal hexosaminidase activity could be considered to be a marker of the dedifferentiated phenotype in monolayer subculture; membrane alkaline phosphatase activity could be used as a marker of non-proliferating cells.

  2. Nuclear deformation and expression change of cartilaginous genes during in vitro expansion of chondrocytes

    SciTech Connect

    Hoshiba, Takashi; Yamada, Tomoe; Lu, Hongxu; Kawazoe, Naoki; Tateishi, Tetsuya; Chen, Guoping

    2008-10-03

    Cartilaginous gene expression decreased when chondrocytes were expanded on cell-culture plates. Understanding the dedifferentiation mechanism may provide valuable insight into cartilage tissue engineering. Here, we demonstrated the relationship between the nuclear shape and gene expression during in vitro expansion culture of chondrocytes. Specifically, the projected nuclear area increased and cartilaginous gene expressions decreased during in vitro expansion culture. When the nuclear deformation was recovered by cytochalasin D treatment, aggrecan expression was up-regulated and type I collagen (Col1a2) expression was down-regulated. These results suggest that nuclear deformation may be one of the mechanisms for chondrocyte dedifferentiation during in vitro expansion culture.

  3. Rapid Chondrocyte Isolation for Tissue Engineering Applications: The Effect of Enzyme Concentration and Temporal Exposure on the Matrix Forming Capacity of Nasal Derived Chondrocytes

    PubMed Central

    Vedicherla, Srujana

    2017-01-01

    Laboratory based processing and expansion to yield adequate cell numbers had been the standard in Autologous Disc Chondrocyte Transplantation (ADCT), Allogeneic Juvenile Chondrocyte Implantation (NuQu®), and Matrix-Induced Autologous Chondrocyte Implantation (MACI). Optimizing cell isolation is a key challenge in terms of obtaining adequate cell numbers while maintaining a vibrant cell population capable of subsequent proliferation and matrix elaboration. However, typical cell yields from a cartilage digest are highly variable between donors and based on user competency. The overall objective of this study was to optimize chondrocyte isolation from cartilaginous nasal tissue through modulation of enzyme concentration exposure (750 and 3000 U/ml) and incubation time (1 and 12 h), combined with physical agitation cycles, and to assess subsequent cell viability and matrix forming capacity. Overall, increasing enzyme exposure time was found to be more detrimental than collagenase concentration for subsequent viability, proliferation, and matrix forming capacity (sGAG and collagen) of these cells resulting in nonuniform cartilaginous matrix deposition. Taken together, consolidating a 3000 U/ml collagenase digest of 1 h at a ratio of 10 ml/g of cartilage tissue with physical agitation cycles can improve efficiency of chondrocyte isolation, yielding robust, more uniform matrix formation. PMID:28337445

  4. Dental implant customization using numerical optimization design and 3-dimensional printing fabrication of zirconia ceramic.

    PubMed

    Cheng, Yung-Chang; Lin, Deng-Huei; Jiang, Cho-Pei; Lin, Yuan-Min

    2017-05-01

    This study proposes a new methodology for dental implant customization consisting of numerical geometric optimization and 3-dimensional printing fabrication of zirconia ceramic. In the numerical modeling, exogenous factors for implant shape include the thread pitch, thread depth, maximal diameter of implant neck, and body size. Endogenous factors are bone density, cortical bone thickness, and non-osseointegration. An integration procedure, including uniform design method, Kriging interpolation and genetic algorithm, is applied to optimize the geometry of dental implants. The threshold of minimal micromotion for optimization evaluation was 100 μm. The optimized model is imported to the 3-dimensional slurry printer to fabricate the zirconia green body (powder is bonded by polymer weakly) of the implant. The sintered implant is obtained using a 2-stage sintering process. Twelve models are constructed according to uniform design method and simulated the micromotion behavior using finite element modeling. The result of uniform design models yields a set of exogenous factors that can provide the minimal micromotion (30.61 μm), as a suitable model. Kriging interpolation and genetic algorithm modified the exogenous factor of the suitable model, resulting in 27.11 μm as an optimization model. Experimental results show that the 3-dimensional slurry printer successfully fabricated the green body of the optimization model, but the accuracy of sintered part still needs to be improved. In addition, the scanning electron microscopy morphology is a stabilized t-phase microstructure, and the average compressive strength of the sintered part is 632.1 MPa. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Laparoscopic Total Extraperitoneal (TEP) Inguinal Hernia Repair Using 3-dimensional Mesh Without Mesh Fixation.

    PubMed

    Aliyazicioglu, Tolga; Yalti, Tunc; Kabaoglu, Burcak

    2017-08-01

    Approximately one fifth of patients suffer from inguinal pain after laparoscopic total extraperitoneal (TEP) inguinal hernia repair. There is existing literature suggesting that the staples used to fix the mesh can cause postoperative inguinal pain. In this study, we describe our experience with laparoscopic TEP inguinal hernia surgery using 3-dimensional mesh without mesh fixation, in our institution. A total of 300 patients who had undergone laparoscopic TEP inguinal hernia repair with 3-dimensional mesh in VKV American Hospital, Istanbul from November 2006 to November 2015 were studied retrospectively. Using the hospital's electronic archive, we studied patients' selected parameters, which are demographic features (age, sex), body mass index, hernia locations and types, duration of operations, preoperative and postoperative complications, duration of hospital stays, cost of surgery, need for analgesics, time elapsed until returning to daily activities and work. A total of 300 patients underwent laparoscopic TEP hernia repair of 437 inguinal hernias from November 2006 to November 2015. Of the 185 patients, 140 were symptomatic. Mean duration of follow-up was 48 months (range, 6 to 104 mo). The mean duration of surgery was 55 minutes for bilateral hernia repair, and 38 minutes for unilateral hernia repair. The mean duration of hospital stay was 0.9 day. There was no conversion to open surgery. In none of the cases the mesh was fixated with either staples or fibrin glue. Six patients (2%) developed seroma that were treated conservatively. One patient had inguinal hernia recurrence. One patient had preperitoneal hematoma. One patient operated due to indirect right-sided hernia developed right-sided hydrocele. One patient had wound dehiscence at the umbilical port entry site. Chronic pain developed postoperatively in 1 patient. Ileus developed in 1 patient. Laparoscopic TEP inguinal repair with 3-dimensional mesh without mesh fixation can be performed as safe as

  6. Collagen, agarose, alginate and Matrigel hydrogels as cell substrates for culture of chondrocytes in vitro: A comparative study.

    PubMed

    Miao, Zhikang; Lu, Zhenhui; Wu, Huayu; Liu, Hui; Li, Muyan; Lei, Danqing; Zheng, Li; Zhao, Jinmin

    2017-09-23

    Autologous chondrocyte implantation (ACI) has emerged as a new approach to cartilage repair through the use of harvested chondrocytes. But the expansion of the chondrocytes from the donor tissue in vitro is restricted by limited cell numbers and dedifferentiation of chondrocytes. In this study, we used four types of hydrogels including agarose, alginate, Matrigel and collagen type I hydrogels to serve as cell substrates and investigated the effect on proliferation and phenotype maintenance of chondrocytes. As a substrate for monolayer culture, collagen facilitated cell expansion and effectively suppressed the dedifferentiation of chondrocytes, as evidenced by fluorescein diacetate / propidium iodide (FDA/PI), hematoxylin-eosin staining (HE), Safranin O, immunofluorescenceassay, biochemistry analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Compared with that in agarose gels, alginate and Matrigel, collagen accelerated cell proliferation and enhanced the expression of cartilage specific genes such as ACAN, SOX9 and COLII more markedly. Furthermore, significantly lower expression of COL I (an indicator of dedifferentiation) and COL X (the chondrocyte hypertrophy marker) was present in collagen group than in other groups. This indicated that collagen substrate can better support chondrocyte growth and maintain cell phenotype, due to that it might serve as a cartilage-like ECM to provide adhesive site for chondrocytes. In summary, collagen hydrogel is a promising cell substrate for chondrocytes culture for ACI. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  7. Embedding compact surfaces into the 3-dimensional Euclidean space with maximum symmetry

    NASA Astrophysics Data System (ADS)

    Wang, Chao; Wang, ShiCheng; Zhang, YiMu; Zimmermann, Bruno

    2017-09-01

    The symmetries of surfaces which can be embedded into the symmetries of the 3-dimensional Euclidean space $\\mathbb{R}^3$ are easier to feel by human's intuition. We give the maximum order of finite group actions on $(\\mathbb{R}^3, \\Sigma)$ among all possible embedded closed/bordered surfaces with given geometric/algebraic genus $>1$ in $\\mathbb{R}^3$. We also identify the topological types of the bordered surfaces realizing the maximum order, and find simple representative embeddings for such surfaces.

  8. Multimodality imaging of intrauterine devices with an emphasis on the emerging role of 3-dimensional ultrasound.

    PubMed

    Reiner, Jeffrey S; Brindle, Kathleen A; Khati, Nadia Juliet

    2012-12-01

    The intrauterine contraceptive device (IUD) is one of the most widely used reversible contraception methods throughout the world. With advancing technology, it has rapidly gained acceptance through its increased effectiveness and practicality compared with more invasive means such as laparoscopic tubal ligation. This pictorial essay will present the IUDs most commonly used today. It will illustrate both normal and abnormal positions of IUDs across all cross-sectional imaging modalities including 2-dimensional ultrasound, computed tomography, and magnetic resonance imaging, with a focus on the emerging role of 3-dimensional ultrasound as the modality of choice.

  9. Incorporating a 3-dimensional printer into the management of early-stage cervical cancer.

    PubMed

    Baek, Min-Hyun; Kim, Dae-Yeon; Kim, Namkug; Rhim, Chae Chun; Kim, Jong-Hyeok; Nam, Joo-Hyun

    2016-08-01

    We used a 3-dimensional (3D) printer to create anatomical replicas of real lesions and tested its application in cervical cancer. Our study patient decided to undergo radical hysterectomy after seeing her 3D model which was then used to plan and simulate this surgery. Using 3D printers to create patient-specific 3D tumor models may aid cervical cancer patients make treatment decisions. This technology will lead to better surgical and oncological outcomes for cervical cancer patients. J. Surg. Oncol. 2016;114:150-152. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Brief communications: visualization of coronary arteries in rats by 3-dimensional real-time contrast echocardiography.

    PubMed

    Ishikura, Fuminobu; Hirayama, Hideo; Iwata, Akiko; Toshida, Tsutomu; Masuda, Kasumi; Otani, Kentaro; Asanuma, Toshihiko; Beppu, Shintaro

    2008-05-01

    Angiogenesis is under intense investigation to advance the treatment of various ischemic diseases. Small animals, such as mice and rats, are often used for this purpose. However, evaluating the structure of coronary arteries in small animals in situ is not easy. We succeeded in visualizing the coronary artery in rats on 3-dimensional real-time contrast echocardiography using a high-frequency transducer. These methods will be applied for more convenient assessment in a new study, examining issues such as angiogenesis using rats in situ.

  11. Introducing a well-ordered volume porosity in 3-dimensional gold microcantilevers

    NASA Astrophysics Data System (ADS)

    Ayela, Cédric; Lalo, Hélène; Kuhn, Alexander

    2013-02-01

    The purpose of the present work is the introduction of a combined bottom-up and top-down approach to generate 3-dimensional gold microcantilevers, where the porosity in the volume of the free-standing microstructure is well-controlled. By combining the elaboration of a colloidal crystal, followed by electrodeposition, with a sacrificial layer process, free-standing macroporous gold cantilevers are fabricated collectively. In order to validate the proposed concept, a simple application to humidity sensing is evaluated using the devices as mass sensors. A large sensitivity of -529 ppm/%RH and low discrepancy are obtained experimentally, confirming the promising application potential of this original architecture.

  12. A 3-dimensional finite-difference method for calculating the dynamic coefficients of seals

    NASA Technical Reports Server (NTRS)

    Dietzen, F. J.; Nordmann, R.

    1989-01-01

    A method to calculate the dynamic coefficients of seals with arbitrary geometry is presented. The Navier-Stokes equations are used in conjunction with the k-e turbulence model to describe the turbulent flow. These equations are solved by a full 3-dimensional finite-difference procedure instead of the normally used perturbation analysis. The time dependence of the equations is introduced by working with a coordinate system rotating with the precession frequency of the shaft. The results of this theory are compared with coefficients calculated by a perturbation analysis and with experimental results.

  13. International "Intercomparison of 3-Dimensional (3D) Radiation Codes" (13RC)

    NASA Technical Reports Server (NTRS)

    Cahalan, Robert F.; Einaudi, Franco (Technical Monitor)

    2000-01-01

    An international "Intercomparison of 3-dimensional (3D) Radiation Codes" 13RC) has been initiated. It is endorsed by the GEWEX Radiation Panel, and funded jointly by the United States Department of Energy ARM program, and by the National Aeronautics and Space Administration Radiation Sciences program. It is a 3-phase effort that has as its goals to: (1) understand the errors and limits of 3D methods; (2) provide 'baseline' cases for future 3D code development; (3) promote sharing of 3D tools; (4) derive guidelines for 3D tool selection; and (5) improve atmospheric science education in 3D radiation.

  14. Design of 3-dimensional complex airplane configurations with specified pressure distribution via optimization

    NASA Technical Reports Server (NTRS)

    Kubrynski, Krzysztof

    1991-01-01

    A subcritical panel method applied to flow analysis and aerodynamic design of complex aircraft configurations is presented. The analysis method is based on linearized, compressible, subsonic flow equations and indirect Dirichlet boundary conditions. Quadratic dipol and linear source distribution on flat panels are applied. In the case of aerodynamic design, the geometry which minimizes differences between design and actual pressure distribution is found iteratively, using numerical optimization technique. Geometry modifications are modeled by surface transpiration concept. Constraints in respect to resulting geometry can be specified. A number of complex 3-dimensional design examples are presented. The software is adopted to personal computers, and as result an unexpected low cost of computations is obtained.

  15. Cells of the synovium in rheumatoid arthritis. Chondrocytes.

    PubMed

    Otero, Miguel; Goldring, Mary B

    2007-01-01

    Rheumatoid arthritis (RA) is one of the inflammatory joint diseases in a heterogeneous group of disorders that share features of destruction of the extracellular matrices of articular cartilage and bone. The underlying disturbance in immune regulation that is responsible for the localized joint pathology results in the release of inflammatory mediators in the synovial fluid and synovium that directly and indirectly influence cartilage homeostasis. Analysis of the breakdown products of the matrix components of joint cartilage in body fluids and quantitative imaging techniques have been used to assess the effects of the inflammatory joint disease on the local remodeling of joint structures. The role of the chondrocyte itself in cartilage destruction in the human rheumatoid joint has been difficult to address but has been inferred from studies in vitro and in animal models. This review covers current knowledge about the specific cellular and biochemical mechanisms that account for the disruption of the integrity of the cartilage matrix in RA.

  16. Potassium channels of pig articular chondrocytes are blocked by propofol.

    PubMed

    Mozrzymas, J W; Visintin, M; Vittur, F; Ruzzier, F

    1994-07-15

    The effect of propofol on the voltage-activated potassium channels in pig articular chondrocytes was investigated. Propofol was found to reversibly block the potassium channels in a dose-dependent manner. The blocking effect was voltage-independent and the Hill coefficient was 1.85 +/- 0.18. No changes either in the slope conductance or in the single channel kinetics were observed. The half-blocking concentration (Ec50) was 6.0 +/- 0.49 microM which is much lower than the concentrations used to observe the scavenging effect of the drug in an artificial synovial fluid. Interestingly, Ec50 found in our experiments is also smaller than the blood concentration of propofol used in anaesthesia. These results show that propofol may strongly affect the potassium channels in some non-excitable cells.

  17. Streptococcus pyogenes degrades extracellular matrix in chondrocytes via MMP-13

    SciTech Connect

    Sakurai, Atsuo; Okahashi, Nobuo; Maruyama, Fumito; Ooshima, Takashi; Hamada, Shigeyuki; Nakagawa, Ichiro

    2008-08-29

    Group A streptococcus (GAS) causes a wide range of human diseases, including bacterial arthritis. The pathogenesis of arthritis is characterized by synovial proliferation and the destruction of cartilage and subchondral bone in joints. We report here that GAS strain JRS4 invaded a chondrogenic cell line ATDC5 and induced the degradation of the extracellular matrix (ECM), whereas an isogenic mutant of JRS4 lacking a fibronectin-binding protein, SAM1, failed to invade the chondrocytes or degrade the ECM. Reverse transcription-PCR and Western blot analysis revealed that the expression of matrix metalloproteinase (MMP)-13 was strongly elevated during the infection with GAS. A reporter assay revealed that the activation of the AP-1 transcription factor and the phosphorylation of c-Jun terminal kinase participated in MMP-13 expression. These results suggest that MMP-13 plays an important role in the destruction of infected joints during the development of septic arthritis.

  18. Development of a novel vitrification method for chondrocyte sheets

    PubMed Central

    2013-01-01

    Background There is considerable interest in using cell sheets for the treatment of various lesions as part of regenerative medicine therapy. Cell sheets can be prepared in temperature-responsive culture dishes and applied to injured tissue. For example, cartilage-derived cell sheets are currently under preclinical testing for use in treatment of knee cartilage injuries. The additional use of cryopreservation technology could increase the range and practicality of cell sheet therapies. To date, however, cryopreservation of cell sheets has proved impractical. Results Here we have developed a novel and effective method for cryopreserving fragile chondrocyte sheets. We modified the vitrification method previously developed for cryopreservation of mammalian embryos to vitrify a cell sheet through use of a minimum volume of vitrification solution containing 20% dimethyl sulfoxide, 20% ethylene glycol, 0.5 M sucrose, and 10% carboxylated poly-L-lysine. The principal feature of our method is the coating of the cell sheet with a viscous vitrification solution containing permeable and non-permeable cryoprotectants prior to vitrification in liquid nitrogen vapor. This method prevented fracturing of the fragile cell sheet even after vitrification and rewarming. Both the macro- and microstructures of the vitrified cell sheets were maintained without damage or loss of major components. Cell survival in the vitrified sheets was comparable to that in non-vitrified samples. Conclusions We have shown here that it is feasible to vitrify chondrocyte cell sheets and that these sheets retain their normal characteristics upon thawing. The availability of a practical cryopreservation method should make a significant contribution to the effectiveness and range of applications of cell sheet therapy. PMID:23886356

  19. Exploration of mechanisms underlying the strain-rate-dependent mechanical property of single chondrocytes

    SciTech Connect

    Nguyen, Trung Dung; Gu, YuanTong

    2014-05-05

    Based on the characterization by Atomic Force Microscopy, we report that the mechanical property of single chondrocytes has dependency on the strain-rates. By comparing the mechanical deformation responses and the Young's moduli of living and fixed chondrocytes at four different strain-rates, we explore the deformation mechanisms underlying this dependency property. We found that the strain-rate-dependent mechanical property of living cells is governed by both of the cellular cytoskeleton and the intracellular fluid when the fixed chondrocytes are mainly governed by their intracellular fluid, which is called the consolidation-dependent deformation behavior. Finally, we report that the porohyperelastic constitutive material model which can capture the consolidation-dependent behavior of both living and fixed chondrocytes is a potential candidature to study living cell biomechanics.

  20. Effects of Selenium on Fusarium Growth and Associated Fermentation Products and the Relationship with Chondrocyte Viability.

    PubMed

    Yin, Hong; Zhang, Yan; Zhang, Feng; Hu, Jin Tao; Zhao, Yu Meng; Cheng, Bo Lun

    2017-02-01

    This study determined the effects of selenium on the growth of Fusarium strains and the effects of products extracted from the fungal cultures on relevant indicators of chondrocytes injury. The results showed that selenium supplementation resulted in differential effects on the mycelial growth of the strains. Levels of the chondrocyte injury indicators, including cell viability, proteoglycan and type II collagen contents and their mRNA expressions, were all reduced to varying degrees when the chondrocytes were incubated with fermentation extracts, the inhibitory effect varied depending on selenium content supplemented to fungal culture media. The results indicated that certain chain relations existed between the content of selenium in the environment, the production of some metabolites by fungi, and the occurrence of chondrocyte damage. The extent of this relationship and the role it plays in Kaschin-Beck disease pathogenesis merit further study. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  1. Irradiated human chondrocytes expressing bone morphogenetic protein 2 promote healing of osteoporotic bone fracture in rats.

    PubMed

    Yi, Youngsuk; Choi, Kyoung Baek; Lim, Chae-Lyul; Hyun, Jong-Pil; Lee, Hyeon-Youl; Lee, Kun Bok; Yun, Lillian; Ayverdi, Asli; Hwang, Sally; Yip, Vivian; Noh, Moon Jong; Lee, Kwan Hee

    2009-10-01

    Bone morphogenetic protein 2 (BMP2) was selected as a transgene to regenerate osteoporotic bone defects after several BMPs were tested using a bone formation study in nude mice. Human chondrocytes were transduced with a BMP2-containing retroviral vector, and single clones were selected. The cells were characterized over numerous passages for growth and BMP2 expression. The single clones were irradiated and tested for viability. BMP2 expression lasted for 3 weeks before dying off completely after approximately 1 month. Irradiated and non-irradiated transduced chondrocytes successfully healed fractures in osteoporotic rats induced by ovariectomy. The osteoinducing effect of irradiated cells was better than that of their non-irradiated counterparts or a chondrocytes-only control. This study showed that delivering BMP2 from the transduced and irradiated chondrocytes could be an effective and safe method of repairing osteoporotic bone fractures.

  2. Mechanical overloading causes mitochondrial superoxide and SOD2 imbalance in chondrocytes resulting in cartilage degeneration.

    PubMed

    Koike, Masato; Nojiri, Hidetoshi; Ozawa, Yusuke; Watanabe, Kenji; Muramatsu, Yuta; Kaneko, Haruka; Morikawa, Daichi; Kobayashi, Keiji; Saita, Yoshitomo; Sasho, Takahisa; Shirasawa, Takuji; Yokote, Koutaro; Kaneko, Kazuo; Shimizu, Takahiko

    2015-06-25

    Mechanical stress and aging are major risk factors of cartilage degeneration. Human studies have previously reported that oxidative damage increased, while SOD2 protein was reciprocally downregulated in osteoarthritic degenerated cartilage. However, it remains unclear whether mitochondrial superoxide imbalance in chondrocytes causes cartilage degeneration. We herein demonstrate that mechanical loading promoted mitochondrial superoxide generation and selective Sod2 downregulation in chondrocytes in vivo and that mitochondrial superoxide inducer also downregulated Sod2 expression in chondrocytes in vitro. A genetically manipulated model revealed that Sod2 deficiency in chondrocytes also resulted in mitochondrial superoxide overproduction and dysfunction, thus leading to cartilage degeneration. Intra-articular injection of a permeable antioxidant effectively suppressed the mechanical loading-induced mitochondrial superoxide generation and cartilage degeneration in mice. Our findings demonstrate that mitochondrial superoxide plays a pivotal role in the development and progression of osteoarthritis, and the mitochondrial superoxide balance may therefore be a promising target for the treatment of cartilage degeneration.

  3. Hyaluronan Protects Bovine Articular Chondrocytes against Cell Death Induced by Bupivacaine under Supraphysiologic Temperatures

    PubMed Central

    Liu, Sen; Zhang, Qing-Song; Hester, William; O’Brien, Michael J.; Savoie, Felix H.; You, Zongbing

    2013-01-01

    Background Bupivacaine and supraphysiologic temperature can independently reduce cell viability of articular chondrocytes. In combination these two deleterious factors could further impair cell viability. Hypothesis Hyaluronan may protect chondrocytes from death induced by bupivacaine at supraphysiologic temperatures. Study Design Controlled laboratory study. Methods Bovine articular chondrocytes were treated with hyaluronan at physiologic (37°C) and supraphysiologic temperatures (45°C and 50°C) for one hour, and then exposed to bupivacaine for one hour at room temperature. Cell viability was assessed at three time points: immediately after treatment, six hours later, and twenty-four hours later using flow cytometry and fluorescence microscopy. The effects of hyaluronan on the levels of sulfated glycosaminoglycan in the chondrocytes were determined using Alcian blue staining. Results (1) Bupivacaine alone did not induce noticeable chondrocyte death at 37°C; (2) bupivacaine and temperature synergistically increased chondrocyte death, that is, when the chondrocytes were conditioned to 45°C and 50°C, 0.25% and 0.5% bupivacaine increased the cell death rate by 131% to 383% in comparison to the phosphate-buffered saline control group; and, (3) addition of hyaluronan reduced chondrocyte death rates to approximately 14% and 25% at 45°C and 50°C, respectively. Hyaluronan’s protective effects were still observed at six and twenty-four hours after bupivacaine treatment at 45°C. However, at 50°C, hyaluronan delayed but did not prevent the cell death caused by bupivacaine. One-hour treatment with hyaluronan significantly increased sulfated glycosaminoglycan levels in the chondrocytes. Conclusions Bupivacaine and supraphysiologic temperature synergistically increase chondrocyte death and hyaluronan may protect articular chondrocytes from death caused by bupivacaine. Clinical Relevance This study provides a rationale to perform pre-clinical and clinical studies to

  4. Chondrocyte Morphology in Stiff and Soft Agarose Gels and the Influence of Fetal Calf Serum.

    PubMed

    Karim, Asima; Hall, Andrew C

    2017-05-01

    Changes to chondrocyte volume/morphology may have deleterious effects on extracellular matrix (ECM) metabolism potentially leading to cartilage deterioration and osteoarthritis (OA). The factors controlling chondrocyte properties are poorly understood, however, pericellular matrix (PCM) weakening may be involved. We have studied the density, volume, morphology, and clustering of cultured bovine articular chondrocytes within stiff (2% w/v) and soft (0.2% w/v) three-dimensional agarose gels. Gels with encapsulated chondrocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM; fetal calf serum (FCS) 1-10%;380 mOsm) for up to 7 days. Chondrocytes were fluorescently labeled after 1, 3, and 7 days with 5-chloromethylfluorescein-diacetate (CMFDA) and propidium iodide (PI) or 1,5-bis{[2-(di-methylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione (DRAQ5) to identify cytoplasmic space or DNA and imaged by confocal laser scanning microscopy (CLSM). Chondrocyte density, volume, morphology, and clustering were quantified using Volocity™ software. In stiff gels after 7 d with 10% FCS, chondrocyte density remained unaffected and morphology was relatively normal with occasional cytoplasmic processes. However, in soft gels by day 1, chondrocyte volume increased (P = 0.0058) and by day 7, density increased (P = 0.0080), along with the percentage of chondrocytes of abnormal morphology (P < 0.0001) and enhanced clustering (P < 0.05), compared to stiff gels. FCS exacerbated changes to density (P < 0.01), abnormal morphology (P < 0.001) and clustering (P < 0.01) compared to lower concentrations at the same gel strength. Reduced gel stiffness and/or increased FCS concentrations promoted chondrocyte proliferation and clustering, increased cell volume, and stimulated abnormal morphology, producing similar changes to those occurring in OA. The increased penetration of factors in FCS into soft gels may be important in the development of

  5. Cryopreservation Effect on Proliferative and Chondrogenic Potential of Human Chondrocytes Isolated from Superficial and Deep Cartilage

    PubMed Central

    Muiños-López, Emma; Rendal-Vázquez, Mª Esther; Hermida-Gómez, Tamara; Fuentes-Boquete, Isaac; Díaz-Prado, Silvia; Blanco, Francisco J

    2012-01-01

    Objectives: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. Materials and Methodology: The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. Results: Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). Conclusions: The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes. PMID:22523526

  6. The involvement and possible mechanism of NR4A1 in chondrocyte apoptosis during osteoarthritis

    PubMed Central

    Shi, Xinge; Ye, Hui; Yao, Xuedong; Gao, Yanzheng

    2017-01-01

    Osteoarthritis (OA) is a joint disease caused by the breakdown of joint cartilage and underlying bone, and places great burdens to daily life of patients. Nuclear orphan receptor nuclear receptor subfamily 4, group A, member 1 (NR4A1) is vital for cell apoptosis, but little is known about its role in OA. This study aims to reveal the expression and function of NR4A1 during OA chondrocyte apoptosis. NR4A1 expression by qRT-PCR and western blot, and chondrocyte apoptosis by TUNEL assay were detected in normal and OA joint cartilage. NR4A1 was located in cartilage sections by immunohistofluorescence. Chondrocytes from normal joint cartilage were cultured in vitro for interleukin 6 (IL6) or tumor necrosis factor (TNF) treatment and si-NR4A1 transfection, after which the possible mechanism involving NR4A1 was analyzed. Results showed that NR4A1 expression and chondrocyte apoptosis were significantly elevated in OA cartilage (P < 0.05 and P < 0.01). NR4A1 was located in nuclei of normal cartilage chondrocytes, but was translocated to mitochondria and co-located with B-cell lymphoma 2 in OA chondrocytes. NR4A1 expression in cultured chondrocytes could be promoted by both IL6 and TNF treatment. si-NR4A1 partly reduced TNF-induced cell apoptosis. Inhibiting p38 by SB203580 could decrease TNF-induced NR4A1 to some extent, while inhibiting JNK could not. So NR4A1 is likely to facilitate OA chondrocyte apoptosis, which is associated with p38 MAPK and mitochondrial apoptosis pathway. This study provides a potential therapeutic target for OA treatment and offers information for regulatory mechanisms in OA. PMID:28337303

  7. A Biosynthetic Scaffold that Facilitates Chondrocyte-Mediated Degradation and Promotes Articular Cartilage Extracellular Matrix Deposition

    PubMed Central

    Sridhar., Balaji V.; Dailing, Eric A.; Brock, J. Logan; Stansbury, Jeffrey W.; Randolph, Mark A.; Anseth, Kristi S.

    2015-01-01

    Articular cartilage remains a significant clinical challenge to repair because of its limited self-healing capacity. Interest has grown in the delivery of autologous chondrocytes to cartilage defects, and combining cell-based therapies with scaffolds that capture aspects of native tissue and allow cell-mediated remodeling could improve outcomes. Currently, scaffold-based therapies with encapsulated chondrocytes permit matrix production; however, resorption of the scaffold often does not match the rate of matrix production by chondrocytes, which can limit functional tissue regeneration. Here, we designed a hybrid biosynthetic system consisting of poly (ethylene glycol) (PEG) endcapped with thiols and crosslinked by norbornene-functionalized gelatin via a thiol-ene photopolymerization. The protein crosslinker was selected to facilitate chondrocyte-mediated scaffold remodeling and matrix deposition. Gelatin was functionalized with norbornene to varying degrees (~4–17 norbornenes/gelatin), and the shear modulus of the resulting hydrogels was characterized (<0.1–0.5 kPa). Degradation of the crosslinked PEG-gelatin hydrogels by chondrocyte-secreted enzymes was confirmed by gel permeation chromatography. Finally, chondrocytes encapsulated in these biosynthetic scaffolds showed significantly increased glycosaminoglycan deposition over just 14 days of culture, while maintaining high levels of viability and producing a distributed matrix. These results indicate the potential of a hybrid PEG-gelatin hydrogel to permit chondrocyte-mediated remodeling and promote articular cartilage matrix production. Tunable scaffolds that can easily permit chondrocyte-mediated remodeling may be useful in designing treatment options for cartilage tissue engineering applications. PMID:26900597

  8. Filamin B Regulates Chondrocyte Proliferation and Differentiation through Cdk1 Signaling

    PubMed Central

    Lian, Gewei; Zhang, Jingping; Hecht, Jonathan L.; Sheen, Volney L.

    2014-01-01

    Humans who harbor loss of function mutations in the actin-associated filamin B (FLNB) gene develop spondylocarpotarsal syndrome (SCT), a disorder characterized by dwarfism (delayed bone formation) and premature fusion of the vertebral, carpal and tarsal bones (premature differentiation). To better understand the cellular and molecular mechanisms governing these seemingly divergent processes, we generated and characterized FlnB knockdown ATDC5 cell lines. We found that FlnB knockdown led to reduced proliferation and enhanced differentiation in chondrocytes. Within the shortened growth plate of postnatal FlnB−/− mice long bone, we observed a similarly progressive decline in the number of rapidly proliferating chondrocytes and premature differentiation characterized by an enlarged prehypertrophic zone, a widened Col2a1+/Col10a1+ overlapping region, but relatively reduced hypertrophic zone length. The reduced chondrocyte proliferation and premature differentiation were, in part, attributable to enhanced G2/M phase progression, where fewer FlnB deficient ATDC5 chondrocytes resided in the G2/M phase of the cell cycle. FlnB loss reduced Cdk1 phosphorylation (an inhibitor of G2/M phase progression) and Cdk1 inhibition in chondrocytes mimicked the null FlnB, premature differentiation phenotype, through a β1-integrin receptor- Pi3k/Akt (a key regulator of chondrocyte differentiation) mediated pathway. In this context, the early prehypertrophic differentiation provides an explanation for the premature differentiation seen in this disorder, whereas the progressive decline in proliferating chondrocytes would ultimately lead to reduced chondrocyte production and shortened bone length. These findings begin to define a role for filamin proteins in directing both cell proliferation and differentiation through indirect regulation of cell cycle associated proteins. PMID:24551245

  9. Cysteine-Mediated Redox Regulation of Cell Signaling in Chondrocytes Stimulated With Fibronectin Fragments

    PubMed Central

    Wood, Scott T.; Long, David L.; Reisz, Julie A.; Yammani, Raghunatha R.; Burke, Elizabeth A.; Klomsiri, Chananat; Poole, Leslie B.; Furdui, Cristina M.; Loeser, Richard F.

    2016-01-01

    Objective Oxidative posttranslational modifications of intracellular proteins can potentially regulate signaling pathways relevant to cartilage destruction in arthritis. In this study, oxidation of cysteine residues to form sulfenic acid (S-sulfenylation) was examined in osteo-arthritic (OA) chondrocytes and investigated in normal chondrocytes as a mechanism by which fragments of fibronectin (FN-f) stimulate chondrocyte catabolic signaling. Methods Chondrocytes isolated from OA and normal human articular cartilage were analyzed using analogs of dimedone that specifically and irreversibly react with protein S-sulfenylated cysteines. Global S-sulfenylation was measured in cell lysates with and without FN-f stimulation by immunoblotting and in fixed cells by confocal microscopy. S-sulfenylation in specific proteins was identified by mass spectroscopy and confirmed by immunoblotting. Src activity was measured in live cells using a fluorescence resonance energy transfer biosensor. Results Proteins in chondrocytes isolated from OA cartilage were found to have elevated basal levels of S-sulfenylation relative to those of chondrocytes from normal cartilage. Treatment of normal chondrocytes with FN-f induced increased levels of S-sulfenylation in multiple proteins, including the tyrosine kinase Src. FN-f treatment also increased the levels of Src activity. Pretreatment with dimedone to alter S-sulfenylation function or with Src kinase inhibitors inhibited FN-f–induced production of matrix metalloproteinase 13. Conclusion These results demonstrate for the first time the presence of oxidative posttranslational modification of proteins in human articular chondrocytes by S-sulfenylation. Due to the ability to regulate the activity of a number of cell signaling pathways, including catabolic mediators induced by fibronectin fragments, S-sulfenylation may contribute to cartilage destruction in OA and warrants further investigation. PMID:26314228

  10. Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA)

    PubMed Central

    Gradišnik, Lidija; Gorenjak, Mario; Vogrin, Matjaž

    2017-01-01

    Background Cartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue. Methods Isolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA) was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2), collagen 1 (COL1) and aggrecan (ACAN) was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production. Results Cartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well. Discussion In this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a common and very

  11. The secreted protein WNT5A regulates condylar chondrocyte proliferation, hypertrophy and migration.

    PubMed

    Ge, Xianpeng; Shi, Ruirui; Ma, Xuchen

    2017-10-01

    Our previous study showed that WNT5A, a member of the noncanonical WNT pathway, is involved in interleukin-1beta induced matrix metalloproteinase expression in temporomandibular joint (TMJ) condylar chondrocytes. The purpose of this study is to further explore the roles of WNT5A in cartilage biology of the TMJ. An early TMJ osteoarthritis-like rat model was constructed by a mechanical method (steady mouth-opening). The gene and protein levels of WNT5A during the condylar cartilage changes were measured. Effects of WNT5A on chondrocyte proliferation, hypertrophy and migration were analyzed after WNT5A gain or loss of function in vitro. A c-Jun N-terminal kinase (JNK) inhibitor SP600125 was used to evaluate the involvement of JNK pathway in these effects of WNT5A. The expression and transcription activity of cell cycle regulators c-MYC and Cyclin D1 were examined to determine the mechanism behind WNT5A regulation of chondrocyte proliferation. WNT5A was significantly upregulated in the condylar cartilage of rats in the early TMJ osteoarthritis-like model. Activating WNT5A facilitated condylar chondrocyte proliferation, hypertrophy and migration. Conversely, inhibiting WNT5A activity in chondrocytes decreased their proliferation, hypertrophy and migration. Blockage of the JNK pathway by its inhibitor, SP600125, impaired these effects of WNT5A on chondrocytes. WNT5A regulated both the expression and transcriptional activity of c-MYC and Cyclin D1 in chondrocytes, both of which were upregulated in condylar cartilage of the rat early TMJ osteoarthritis. WNT5A regulates condylar chondrocyte proliferation, hypertrophy and migration. These findings provide new insights into the role of WNT5A signaling in TMJ cartilage biology and its potential in future therapy for TMJ degenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Effect of ascorbate and two different media on canine chondrocytes in three-dimensional culture.

    PubMed

    Priddy, N H; Cook, J L; Kreeger, J M; Tomlinson, J L; Steffen, D J

    2001-01-01

    The objective of this study was to determine the effects of ascorbate and two different culture media on cell morphology and extracellular matrix formation of canine chondrocytes grown in a three-dimensional (3-D) culture system. Articular cartilage harvested from the humeral head of three adult canine cadavers was used to obtain chondrocytes for primary culture. Subcultured chondrocytes were seeded in a 3-D medium of RPMI-1640 (R), RPMI-1640 with 50 microg/mL ascorbate (RA), Ham's F-12 (F), or Ham's F-12 with 50 microg/mL ascorbate (FA) in agarose. Samples were harvested at 5, 10, 15, and 20 days of 3-D culture and analyzed for histologic appearance and proteoglycan staining, electron microscopic appearance, dimethylmethylene blue assay for glycosaminoglycan (GAG) content, and immunohistochemical staining for collagen type II production. Chondrocytes in all four groups maintained appropriate morphology and produced matrix over the entire study period. Chondrocytes from groups R and RA produced more GAG and collagen type II than did those from groups F and FA on days 10 (P = .00791) and 15 (P = .0173). Chondrocytes from group RA produced more GAG on days 5 (P = .0154) and 20 (P = .0044) than did those in groups R, F, and FA. With respect to matrix production, RPMI-1640 is superior to Ham's F-12 for 3-D culture of canine chondrocytes. The addition of ascorbate at 50 microg/mL to RPMI-1640 did have a positive effect on the production of GAG but had minimal effect on type II collagen production. Determining the most ideal in vitro microenvironment for canine chondrocytes grown in a 3-D culture system has important implications to the in vivo application of this technique.

  13. Crosstalk between FLS and chondrocytes is regulated by HIF-2α-mediated cytokines in arthritis.

    PubMed

    Huh, Yun Hyun; Lee, Gyuseok; Song, Won-Hyun; Koh, Jeong-Tae; Ryu, Je-Hwang

    2015-12-04

    Rheumatoid arthritis (RA) and osteoarthritis (OA), two common types of arthritis, affect the joints mainly by targeting the synovium and cartilage. Increasing evidence indicates that a significant network connects synovitis and cartilage destruction during the progression of arthritis. We recently demonstrated that hypoxia-inducible factor (HIF)-2α causes RA and OA by regulating the expression of catabolic factors in fibroblast-like synoviocytes (FLS) or chondrocytes. To address the reciprocal influences of HIF-2α on FLS and chondrocytes, we applied an in vitro co-culture system using a transwell apparatus. When co-cultured with HIF-2α-overexpressing chondrocytes, FLS exhibited increased expression of matrix metalloproteinases and inflammatory mediators, similar to the effects induced by tumor-necrosis factor (TNF)-α treatment of FLS. Moreover, chondrocytes co-cultured with HIF-2α-overexpressing FLS exhibited upregulation of Mmp3 and Mmp13, which is similar to the effects induced by interleukin (IL)-6 treatment of chondrocytes. We confirmed these differential HIF-2α-induced effects via distinct secretory mediators using Il6-knockout cells and a TNF-α-blocking antibody. The FLS-co-culture-induced gene expression changes in chondrocytes were significantly abrogated by IL-6 deficiency, whereas TNF-α neutralization blocked the alterations in gene expression associated with co-culture of FLS with chondrocytes. Our results further suggested that the observed changes might reflect the HIF-2α-induced upregulation of specific receptors for TNF-α (in FLS) and IL-6 (in chondrocytes). This study broadens our understanding of the possible regulatory mechanisms underlying the crosstalk between the synovium and cartilage in the presence of HIF-2α, and may suggest potential new anti-arthritis therapies.

  14. Apoptosis and mitochondrial dysfunction in human chondrocytes following exposure to lidocaine, bupivacaine, and ropivacaine.

    PubMed

    Grishko, Valentina; Xu, Min; Wilson, Glenn; Pearsall, Albert W

    2010-03-01

    Several mechanisms have been proposed to explain toxicity of local anesthetics to chondrocytes, including the blockade of potassium channels and mitochondrial injury. The purposes of this investigation were to study the effects of lidocaine, bupivacaine, and ropivacaine on human chondrocyte viability and mitochondrial function in vitro and to characterize the type of cell death elicited following exposure. Primary chondrocyte cultures from patients with osteoarthritis undergoing knee replacement were treated with saline solution and the following concentrations of local anesthetics: 2%, 1%, and 0.5% lidocaine, 0.5% and 0.25% bupivacaine, and 0.5% and 0.2% ropivacaine for one hour. Cell viability and apoptosis were measured by flow cytometry at twenty-four hours and 120 hours after treatment. Nuclear staining and caspase 3 and 9 cleavage assays (Western blot) were used to further establish the induction of apoptosis. Mitochondrial dysfunction was evaluated by the accumulation of mitochondrial DNA damage (quantitative Southern blot), changes in adenosine triphosphate production (bioluminescence kit), and mitochondrial protein levels (Western blot analysis). Exposure of primary human chondrocytes to a 2% concentration of lidocaine caused massive necrosis of chondrocytes after twenty-four hours, 1% lidocaine and 0.5% bupivacaine caused a detectable, but not significant, decrease in viability after twenty-four hours, while 0.5% lidocaine, 0.25% bupivacaine, and both concentrations of ropivacaine (0.5% and 0.2%) did not affect chondrocyte viability. Flow cytometry analysis of chondrocytes 120 hours after drug treatment revealed a significant decrease in viability (p < 0.05) with a concomitant increase in the number of apoptotic cells at all concentrations of lidocaine, bupivacaine, and ropivacaine analyzed, except 0.2% ropivacaine. Apoptosis was verified by observation of condensed and fragmented nuclei and a decrease in procaspase 3 and 9 levels. Local anesthetics

  15. Dynamic compression of chondrocyte-agarose constructs reveals new candidate mechanosensitive genes.

    PubMed

    Bougault, Carole; Aubert-Foucher, Elisabeth; Paumier, Anne; Perrier-Groult, Emeline; Huot, Ludovic; Hot, David; Duterque-Coquillaud, Martine; Mallein-Gerin, Frédéric

    2012-01-01

    Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond

  16. Dynamic Compression of Chondrocyte-Agarose Constructs Reveals New Candidate Mechanosensitive Genes

    PubMed Central

    Bougault, Carole; Aubert-Foucher, Elisabeth; Paumier, Anne; Perrier-Groult, Emeline; Huot, Ludovic; Hot, David; Duterque-Coquillaud, Martine; Mallein-Gerin, Frédéric

    2012-01-01

    Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond

  17. Role of insulin-transferrin-selenium in auricular chondrocyte proliferation and engineered cartilage formation in vitro.

    PubMed

    Liu, Xia; Liu, Jinchun; Kang, Ning; Yan, Li; Wang, Qian; Fu, Xin; Zhang, Yuanyuan; Xiao, Ran; Cao, Yilin

    2014-01-21

    The goal of this study is to determine the effects of Insulin-Transferrin-Selenium (ITS) on proliferation of auricular chondrocytes and formation of engineered cartilage in vitro. Pig auricular monolayer chondrocytes and chondrocyte pellets were cultured in media containing 1% ITS at different concentrations of fetal bovine serum (FBS, 10%, 6%, 2%, 0%), or 10% FBS alone as a control for four weeks. Parameters including cell proliferation in monolayer, wet weight, collagen type I/II/X (Col I, II, X) and glycosaminoglycan (GAG) expression, GAG content of pellets and gene expression associated with cartilage formation/dedifferentiation (lost cartilage phenotype)/hypertrophy within the chondrocyte pellets were assessed. The results showed that chondrocytes proliferation rates increased when FBS concentrations increased (2%, 6%, 10% FBS) in ITS supplemented groups. In addition, 1% ITS plus 10% FBS significantly promoted cell proliferation than 10% FBS alone. No chondrocytes grew in ITS alone medium. 1% ITS plus 10% FBS enhanced cartilage formation in terms of size, wet weight, cartilage specific matrices, and homogeneity, compared to 10% FBS alone group. Furthermore, ITS prevented engineered cartilage from dedifferentiation (i.e., higher index of Col II/Col I mRNA expression and expression of aggrecan) and hypertrophy (i.e., lower mRNA expression of Col X and MMP13). In conclusion, our results indicated that ITS efficiently enhanced auricular chondrocytes proliferation, retained chondrogenic phenotypes, and promoted engineered cartilage formation when combined with FBS, which is potentially used as key supplementation in auricular chondrocytes and engineered cartilage culture.

  18. Stimulation by concanavalin A of cartilage-matrix proteoglycan synthesis in chondrocyte cultures

    SciTech Connect

    Yan, W.Q.; Nakashima, K.; Iwamoto, M.; Kato, Y. )

    1990-06-15

    The effect of concanavalin A on proteoglycan synthesis by rabbit costal and articular chondrocytes was examined. Chondrocytes were seeded at low density and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of concanavalin A to the culture medium induced a morphologic alteration of the fibroblastic cells to spherical chondrocytes and increased by 3- to 4-fold incorporation of (35S)sulfate and (3H)glucosamine into large chondroitin sulfate proteoglycan that was characteristically found in cartilage. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, as chemical analyses showed a 4-fold increase in the accumulation of macromolecules containing hexuronic acid in concanavalin A-maintained cultures. Furthermore, the effect of concanavalin A on (35S)sulfate incorporation into proteoglycans was greater than that of various growth factors or hormones. However, concanavalin A had smaller effects on (35S)sulfate incorporation into small proteoglycans and (3H)glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant glycosaminoglycans. Since other lectins tested, such as wheat germ agglutinin, lentil lectin, and phytohemagglutinin, had little effect on (35S)sulfate incorporation into proteoglycans, the concanavalin A action on chondrocytes seems specific. Although concanavalin A decreased (3H)thymidine incorporation in chondrocytes, the stimulation of proteoglycan synthesis could be observed in chondrocytes exposed to the inhibitor of DNA synthesis, cytosine arabinoside. These results indicate that concanavalin A is a potent modulator of proteoglycan synthesis by chondrocytes.

  19. Femtosecond laser assisted 3-dimensional freeform fabrication of metal microstructures in fused silica (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Ebrahim, Fatmah; Charvet, Raphaël.; Dénéréaz, Cyril; Mortensen, Andreas; Bellouard, Yves

    2017-03-01

    Femtosecond laser exposure of fused silica combined with chemical etching has opened up new opportunities for three-dimensional freeform processing of micro-structures that can form complex micro-devices of silica, integrating optical, mechanical and/or fluidic functionalities. Here, we demontrate an expansion of this process with an additional fabrication step that enables the integration of three-dimensional embedded metallic structures out of useful engineering metals such as silver, gold, copper as well as some of their alloys. This additional step is an adaptation of the pressure infiltration for the insertion of high conductivity, high melting point metals and alloys into topologically complex, femtosecond laser-machined cavities in fused silica. This produces truly 3-dimensional microstructures, including microcoils and needles, within the bulk of glass substrates. Combining this added capability with the existing possibilities of femtosecond laser micromachining (i.e. direct written waveguides, microchannels, resonators, etc.) opens up a host of potential applications for the contactless fabrication of highly integrated monolithic devices that include conductive element of all kind. We present preliminary results from this new fabrication process, including prototype devices that incorporate 3D electrodes with aspect ratios of 1:100 and a feature size resolution down to 2μm. We demonstrate the generation of high electric field gradients (of the order of 1013 Vm-2) in these devices due to the 3-dimensional topology of fabricated microstructures.

  20. Particle trajectory computation on a 3-dimensional engine inlet. Final Report Ph.D. Thesis

    NASA Technical Reports Server (NTRS)

    Kim, J. J.

    1986-01-01

    A 3-dimensional particle trajectory computer code was developed to compute the distribution of water droplet impingement efficiency on a 3-dimensional engine inlet. The computed results provide the essential droplet impingement data required for the engine inlet anti-icing system design and analysis. The droplet trajectories are obtained by solving the trajectory equation using the fourth order Runge-Kutta and Adams predictor-corrector schemes. A compressible 3-D full potential flow code is employed to obtain a cylindrical grid definition of the flowfield on and about the engine inlet. The inlet surface is defined mathematically through a system of bi-cubic parametric patches in order to compute the droplet impingement points accurately. Analysis results of the 3-D trajectory code obtained for an axisymmetric droplet impingement problem are in good agreement with NACA experimental data. Experimental data are not yet available for the engine inlet impingement problem analyzed. Applicability of the method to solid particle impingement problems, such as engine sand ingestion, is also demonstrated.

  1. Crossover from 2-dimensional to 3-dimensional aggregations of clusters on square lattice substrates

    NASA Astrophysics Data System (ADS)

    Cheng, Yi; Zhu, Yu-Hong; Pan, Qi-Fa; Yang, Bo; Tao, Xiang-Ming; Ye, Gao-Xiang

    2015-11-01

    A Monte Carlo study on the crossover from 2-dimensional to 3-dimensional aggregations of clusters is presented. Based on the traditional cluster-cluster aggregation (CCA) simulation, a modified growth model is proposed. The clusters (including single particles and their aggregates) diffuse with diffusion step length l (1 ≤ l ≤ 7) and aggregate on a square lattice substrate. If the number of particles contained in a cluster is larger than a critical size sc, the particles at the edge of the cluster have a possibility to jump onto the upper layer, which results in the crossover from 2-dimensional to 3-dimensional aggregations. Our simulation results are in good agreement with the experimental findings. Project supported by the National Natural Science Foundation of China (Grant Nos. 11374082 and 11074215), the Science Foundation of Zhejiang Province Department of Education, China (Grant No. Y201018280), the Fundamental Research Funds for Central Universities, China (Grant No. 2012QNA3010), and the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20100101110005).

  2. Virtual temporal bone: an interactive 3-dimensional learning aid for cranial base surgery.

    PubMed

    Kockro, Ralf A; Hwang, Peter Y K

    2009-05-01

    We have developed an interactive virtual model of the temporal bone for the training and teaching of cranial base surgery. The virtual model was based on the tomographic data of the Visible Human Project. The male Visible Human's computed tomographic data were volumetrically reconstructed as virtual bone tissue, and the individual photographic slices provided the basis for segmentation of the middle and inner ear structures, cranial nerves, vessels, and brainstem. These structures were created by using outlining and tube editing tools, allowing structural modeling either directly on the basis of the photographic data or according to information from textbooks and cadaver dissections. For training and teaching, the virtual model was accessed in the previously described 3-dimensional workspaces of the Dextroscope or Dextrobeam (Volume Interactions Pte, Ltd., Singapore), whose interfaces enable volumetric exploration from any perspective and provide virtual tools for drilling and measuring. We have simulated several cranial base procedures including approaches via the floor of the middle fossa and the lateral petrous bone. The virtual model suitably illustrated the core facts of anatomic spatial relationships while simulating different stages of bone drilling along a variety of surgical corridors. The system was used for teaching during training courses to plan and discuss operative anatomy and strategies. The Virtual Temporal Bone and its surrounding 3-dimensional workspace provide an effective way to study the essential surgical anatomy of this complex region and to teach and train operative strategies, especially when used as an adjunct to cadaver dissections.

  3. 3-Dimensional quantitative detection of nanoparticle content in biological tissue samples after local cancer treatment

    NASA Astrophysics Data System (ADS)

    Rahn, Helene; Alexiou, Christoph; Trahms, Lutz; Odenbach, Stefan

    2014-06-01

    X-ray computed tomography is nowadays used for a wide range of applications in medicine, science and technology. X-ray microcomputed tomography (XμCT) follows the same principles used for conventional medical CT scanners, but improves the spatial resolution to a few micrometers. We present an example of an application of X-ray microtomography, a study of 3-dimensional biodistribution, as along with the quantification of nanoparticle content in tumoral tissue after minimally invasive cancer therapy. One of these minimal invasive cancer treatments is magnetic drug targeting, where the magnetic nanoparticles are used as controllable drug carriers. The quantification is based on a calibration of the XμCT-equipment. The developed calibration procedure of the X-ray-μCT-equipment is based on a phantom system which allows the discrimination between the various gray values of the data set. These phantoms consist of a biological tissue substitute and magnetic nanoparticles. The phantoms have been studied with XμCT and have been examined magnetically. The obtained gray values and nanoparticle concentration lead to a calibration curve. This curve can be applied to tomographic data sets. Accordingly, this calibration enables a voxel-wise assignment of gray values in the digital tomographic data set to nanoparticle content. Thus, the calibration procedure enables a 3-dimensional study of nanoparticle distribution as well as concentration.

  4. Grain boundary segregation in boron added interstitial free steels studied by 3-dimensional atom probe

    SciTech Connect

    Seto, K.; Larson, D.J.; Warren, P.J.; Smith, G.D.W.

    1999-04-09

    The development of deep-drawable sheet steels is of particular significance for the automotive industry. Titanium and/or niobium added extra-low carbon interstitial free (IF) steels are key materials. The virtually complete removal of carbon and nitrogen should lead to superior forming properties. However, the lack of solute carbon at grain boundaries significantly decreases the bonding force at the interfaces, which often causes intergranular brittle fracture when deeply drawn steel sheets are subjected to impact deformation at low temperature. This phenomenon is called secondary working embrittlement (SWE), and is a major problem when solute atoms such as phosphorus, manganese or silicon are added to increase the tensile strength of the steels. Small amounts of boron, which does not affect the formability of the steels significantly, are usually added as a remedial measure in such cases. The 3-dimensional atom probe (3DAP) combined with field ion microscopy (FIM) has the ability to produce 3-dimensional images from regions approximately 20nm*20nm*100nm in size, and identify each atomic species and the relative location of each atom with nearly lattice resolution. In this study, a combination of these methods was applied to produce FIM tips of IF steel containing grain boundaries. The authors report here the first observations of the segregation of boron in IF steels using 3DAP.

  5. An electro-dynamic 3-dimensional vibration test bed for engineering testing

    NASA Astrophysics Data System (ADS)

    Saadatzi, Mohammadsadegh; Saadatzi, Mohammad Nasser; Ahmed, Riaz; Banerjee, Sourav

    2017-04-01

    Primary objective of the work is to design, fabrication and testing of a 3-dimensional Mechanical vibration test bed. Vibration testing of engineering prototype devices in mechanical and industrial laboratories is essential to understand the response of the envisioned model under physical excitation conditions. Typically, two sorts of vibration sources are available in physical environment, acoustical and mechanical. Traditionally, test bed to simulate unidirectional acoustic or mechanical vibration is used in engineering laboratories. However, a device may encounter multiple uncoupled and/or coupled loading conditions. Hence, a comprehensive test bed in essential that can simulate all possible sorts of vibration conditions. In this article, an electrodynamic vibration exciter is presented which is capable of simulating 3-dimensional uncoupled (unidirectional) and coupled excitation, in mechanical environments. The proposed model consists of three electromagnetic shakers (for mechanical excitation). A robust electrical control circuit is designed to regulate the components of the test bed through a self-developed Graphical User Interface. Finally, performance of the test bed is tested and validated using commercially available piezoelectric sensors.

  6. Comparison of nonnavigated and 3-dimensional image-based computer navigated balloon kyphoplasty.

    PubMed

    Sembrano, Jonathan N; Yson, Sharon C; Polly, David W; Ledonio, Charles Gerald T; Nuckley, David J; Santos, Edward R G

    2015-01-01

    Balloon kyphoplasty is a common treatment for osteoporotic and pathologic compression fractures. Advantages include minimal tissue disruption, quick recovery, pain relief, and in some cases prevention of progressive sagittal deformity. The benefit of image-based navigation in kyphoplasty has not been established. The goal of this study was to determine whether there is a difference between fluoroscopy-guided balloon kyphoplasty and 3-dimensional image-based navigation in terms of needle malposition rate, cement leakage rate, and radiation exposure time. The authors compared navigated and nonnavigated needle placement in 30 balloon kyphoplasty procedures (47 levels). Intraoperative 3-dimensional image-based navigation was used for needle placement in 21 cases (36 levels); conventional 2-dimensional fluoroscopy was used in the other 9 cases (11 levels). The 2 groups were compared for rates of needle malposition and cement leakage as well as radiation exposure time. Three of 11 (27%) nonnavigated cases were complicated by a malpositioned needle, and 2 of these had to be repositioned. The navigated group had a significantly lower malposition rate (1 of 36; 3%; P=.04). The overall rate of cement leakage was also similar in both groups (P=.29). Radiation exposure time was similar in both groups (navigated, 98 s/level; nonnavigated, 125 s/level; P=.10). Navigated kyphoplasty procedures did not differ significantly from nonnavigated procedures except in terms of needle malposition rate, where navigation may have decreased the need for needle repositioning.

  7. A 3-dimensional model for teaching local flaps using porcine skin.

    PubMed

    Hassan, Zahid; Hogg, Fiona; Graham, Ken

    2014-10-01

    The European Working Time Directive and streamlined training has led to reduced training time. Surgery, as an experience-dependent craft specialty is affected more than other medical specialties. Trainees want to maximize all training opportunities in the clinical setting, and having predeveloped basic skills acquired on a simulated model can facilitate this.Here we describe the use of a novel model to design and raise local flaps in the face and scalp regions. The model consists of mannequin heads draped with porcine skin which is skewered with pins at strategic points to give a 3-dimensional model which closely resembles a cadaveric head.The advantages of this model are that it is life size and incorporates all the relevant anatomical features, which can be drawn on if required.This model was used on a recent course, Intermediate Skills in Plastic Surgery: Flaps Around the Face, at the Royal College of Surgeons England. The trainees found that practicing on the porcine skin gave them an opportunity to master the basics of flap design and implementation.In summary, this innovative 3-dimensional training model has received high levels of satisfaction and is currently as close as we can get to cadaveric dissection without the constraints and cost of using human tissue.

  8. Simple parameter estimation for complex models — Testing evolutionary techniques on 3-dimensional biogeochemical ocean models

    NASA Astrophysics Data System (ADS)

    Mattern, Jann Paul; Edwards, Christopher A.

    2017-01-01

    Parameter estimation is an important part of numerical modeling and often required when a coupled physical-biogeochemical ocean model is first deployed. However, 3-dimensional ocean model simulations are computationally expensive and models typically contain upwards of 10 parameters suitable for estimation. Hence, manual parameter tuning can be lengthy and cumbersome. Here, we present four easy to implement and flexible parameter estimation techniques and apply them to two 3-dimensional biogeochemical models of different complexities. Based on a Monte Carlo experiment, we first develop a cost function measuring the model-observation misfit based on multiple data types. The parameter estimation techniques are then applied and yield a substantial cost reduction over ∼ 100 simulations. Based on the outcome of multiple replicate experiments, they perform on average better than random, uninformed parameter search but performance declines when more than 40 parameters are estimated together. Our results emphasize the complex cost function structure for biogeochemical parameters and highlight dependencies between different parameters as well as different cost function formulations.

  9. Automated 3-Dimensional Brain Atlas Fitting to Microelectrode Recordings from Deep Brain Stimulation Surgeries

    PubMed Central

    Luján, J. Luis; Noecker, Angela M.; Butson, Christopher R.; Cooper, Scott E.; Walter, Benjamin L.; Vitek, Jerrold L.; McIntyre, Cameron C.

    2009-01-01

    Objective Deep brain stimulation (DBS) surgeries commonly rely on brain atlases and microelectrode recordings (MER) to help identify the target location for electrode implantation. We present an automated method for optimally fitting a 3-dimensional brain atlas to intraoperative MER and predicting a target DBS electrode location in stereotactic coordinates for the patient. Methods We retrospectively fit a 3-dimensional brain atlas to MER points from 10 DBS surgeries targeting the subthalamic nucleus (STN). We used a constrained optimization algorithm to maximize the MER points correctly fitted (i.e., contained) within the appropriate atlas nuclei. We compared our optimization approach to conventional anterior commissure-posterior commissure (AC/PC) scaling, and to manual fits performed by four experts. A theoretical DBS electrode target location in the dorsal STN was customized to each patient as part of the fitting process and compared to the location of the clinically defined therapeutic stimulation contact. Results The human expert and computer optimization fits achieved significantly better fits than the AC/PC scaling (80, 81, and 41% of correctly fitted MER, respectively). However, the optimization fits were performed in less time than the expert fits and converged to a single solution for each patient, eliminating interexpert variance. Conclusions and Significance DBS therapeutic outcomes are directly related to electrode implantation accuracy. Our automated fitting techniques may aid in the surgical decision-making process by optimally integrating brain atlas and intraoperative neurophysiological data to provide a visual guide for target identification. PMID:19556832

  10. High resolution 3-Dimensional imaging of the human cardiac conduction system from microanatomy to mathematical modeling.

    PubMed

    Stephenson, Robert S; Atkinson, Andrew; Kottas, Petros; Perde, Filip; Jafarzadeh, Fatemeh; Bateman, Mike; Iaizzo, Paul A; Zhao, Jichao; Zhang, Henggui; Anderson, Robert H; Jarvis, Jonathan C; Dobrzynski, Halina

    2017-08-03

    Cardiac arrhythmias and conduction disturbances are accompanied by structural remodelling of the specialised cardiomyocytes known collectively as the cardiac conduction system. Here, using contrast enhanced micro-computed tomography, we present, in attitudinally appropriate fashion, the first 3-dimensional representations of the cardiac conduction system within the intact human heart. We show that cardiomyocyte orientation can be extracted from these datasets at spatial resolutions approaching the single cell. These data show that commonly accepted anatomical representations are oversimplified. We have incorporated the high-resolution anatomical data into mathematical simulations of cardiac electrical depolarisation. The data presented should have multidisciplinary impact. Since the rate of depolarisation is dictated by cardiac microstructure, and the precise orientation of the cardiomyocytes, our data should improve the fidelity of mathematical models. By showing the precise 3-dimensional relationships between the cardiac conduction system and surrounding structures, we provide new insights relevant to valvar replacement surgery and ablation therapies. We also offer a practical method for investigation of remodelling in disease, and thus, virtual pathology and archiving. Such data presented as 3D images or 3D printed models, will inform discussions between medical teams and their patients, and aid the education of medical and surgical trainees.

  11. A new 3-dimensional dynamic quantitative analysis system of facial motion: an establishment and reliability test.

    PubMed

    Feng, Guodong; Zhao, Yang; Tian, Xu; Gao, Zhiqiang

    2014-01-01

    This study aimed to establish a 3-dimensional dynamic quantitative facial motion analysis system, and then determine its accuracy and test-retest reliability. The system could automatically reconstruct the motion of the observational points. Standardized T-shaped rod and L-shaped rods were used to evaluate the static and dynamic accuracy of the system. Nineteen healthy volunteers were recruited to test the reliability of the system. The average static distance error measurement was 0.19 mm, and the average angular error was 0.29°. The measuring results decreased with the increase of distance between the cameras and objects, 80 cm of which was considered to be optimal. It took only 58 seconds to perform the full facial measurement process. The average intra-class correlation coefficient for distance measurement and angular measurement was 0.973 and 0.794 respectively. The results demonstrated that we successfully established a practical 3-dimensional dynamic quantitative analysis system that is accurate and reliable enough to meet both clinical and research needs.

  12. Influence of different setups of the Frankfort horizontal plane on 3-dimensional cephalometric measurements.

    PubMed

    Santos, Rodrigo Mologni Gonçalves Dos; De Martino, José Mario; Haiter Neto, Francisco; Passeri, Luis Augusto

    2017-08-01

    The Frankfort horizontal (FH) is a plane that intersects both porions and the left orbitale. However, other combinations of points have also been used to define this plane in 3-dimensional cephalometry. These variations are based on the hypothesis that they do not affect the cephalometric analysis. We investigated the validity of this hypothesis. The material included cone-beam computed tomography data sets of 82 adult subjects with Class I molar relationship. A third-party method of cone-beam computed tomography-based 3-dimensional cephalometry was performed using 7 setups of the FH plane. Six lateral cephalometric hard tissue measurements relative to the FH plane were carried out for each setup. Measurement differences were calculated for each pair of setups of the FH plane. The number of occurrences of differences greater than the limits of agreement was counted for each of the 6 measurements. Only 3 of 21 pairs of setups had no occurrences for the 6 measurements. No measurement had no occurrences for the 21 pairs of setups. Setups based on left or right porion and both orbitales had the greatest number of occurrences for the 6 measurements. This investigation showed that significant and undesirable measurement differences can be produced by varying the definition of the FH plane. Copyright © 2017 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.

  13. Quantitative 3-Dimensional Imaging of Murine Neointimal and Atherosclerotic Lesions by Optical Projection Tomography

    PubMed Central

    Kirkby, Nicholas S.; Low, Lucinda; Seckl, Jonathan R.; Walker, Brian R.; Webb, David J.; Hadoke, Patrick W. F.

    2011-01-01

    Objective Traditional methods for the analysis of vascular lesion formation are labour intensive to perform - restricting study to ‘snapshots’ within each vessel. This study was undertaken to determine the suitability of optical projection tomographic (OPT) imaging for the 3-dimensional representation and quantification of intimal lesions in mouse arteries. Methods and Results Vascular injury was induced by wire-insertion or ligation of the mouse femoral artery or administration of an atherogenic diet to apoE-deficient mice. Lesion formation was examined by OPT imaging of autofluorescent emission. Lesions could be clearly identified and distinguished from the underlying vascular wall. Planimetric measurements of lesion area correlated well with those made from histological sections subsequently produced from the same vessels (wire-injury: R2 = 0.92; ligation-injury: R2 = 0.89; atherosclerosis: R2 = 0.85), confirming both the accuracy of this methodology and its non-destructive nature. It was also possible to record volumetric measurements of lesion and lumen and these were highly reproducible between scans (coefficient of variation = 5.36%, 11.39% and 4.79% for wire- and ligation-injury and atherosclerosis, respectively). Conclusions These data demonstrate the eminent suitability of OPT for imaging of atherosclerotic and neointimal lesion formation, providing a much needed means for the routine 3-dimensional analysis of vascular morphology in studies of this type. PMID:21379578

  14. A New 3-Dimensional Dynamic Quantitative Analysis System of Facial Motion: An Establishment and Reliability Test

    PubMed Central

    Feng, Guodong; Zhao, Yang; Tian, Xu; Gao, Zhiqiang

    2014-01-01

    This study aimed to establish a 3-dimensional dynamic quantitative facial motion analysis system, and then determine its accuracy and test-retest reliability. The system could automatically reconstruct the motion of the observational points. Standardized T-shaped rod and L-shaped rods were used to evaluate the static and dynamic accuracy of the system. Nineteen healthy volunteers were recruited to test the reliability of the system. The average static distance error measurement was 0.19 mm, and the average angular error was 0.29°. The measuring results decreased with the increase of distance between the cameras and objects, 80 cm of which was considered to be optimal. It took only 58 seconds to perform the full facial measurement process. The average intra-class correlation coefficient for distance measurement and angular measurement was 0.973 and 0.794 respectively. The results demonstrated that we successfully established a practical 3-dimensional dynamic quantitative analysis system that is accurate and reliable enough to meet both clinical and research needs. PMID:25390881

  15. Endothelial cells assemble into a 3-dimensional prevascular network in a bone tissue engineering construct.

    PubMed

    Rouwkema, Jeroen; de Boer, Jan; Van Blitterswijk, Clemens A

    2006-09-01

    To engineer tissues with clinically relevant dimensions, one must overcome the challenge of rapidly creating functional blood vessels to supply cells with oxygen and nutrients and to remove waste products. We tested the hypothesis that endothelial cells, cocultured with osteoprogenitor cells, can organize into a prevascular network in vitro. When cultured in a spheroid coculture model with human mesenchymal stem cells, human umbilical vein endothelial cells (HUVECs) form a 3-dimensional prevascular network within 10 days of in vitro culture. The formation of the prevascular network was promoted by seeding 2% or fewer HUVECs. Moreover, the addition of endothelial cells resulted in a 4-fold upregulation of the osteogenic marker alkaline phosphatase. The addition of mouse embryonic fibroblasts did not result in stabilization of the prevascular network. Upon implantation, the prevascular network developed further and structures including lumen could be seen regularly. However, anastomosis with the host vasculature was limited. We conclude that endothelial cells are able to form a 3-dimensional (3D) prevascular network in vitro in a bone tissue engineering setting. This finding is a strong indication that in vitro prevascularization is a promising strategy to improve implant vascularization in bone tissue engineering.

  16. Gene transfer with HSP 70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis.

    PubMed

    Grossin, Laurent; Cournil-Henrionnet, Christel; Pinzano, Astrid; Gaborit, Nadège; Dumas, Dominique; Etienne, Stéphanie; Stoltz, Jean François; Terlain, Bernard; Netter, Patrick; Mir, Lluis M; Gillet, Pierre

    2006-01-01

    Osteoarthritis is characterized by a gradual degradation of extracellular matrix, resulting from an excess of chondrocyte cell death, mainly due to an increase in apoptotis. Recent studies have revealed the essential role of HSP70 in protecting cells from stressful stimuli. Therefore, overexpressing HSP70 in chondrocytes could represent a good strategy to prevent extracellular matrix destruction. To this end, we have developed a vector carrying HSP70/GFP, and transduced chondrocytes were thus more resistant to cell death induced by mono-iodoacetate (MIA). To overcome the barrier-effect of matrix, we investigated the efficacy of plasmid delivery by electroporation (EP) in rat patellar cartilage. Two days after EP, 50% of patellar chondrocytes were HSP/GFP+. After 3 months, long-term expression of transgene was only depicted in the deep layer (20-30% positive cells). HSP70 overexpression inhibited the natural endochondral ossification in the deep layer, thus leading to a lesser decrease in chondrocyte distribution. Moreover, overexpression of HSP70, after a preventive EP transfer in rat patella, was sufficient to decrease the severity of osteoarthritis-induced lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of HSP70, through EP delivery, could protect chondrocytes from cellular injuries and thus might be a novel chondroprotective modality in rat OA.

  17. Culture of Primary Bovine Chondrocytes on a Continuously Expanding Surface Inhibits Dedifferentiation

    PubMed Central

    Rosenzweig, Derek H.; Matmati, Mourad; Khayat, Ghazaleh; Chaudhry, Sidharth; Hinz, Boris

    2012-01-01

    Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and α-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies. PMID:22738340

  18. Quantitative analysis of gene expression in human articular chondrocytes assigned for autologous implantation.

    PubMed

    Barlic, Ariana; Drobnic, Matej; Malicev, Elvira; Kregar-Velikonja, Nevenka

    2008-06-01

    Autologous chondrocyte implantation (ACI) relies on the implantation of in vitro expanded cells. The aim was to study the dedifferentiation of human articular chondrocytes under different cultivating conditions [days 0-10 in the primary culture (P0); passages in a monolayer from P0 to P3; monolayer vs. alginate and monolayer vs. alginate/agarose hydrogels] using real-time PCR analysis. The relative gene expressions for collagen type I and II, aggrecan and versican were quantified and the corresponding differentiation indexes (Col2/Col1, Agr/Ver) were calculated. The values of both differentiation indexes decreased exponentially with time in the P0 monolayer culture, and continued with a significant decrease over the subsequent monolayer passages. On the contrary, the chondrocytes seeded in either of the hydrogels significantly increased the indexes compared to their parallel monolayer cultures. These results indicate that alginate and alginate/agarose hydrogels offer an appropriate environment for human articular chondrocytes to redifferentiate after being expanded in vitro. Therefore the three-dimensional (3D) hydrogel chondrocyte cultures present not only surgical, but also biological advantage over the classic suspension-periosteum chondrocyte implantation. (c) 2008 Orthopaedic Research Society.

  19. Formation of cartilage in vivo with immobilized autologous rabbit auricular cultured chondrocytes in collagen matrices.

    PubMed

    Sanz, Ernesto; Peñas, Lucas; Lequerica, Juan L

    2007-05-01

    The availability of generated cartilage de novo is one of the needs of reconstructive surgery. In this study, the authors constructed a matrix formed by autologous immobilized chondrocytes using collagen gel as a scaffold. Furthermore, the ability of these matrices to engraft and generate new cartilage was examined. Biopsy specimens of elastic cartilage were surgically obtained from the ears of eight New Zealand White rabbits. After collagenase II digestion of cartilage, chondrocytes were isolated and propagated in culture medium. Chondrocytes were immobilized into bovine collagen lattices and implanted, replacing pieces of removed native cartilage. Five weeks after implantation, the rabbits were killed and the ears were examined macroscopically and analyzed by means of histochemical methods. The results show the formation of new cartilage from implanted lattices with chondrocytes. Gross analysis of the ears shows similarities in appearance, consistency, texture, and histology between native and new cartilage. Fluorescence of the nucleus from bisbenzimide-labeled chondrocytes was detected in newly formed tissue, pointing out its in vitro culture origin. No signs of an inflammatory reaction attributable to implants were found in either the control or the chondrocyte lattices. The authors suggest that this approach is of value for future clinical use.

  20. Culture of primary bovine chondrocytes on a continuously expanding surface inhibits dedifferentiation.

    PubMed

    Rosenzweig, Derek H; Matmati, Mourad; Khayat, Ghazaleh; Chaudhry, Sidharth; Hinz, Boris; Quinn, Thomas M

    2012-12-01

    Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and α-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies.

  1. In vitro isolation and cultivation of human chondrocytes for osteoarthritis renovation.

    PubMed

    Xu, Jiaming; Zhang, Changqing

    2014-08-01

    The purpose of this study was to evaluate the repair effects of chondrocytes that were cultured in vitro on osteoarthritis (OA). Chondrocytes were isolated from fetal rabbits and cultured in Biosilon microcarriers. Sixty rabbits were randomly divided into three groups equally (blank group, model group, treatment group). The rabbit knee OA model was established by inducing papain. Rabbits in the treatment group were injected with the chondrocytes that were cultured in vitro. Hematoxylin-eosin (HE) staining and gross morphologic observation were conducted. Expression level of cytokines such as IL-1bβ, IL-6, and TNF-α in cartilage synovial cells was also analyzed by an ELISA assay. The cultured chondrocyte was validated by a positive stain of type II collagen and vimentin by immunofluorescence. Compared to the model group, the articular cartilage of the rabbit knee in the treatment group showed a normal color, smooth surface, and none of malacia and coloboma. HE staining indicated that the articular surface of the treatment group tended to be smooth and flat; the matrix stained tinge and the cartilage destruction and fiber hyperplasia of the synovia were lightened. The expression levels of IL-1bβ, IL-6, and TNF-α also declined in the treatment group. OA symptoms were improved by treating with chondrocytes. In summary, the animal experiment in the present study indicated that chondrocyte injection played an active effect on renovation of OA.

  2. Chondrocyte Culture in Three Dimensional Alginate Sulfate Hydrogels Promotes Proliferation While Maintaining Expression of Chondrogenic Markers

    PubMed Central

    Mhanna, Rami; Kashyap, Aditya; Palazzolo, Gemma; Vallmajo-Martin, Queralt; Becher, Jana; Möller, Stephanie; Schnabelrauch, Matthias

    2014-01-01

    The loss of expression of chondrogenic markers during monolayer expansion remains a stumbling block for cell-based treatment of cartilage lesions. Here, we introduce sulfated alginate hydrogels as a cartilage biomimetic biomaterial that induces cell proliferation while maintaining the chondrogenic phenotype of encapsulated chondrocytes. Hydroxyl groups of alginate were converted to sulfates by incubation with sulfur trioxide–pyridine complex (SO3/pyridine), yielding a sulfated material cross-linkable with calcium chloride. Passage 3 bovine chondrocytes were encapsulated in alginate and alginate sulfate hydrogels for up to 35 days. Cell proliferation was five-fold higher in alginate sulfate compared with alginate (p=0.038). Blocking beta1 integrins in chondrocytes within alginate sulfate hydrogels significantly inhibited proliferation (p=0.002). Sulfated alginate increased the RhoA activity of chondrocytes compared with unmodified alginate, an increase that was blocked by β1 blocking antibodies (p=0.017). Expression and synthesis of type II collagen, type I collagen, and proteoglycan was not significantly affected by the encapsulation material evidenced by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Alginate sulfate constructs showed an opaque appearance in culture, whereas the unmodified alginate samples remained translucent. In conclusion, alginate sulfate provides a three dimensional microenvironment that promotes both chondrocyte proliferation and maintenance of the chondrogenic phenotype and represents an important advance for chondrocyte-based cartilage repair therapies providing a material in which cell expansion can be done in situ. PMID:24320935

  3. Effects of manganese deficiency on chondrocyte development in tibia growth plate of Arbor Acres chicks.

    PubMed

    Wang, Jian; Wang, Zhen Yong; Wang, Zhao Jun; Liu, Ran; Liu, Shao Qiong; Wang, Lin

    2015-01-01

    The aim of this study was to investigate the effects of manganese (Mn) deficiency on chondrocyte development in tibia growth plate. Ninety 1-day-old Arbor Acres chicks were randomly divided into three groups and fed on control diet (60 mg Mn/kg diet) and manganese deficient diets (40 mg Mn/kg diet, manganese deficiency group I; 8.7 mg Mn/kg diet, manganese deficiency group II), respectively. The width of the proliferative zone of growth plate was measured by the microscope graticule. Chondrocyte apoptosis was estimated by TUNEL staining. Gene expression of p21 and Bcl-2, and expression of related proteins were analyzed by quantitative real time reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Compared with the control group, manganese deficiency significantly decreased the proliferative zone width and Bcl-2 mRNA expression level, while significantly increased the apoptotic rates and the expression level of p21 gene in chondrocytes. The results indicate that manganese deficiency had a negative effect on chondrocyte development, which was mediated by the inhibition of chondrocyte proliferation and promotion of chondrocyte apoptosis.

  4. Toxicity of antiseptics against chondrocytes: what is best for the cartilage in septic joint surgery?

    PubMed

    Röhner, Eric; Kolar, Paula; Seeger, Joern B; Arnholdt, Joerg; Thiele, Kathi; Perka, Carsten; Matziolis, Georg

    2011-11-01

    In septic joint surgery, the most frequently used antiseptics are polyhexanide, hydrogen peroxide and taurolidine. The aim of this study was to examine the effects of these antiseptics on viability of human chondrocytes. Our hypothesis was that antiseptics and supplemental irrigation with sodium chloride lavage are less toxic on human chondrocytes than treatment with antiseptics only. Primary human chondrocytes were isolated and cultured from six donated human knee joints. Polyhexanide, hydrogen peroxide or taurolidine were added to the cultures. Toxicity analysis was performed by visualisation of cell structure using light microscopy and LDH activity. The determination of vital cells and total cell numbers of chondrocytes treated with antiseptics partly followed by irrigation with sodium chloride solution was performed by using Casy Cell-Counter. Light microscopic data revealed a defect in cell structure after addition of antiseptics. We showed a significant increase of LDH enzyme activity after the treatment with polyhexanide or taurolidine. After treatment with antiseptics followed by sodium chloride solution a significant increase of vital and total cell numbers resulted in comparison with the chondrocytes that were only treated with antiseptics. The data show that treatment with polyhexanid, hydrogen peroxide or taurolidine induces cell death of human chondroctes in vitro. The application of sodium chloride solution after the treatment with polyhexanide and hydrogen peroxide possibly has a protective effect on chondrocyte viability.

  5. Opiates do not violate the viability and proliferative activity of human articular chondrocytes.

    PubMed

    Chechik, Ofir; Arbel, Ron; Salai, Moshe; Gigi, Roy; Beilin, Mark; Flaishon, Ron; Sever, Ronen; Khashan, Morsi; Ben-Tov, Tomer; Gal-Levy, Ronit; Yayon, Avner; Blumenstein, Sara

    2014-09-01

    Articular cartilage injuries present a challenge for the clinician. Autologous chondrocyte implantation embedded in scaffolds are used to treat cartilage defects with favorable outcomes. Autologous serum is often used as a medium for chondrocyte cell culture during the proliferation phase of the process of such products. A previous report showed that opiate analgesics (fentanyl, alfentanil and diamorphine) in the sera have a significant inhibitory effect on chondrocyte proliferation. In order to determine if opiates in serum inhibit chondrocyte proliferation, twenty two patients who underwent knee arthroscopy and were anesthetized with either fentanyl or remifentanil were studied. Blood was drawn before and during opiate administration and up to 2 h after its discontinuation. The sera were used as medium for in vitro proliferation of both cryopreserved and freshly isolated chondrocytes, and the number and viability of cells were measured. There was no difference in the yield or cell viability between the serum samples of patients anesthetized with fentanyl when either fresh or cryopreserved human articular chondrocytes (hACs) were used. Some non-significant reduction in the yield of cells was observed in the serum samples of patients anesthetized with remifentanil when fresh hAC were used. We conclude that Fentanyl in human autologous serum does not inhibit in vitro hAC proliferation. Remifentanil may show minimal inhibitory effect on in vitro fresh hAC proliferation.

  6. Calcium signaling in response to fluid flow by chondrocytes in 3D alginate culture.

    PubMed

    Degala, Satish; Williams, Rebecca; Zipfel, Warren; Bonassar, Lawrence J

    2012-05-01

    Quantifying the effects of mechanical loading on the metabolic response of chondrocytes is difficult due to complicated structure of cartilage ECM and the coupled nature of the mechanical stimuli presented to the cells. In this study we describe the effects of fluid flow, particularly hydrostatic pressure and wall shear stress, on the Ca(2+) signaling response of bovine articular chondrocytes in 3D culture. Using well-established alginate hydrogel system to maintain spherical chondrocyte morphology, we altered solid volume fraction to change scaffold mechanics. Fluid velocities in the bulk of the scaffolds were directly measured via an optical technique and scaffold permeability and aggregate modulus was characterized to quantify the mechanical stimuli presented to cells. Ca(2+) signaling response to direct perfusion of chondrocyte-seeded scaffolds increased monotonically with flow rate and was found more directly dependent on fluid velocity rather than shear stress or hydrostatic pressure. Chondrocytes in alginate scaffolds responded to fluid flow at velocities and shear stresses 2-3 orders of magnitude lower than seen in previous monolayer studies. Our data suggest that flow-induced Ca(2+) signaling response of chondrocytes in alginate culture may be due to mechanical signaling pathways, which is influenced by the 3D nature of cell shape.

  7. Passaged Adult Chondrocytes Can Form Engineered Cartilage with Functional Mechanical Properties: A Canine Model

    PubMed Central

    Ng, Kenneth W.; Lima, Eric G.; Bian, Liming; O'Conor, Christopher J.; Jayabalan, Prakash S.; Stoker, Aaron M.; Kuroki, Keiichi; Cook, Cristi R.; Ateshian, Gerard A.; Cook, James L.

    2010-01-01

    It was hypothesized that previously optimized serum-free culture conditions for juvenile bovine chondrocytes could be adapted to generate engineered cartilage with physiologic mechanical properties in a preclinical, adult canine model. Primary or passaged (using growth factors) adult chondrocytes from three adult dogs were encapsulated in agarose, and cultured in serum-free media with transforming growth factor-β3. After 28 days in culture, engineered cartilage formed by primary chondrocytes exhibited only small increases in glycosaminoglycan content. However, all passaged chondrocytes on day 28 elaborated a cartilage matrix with compressive properties and glycosaminoglycan content in the range of native adult canine cartilage values. A preliminary biocompatibility study utilizing chondral and osteochondral constructs showed no gross or histological signs of rejection, with all implanted constructs showing excellent integration with surrounding cartilage and subchondral bone. This study demonstrates that adult canine chondrocytes can form a mechanically functional, biocompatible engineered cartilage tissue under optimized culture conditions. The encouraging findings of this work highlight the potential for tissue engineering strategies using adult chondrocytes in the clinical treatment of cartilage defects. PMID:19845465

  8. Expression of osteogenic proteins during the intrasplenic transplantation of Meckel's chondrocytes: A histochemical and immunohistochemical study.

    PubMed

    Ishizeki, Kiyoto; Kagiya, Tadayoshi; Fujiwara, Naoki; Otsu, Keishi; Harada, Hidemitsu

    2009-03-01

    Meckel's chondrocytes, derived from the ectomesenchyme, have the potential to transform into other phenotypes. In this study, we transplanted cell pellets of Meckel's chondrocytes into isogenic mouse spleens and analyzed their phenotypic transformation into osteogenic cells using histological and immunohistochemical methods. With the increasing duration of transplantation, chondrocytes were incorporated into splenic tissues and formed a von Kossa-positive calcified matrix containing calcium and phosphoric acid, similar to that of intact bone. Type I, II, and X collagens, and the bone-marker proteins osteocalcin, osteopontin, osteonectin, and bone morphogenetic protein-2 (BMP-2) were immunolocalized in the matrix formed by the transplanted chondrocytes. Osteopontin and osteonectin were detected in the calcified matrix at earlier stages than osteocalcin and BMP-2. Type II collagen was expressed during the first week of transplantation, and type X collagen-positive cells appeared scattered during the initial stage of calcification, these collagens being later replaced by type I collagen formed by osteocyte-like cells. Electron microscopic observations revealed that chondrocytes surrounded by the calcified matrix transformed into spindle-shaped osteocytic cells accompanying the formation of bone-type thick-banded collagen fibrils. These results suggest that phenotypic switching of Meckel's chondrocytes can occur under in vivo conditions at a cellular morphological level.

  9. Non-woven PGA/PVA fibrous mesh as an appropriate scaffold for chondrocyte proliferation.

    PubMed

    Rampichová, M; Koštáková, E; Filová, E; Prosecká, E; Plencner, M; Ocheretná, L; Lytvynets, A; Lukáš, D; Amler, E

    2010-01-01

    Non-woven textile mesh from polyglycolic acid (PGA) was found as a proper material for chondrocyte adhesion but worse for their proliferation. Neither hyaluronic acid nor chitosan nor polyvinyl alcohol (PVA) increased chondrocyte adhesion. However, chondrocyte proliferation suffered from acidic byproducts of PGA degradation. However, the addition of PVA and/or chitosan into a wet-laid non-woven textile mesh from PGA improved chondrocyte proliferation seeded in vitro on the PGA-based composite scaffold namely due to a diminished acidification of their microenvironment. This PVA/PGA composite mesh used in combination with a proper hydrogel minimized the negative effect of PGA degradation without dropping positive parameters of the PGA wet-laid non-woven textile mesh. In fact, presence of PVA and/or chitosan in the PGA-based wet-laid non-woven textile mesh even advanced the PGA-based wet-laid non-woven textile mesh for chondrocyte seeding and artificial cartilage production due to a positive effect of PVA in such a scaffold on chondrocyte proliferation.

  10. Chitosan Enriched Three-Dimensional Matrix Reduces Inflammatory and Catabolic Mediators Production by Human Chondrocytes

    PubMed Central

    Oprenyeszk, Frederic; Sanchez, Christelle; Dubuc, Jean-Emile; Maquet, Véronique; Henrist, Catherine; Compère, Philippe; Henrotin, Yves

    2015-01-01

    This in vitro study investigated the metabolism of human osteoarthritic (OA) chondrocytes encapsulated in a spherical matrix enriched of chitosan. Human OA chondrocytes were encapsulated and cultured for 28 days either in chitosan-alginate beads or in alginate beads. The beads were formed by slowly passing dropwise either the chitosan 0.6%–alginate 1.2% or the alginate 1.2% solution through a syringe into a 102 mM CaCl2 solution. Beads were analyzed histologically after 28 days. Interleukin (IL)-6 and -8, prostaglandin (PG) E2, matrix metalloproteinases (MMPs), hyaluronan and aggrecan were quantified directly in the culture supernatant by specific ELISA and nitric oxide (NO) by using a colorimetric method based on the Griess reaction. Hematoxylin and eosin staining showed that chitosan was homogeneously distributed through the matrix and was in direct contact with chondrocytes. The production of IL-6, IL-8 and MMP-3 by chondrocytes significantly decreased in chitosan-alginate beads compared to alginate beads. PGE2 and NO decreased also significantly but only during the first three days of culture. Hyaluronan and aggrecan production tended to increase in chitosan-alginate beads after 28 days of culture. Chitosan-alginate beads reduced the production of inflammatory