Collagen-grafted porous HDPE/PEAA scaffolds for bone reconstruction.

PubMed

Kim, Chang-Shik; Jung, Kyung-Hye; Kim, Hun; Kim, Chan-Bong; Kang, Inn-Kyu

2016-01-01

After tumor resection, bone reconstruction such as skull base reconstruction using interconnected porous structure is absolutely necessary. In this study, porous scaffolds for bone reconstruction were prepared using heat-pressing and salt-leaching methods. High-density polyethylene (HDPE) and poly(ethylene-co-acrylic acid) (PEAA) were chosen as the polymer composites for producing a porous scaffold of high mechanical strength and having high reactivity with biomaterials such as collagen, respectively. The porous structure was observed through surface images, and its intrusion volume and porosity were measured. Owing to the carboxylic acids on PEAA, collagen was successfully grafted onto the porous HDPE/PEAA scaffold, which was confirmed by FT-IR spectroscopy and electron spectroscopy for chemical analysis. Osteoblasts were cultured on the collagen-grafted porous scaffold, and their adhesion, proliferation, and differentiation were investigated. The high viability and growth of the osteoblasts suggest that the collagen-grafted porous HDPE/PEAA is a promising scaffold material for bone generation.

Collagen Self-Assembly on Orthopedic Magnesium Biomaterials Surface and Subsequent Bone Cell Attachment

PubMed Central

Zhao, Nan; Zhu, Donghui

2014-01-01

Magnesium (Mg) biomaterials are a new generation of biodegradable materials and have promising potential for orthopedic applications. After implantation in bone tissues, these materials will directly interact with extracellular matrix (ECM) biomolecules and bone cells. Type I collagen, the major component of bone ECM, forms the architecture scaffold that provides physical support for bone cell attachment. However, it is still unknown how Mg substrate affects collagen assembly on top of it as well as subsequent cell attachment and growth. Here, we studied the effects of collagen monomer concentration, pH, assembly time, and surface roughness of two Mg materials (pure Mg and AZ31) on collagen fibril formation. Results showed that formation of fibrils would not initiate until the monomer concentration reached a certain level depending on the type of Mg material. The thickness of collagen fibril increased with the increase of assembly time. The structures of collagen fibrils formed on semi-rough surfaces of Mg materials have a high similarity to that of native bone collagen. Next, cell attachment and growth after collagen assembly were examined. Materials with rough surface showed higher collagen adsorption but compromised bone cell attachment. Interestingly, surface roughness and collagen structure did not affect cell growth on AZ31 for up to a week. Findings from this work provide some insightful information on Mg-tissue interaction at the interface and guidance for future surface modifications of Mg biomaterials. PMID:25303459

Bone Collagen: New Clues to its Mineralization Mechanism From Recessive Osteogenesis Imperfecta

PubMed Central

Eyre, David R.; Ann Weis, Mary

2013-01-01

Until 2006 the only mutations known to cause osteogenesis imperfecta (OI) were in the two genes coding for type I collagen chains. These dominant mutations affecting the expression or primary sequence of collagen α1(I) and α2(I) chains account for over 90% of OI cases. Since then a growing list of mutant genes causing the 5–10% of recessive cases has rapidly emerged. They include CRTAP, LEPRE1 and PPIB, which encode three proteins forming the prolyl 3-hydroxylase complex; PLOD2 and FKBP10, which encode respectively lysyl hydroxylase 2 and a foldase required for its activity in forming mature cross-links in bone collagen; SERPIN H1, which encodes the collagen chaperone HSP47; SERPIN F1, which encodes pigment epithelium-derived factor required for osteoid mineralization; and BMP1, which encodes the type I procollagen C-propeptidase. All cause fragile bone in infancy, which can include over-mineralization or under-mineralization defects as well as abnormal collagen post-translational modifications. Consistently both dominant and recessive variants lead to abnormal cross-linking chemistry in bone collagen. These recent discoveries strengthen the potential for a common pathogenic mechanism of misassembled collagen fibrils. Of the new genes identified, eight encode proteins required for collagen post-translational modification, chaperoning of newly synthesized collagen chains into native molecules or transport through the endoplasmic reticulum and Golgi for polymerization, cross-linking and mineralization. In reviewing these findings, we conclude that a common theme is emerging in the pathogenesis of brittle bone disease of mishandled collagen assembly with important insights on post-translational features of bone collagen that have evolved to optimize it as a biomineral template. PMID:23508630

Distribution of Type I Collagen Morphologies in Bone: Relation to Estrogen Depletion

PubMed Central

Wallace, Joseph M.; Erickson, Blake; Les, Clifford M.; Orr, Bradford G.; Holl, Mark M. Banaszak

2009-01-01

Bone is an amazing material evolved by nature to elegantly balance structural and metabolic needs in the body. Bone health is an integral part of overall health, but our lack of understanding of the ultrastructure of healthy bone precludes us from knowing how disease may impact nanoscale properties in this biological material. Here, we show that quantitative assessments of a distribution of Type I collagen fibril morphologies can be made using atomic force microscopy (AFM). We demonstrate that normal bone contains a distribution of collagen fibril morphologies and that changes in this distribution can be directly related to disease state. Specifically, by monitoring changes in the collagen fibril distribution of sham-operated and estrogen-depleted sheep, we have shown the ability to detect estrogen-deficiency-induced changes in Type I collagen in bone. This discovery provides new insight into the ultrastructure of bone as a tissue and the role of material structure in bone disease. The observation offers the possibility of a much-needed in vitro procedure to complement the current methods used to diagnose osteoporosis and other bone disease. PMID:19932773

Guided Bone Regeneration Using Collagen Scaffolds, Growth Factors, and Periodontal Ligament Stem Cells for Treatment of Peri-Implant Bone Defects In Vivo.

PubMed

Kämmerer, Peer W; Scholz, Malte; Baudisch, Maria; Liese, Jan; Wegner, Katharina; Frerich, Bernhard; Lang, Hermann

2017-01-01

The aim of the study was an evaluation of different approaches for guided bone regeneration (GBR) of peri-implant defects in an in vivo animal model. In minipigs ( n = 15), peri-implant defects around calcium phosphate- (CaP-; n = 46) coated implants were created and randomly filled with (1) blank, (2) collagen/hydroxylapatite/ β -tricalcium phosphate scaffold (CHT), (3) CHT + growth factor cocktail (GFC), (4) jellyfish collagen matrix, (5) jellyfish collagen matrix + GFC, (6) collagen powder, and (7) collagen powder + periodontal ligament stem cells (PDLSC). Additional collagen membranes were used for coverage of the defects. After 120 days of healing, bone growth was evaluated histologically (bone to implant contact (BIC;%)), vertical bone apposition (VBA; mm), and new bone height (NBH; %). In all groups, new bone formation was seen. Though, when compared to the blank group, no significant differences were detected for all parameters. BIC and NBH in the group with collagen matrix as well as the group with the collagen matrix + GFC were significantly less when compared to the collagen powder group (all: p < 0.003). GBR procedures, in combination with CaP-coated implants, will lead to an enhancement of peri-implant bone growth. There was no additional significant enhancement of osseous regeneration when using GFC or PDLSC.

Glycation Contributes to Interaction Between Human Bone Alkaline Phosphatase and Collagen Type I.

PubMed

Halling Linder, Cecilia; Enander, Karin; Magnusson, Per

2016-03-01

Bone is a biological composite material comprised primarily of collagen type I and mineral crystals of calcium and phosphate in the form of hydroxyapatite (HA), which together provide its mechanical properties. Bone alkaline phosphatase (ALP), produced by osteoblasts, plays a pivotal role in the mineralization process. Affinity contacts between collagen, mainly type II, and the crown domain of various ALP isozymes were reported in a few in vitro studies in the 1980s and 1990s, but have not attracted much attention since, although such interactions may have important implications for the bone mineralization process. The objective of this study was to investigate the binding properties of human collagen type I to human bone ALP, including the two bone ALP isoforms B1 and B2. ALP from human liver, human placenta and E. coli were also studied. A surface plasmon resonance-based analysis, supported by electrophoresis and blotting, showed that bone ALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. Further, the B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform. Human bone and liver ALP (with identical amino acid composition) displayed pronounced differences in binding, revealing that post-translational glycosylation properties govern these interactions to a large extent. In conclusion, this study presents the first evidence that glycosylation differences in human ALPs are of crucial importance for protein-protein interactions with collagen type I, although the presence of the ALP crown domain may also be necessary. Different binding affinities among the bone ALP isoforms may influence the mineral-collagen interface, mineralization kinetics, and degree of bone matrix mineralization, which are important factors determining the material properties of bone.

Alterations of collagen matrix in weight-bearing bones during skeletal unloading

NASA Technical Reports Server (NTRS)

Shiiba, M.; Arnaud, S. B.; Tanzawa, H.; Uzawa, K.; Yamauchi, M.

2001-01-01

Skeletal unloading induces loss of bone mineral density in weight-bearing bones. The objectives of this study were to characterize the post-translational modifications of collagen of weight-bearing bones subjected to hindlimb unloading for 8 weeks. In unloaded bones, tibiae and femurs, while the overall amino acid composition was essentially identical in the unloaded and control tibiae and femurs, the collagen cross-link profile showed significant differences. Two major reducible cross-links (analyzed as dihydroxylysinonorleucine and hydroxylysinonorleucine) were increased in the unloaded bones. In addition, the ratios of the former to the latter as well as pyridinoline to deoxypyridinoline were significantly decreased in the unloaded bones indicating a difference in the extent of lysine hydroxylation at the cross-linking sites between these two groups. These results indicate that upon skeletal unloading the relative pool of newly synthesized collagen is increased and it is post-translationally altered. The alteration could be associated with impaired osteoblastic differentiation induced by skeletal unloading that results in a mineralization defect.

Computational segmentation of collagen fibers in bone matrix indicates bone quality in ovariectomized rat spine.

PubMed

Daghma, Diaa Eldin S; Malhan, Deeksha; Simon, Paul; Stötzel, Sabine; Kern, Stefanie; Hassan, Fathi; Lips, Katrin Susanne; Heiss, Christian; El Khassawna, Thaqif

2018-05-01

Bone loss varies according to disease and age and these variations affect bone cells and extracellular matrix. Osteoporosis rat models are widely investigated to assess mechanical and structural properties of bone; however, bone matrix proteins and their discrepant regulation of diseased and aged bone are often overlooked. The current study considered the spine matrix properties of ovariectomized rats (OVX) against control rats (Sham) at 16 months of age. Diseased bone showed less compact structure with inhomogeneous distribution of type 1 collagen (Col1) and changes in osteocyte morphology. Intriguingly, demineralization patches were noticed in the vicinity of blood vessels in the OVX spine. The organic matrix structure was investigated using computational segmentation of collagen fibril properties. In contrast to the aged bone, diseased bone showed longer fibrils and smaller orientation angles. The study shows the potential of quantifying transmission electron microscopy images to predict the mechanical properties of bone tissue.

The Mineral–Collagen Interface in Bone

PubMed Central

2015-01-01

The interface between collagen and the mineral reinforcement phase, carbonated hydroxyapatite (cAp), is essential for bone’s remarkable functionality as a biological composite material. The very small dimensions of the cAp phase and the disparate natures of the reinforcement and matrix are essential to the material’s performance but also complicate study of this interface. This article summarizes what is known about the cAp-collagen interface in bone and begins with descriptions of the matrix and reinforcement roles in composites, of the phases bounding the interface, of growth of cAp growing within the collagen matrix, and of the effect of intra- and extrafibrilar mineral on determinations of interfacial properties. Different observed interfacial interactions with cAp (collagen, water, non-collagenous proteins) are reviewed; experimental results on interface interactions during loading are reported as are their influence on macroscopic mechanical properties; conclusions of numerical modeling of interfacial interactions are also presented. The data suggest interfacial interlocking (bending of collagen molecules around cAp nanoplatelets) and water-mediated bonding between collagen and cAp are essential to load transfer. The review concludes with descriptions of areas where new research is needed to improve understanding of how the interface functions. PMID:25824581

Spectroscopic characterization of collagen cross-links in bone

NASA Technical Reports Server (NTRS)

Paschalis, E. P.; Verdelis, K.; Doty, S. B.; Boskey, A. L.; Mendelsohn, R.; Yamauchi, M.

2001-01-01

Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross-linking pattern. The intermolecular cross-linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross-linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6-deficient chick homogenized bone and their age- and sex-matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at approximately 1660 cm(-1) and approximately 1690 cm(-1) was related to collagen cross-links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH-DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross-links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (approximately 5 microm) calcified tissue sections with a spatial resolution of approximately 7 microm.

Effects of increased collagen-matrix density on the mechanical properties and in vivo absorbability of hydroxyapatite-collagen composites as artificial bone materials.

PubMed

Yunoki, Shunji; Sugiura, Hiroaki; Ikoma, Toshiyuki; Kondo, Eiji; Yasuda, Kazunori; Tanaka, Junzo

2011-02-01

The aim of this study was to evaluate the effects of increased collagen-matrix density on the mechanical properties and in vivo absorbability of porous hydroxyapatite (HAp)-collagen composites as artificial bone materials. Seven types of porous HAp-collagen composites were prepared from HAp nanocrystals and dense collagen fibrils. Their densities and HAp/collagen weight ratios ranged from 122 to 331 mg cm⁻³ and from 20/80 to 80/20, respectively. The flexural modulus and strength increased with an increase in density, reaching 2.46 ± 0.48 and 0.651 ± 0.103 MPa, respectively. The porous composites with a higher collagen-matrix density exhibited much higher mechanical properties at the same densities, suggesting that increasing the collagen-matrix density is an effective way of improving the mechanical properties. It was also suggested that other structural factors in addition to collagen-matrix density are required to achieve bone-like mechanical properties. The in vivo absorbability of the composites was investigated in bone defects of rabbit femurs, demonstrating that the absorption rate decreased with increases in the composite density. An exhaustive increase in density is probably limited by decreases in absorbability as artificial bones.

Guided Bone Regeneration Using Collagen Scaffolds, Growth Factors, and Periodontal Ligament Stem Cells for Treatment of Peri-Implant Bone Defects In Vivo

PubMed Central

Scholz, Malte; Baudisch, Maria; Liese, Jan; Frerich, Bernhard; Lang, Hermann

2017-01-01

Introduction The aim of the study was an evaluation of different approaches for guided bone regeneration (GBR) of peri-implant defects in an in vivo animal model. Materials and Methods In minipigs (n = 15), peri-implant defects around calcium phosphate- (CaP-; n = 46) coated implants were created and randomly filled with (1) blank, (2) collagen/hydroxylapatite/β-tricalcium phosphate scaffold (CHT), (3) CHT + growth factor cocktail (GFC), (4) jellyfish collagen matrix, (5) jellyfish collagen matrix + GFC, (6) collagen powder, and (7) collagen powder + periodontal ligament stem cells (PDLSC). Additional collagen membranes were used for coverage of the defects. After 120 days of healing, bone growth was evaluated histologically (bone to implant contact (BIC;%)), vertical bone apposition (VBA; mm), and new bone height (NBH; %). Results In all groups, new bone formation was seen. Though, when compared to the blank group, no significant differences were detected for all parameters. BIC and NBH in the group with collagen matrix as well as the group with the collagen matrix + GFC were significantly less when compared to the collagen powder group (all: p < 0.003). Conclusion GBR procedures, in combination with CaP-coated implants, will lead to an enhancement of peri-implant bone growth. There was no additional significant enhancement of osseous regeneration when using GFC or PDLSC. PMID:28951742

Onlay bone augmentation on mouse calvarial bone using a hydroxyapatite/collagen composite material with total blood or platelet-rich plasma.

PubMed

Ohba, Seigo; Sumita, Yoshinori; Umebayashi, Mayumi; Yoshimura, Hitoshi; Yoshida, Hisato; Matsuda, Shinpei; Kimura, Hideki; Asahina, Izumi; Sano, Kazuo

2016-01-01

The aim of this study was to assess newly formed onlay bone on mouse calvarial bone using a new artificial bone material, a hydroxyapatite/collagen composite, with total blood or platelet-rich plasma. The hydroxyapatite/collagen composite material with normal saline, total blood or platelet-rich plasma was transplanted on mouse calvarial bone. The mice were sacrificed and the specimens were harvested four weeks after surgery. The newly formed bone area was measured on hematoxylin and eosin stained specimens using Image J software. The hydroxyapatite/collagen composite materials with total blood or platelet-rich plasma induced a significantly greater amount of newly formed bone than that with normal saline. Moreover, bone marrow was observed four weeks after surgery in the transplanted materials with total blood or platelet-rich plasma but not with normal saline. However, there were no significant differences in the amount of newly formed bone between materials used with total blood versus platelet-rich plasma. The hydroxyapatite/collagen composite material was valid for onlay bone augmentation and this material should be soaked in total blood or platelet-rich plasma prior to transplantation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Treatment with eldecalcitol positively affects mineralization, microdamage, and collagen crosslinks in primate bone.

PubMed

Saito, Mitsuru; Grynpas, Marc D; Burr, David B; Allen, Matthew R; Smith, Susan Y; Doyle, Nancy; Amizuka, Norio; Hasegawa, Tomoka; Kida, Yoshikuni; Marumo, Keishi; Saito, Hitoshi

2015-04-01

Eldecalcitol (ELD), an active form of vitamin D analog approved for the treatment of osteoporosis in Japan, increases lumbar spine bone mineral density (BMD), suppresses bone turnover markers, and reduces fracture risk in patients with osteoporosis. We have previously reported that treatment with ELD for 6 months improved the mechanical properties of the lumbar spine in ovariectomized (OVX) cynomolgus monkeys. ELD treatment increased lumbar BMD, suppressed bone turnover markers, and reduced histomorphometric parameters of both bone formation and resorption in vertebral trabecular bone. In this study, we elucidated the effects of ELD on bone quality (namely, mineralization, microarchitecture, microdamage, and bone collagen crosslinks) in OVX cynomolgus monkeys in comparison with OVX-vehicle control monkeys. Density fractionation of bone powder prepared from lumbar vertebrae revealed that ELD treatment shifted the distribution profile of bone mineralization to a higher density, and backscattered electron microscopic imaging showed improved trabecular bone connectivity in the ELD-treated groups. Higher doses of ELD more significantly reduced the amount of microdamage compared to OVX-vehicle controls. The fractionated bone powder samples were divided according to their density, and analyzed for collagen crosslinks. Enzymatic crosslinks were higher in both the high-density (≥2.0 mg/mL) and low-density (<2.0 mg/mL) fractions from the ELD-treated groups than in the corresponding fractions in the OVX-vehicle control groups. On the other hand, non-enzymatic crosslinks were lower in both the high- and low-density fractions. These observations indicated that ELD treatment stimulated the enzymatic reaction of collagen crosslinks and bone mineralization, but prevented non-enzymatic reaction of collagen crosslinks and accumulation of bone microdamage. Bone anti-resorptive agents such as bisphosphonates slow down bone remodeling so that bone mineralization, bone microdamage

Variability in the preservation of the isotopic composition of collagen from fossil bone

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

Tuross, N.; Fogel, M.L.; Hare, P.E.

1988-04-01

Collagen from bone was prepared by several methods. For modern and well-preserved bone the {delta}{sup 13}C and {delta}{sup 15}N of collagen replicas obtained after HCl or EDTA demineralization were similar to those obtained with a gelatinization procedure. However, in more poorly preserved fossil bone the {delta}{sup 13}C and {delta}{sup 15}N varied among the different protein extracts. The yield of collagen obtained with EDTA demineralization was consistently higher than extraction procedures that used HCl. The {delta}{sup 13}C of individual amino acids separated from the collagen of modern and fossil whale bone varied up to 17{per thousand}, and the {delta}{sup 15}N frommore » the same amino acids ranged over 47{per thousand}. The {delta}{sup 13}C and {delta}{sup 15}N of most amino acids clustered closely to the average of the HCl insoluble collagen. The {delta}{sup 13}C of the major amino acid in collagen, glycine, differed from the average HCl insoluble collagen by approximately 8{per thousand} in the fossil whale and 14{per thousand} in the modern whale. The {delta}{sup 15}N of glycine differed from the average HCl insoluble values by approximately 4{per thousand} in the fossil whale and 7{per thousand} in the modern whale. Thus, diagenetic changes that alter the ratio of glycine to other amino acids in bone can be expected to perturb the values for carbon and nitrogen isotopes.« less

Quantitative regulation of bone-mimetic, oriented collagen/apatite matrix structure depends on the degree of osteoblast alignment on oriented collagen substrates.

PubMed

Matsugaki, Aira; Isobe, Yoshihiro; Saku, Taro; Nakano, Takayoshi

2015-02-01

Bone tissue has a specific anisotropic morphology derived from collagen fiber alignment and the related apatite crystal orientation as a bone quality index. However, the precise mechanism of cellular regulation of the crystallographic orientation of apatite has not been clarified. In this study, anisotropic construction of cell-produced mineralized matrix in vitro was established by initiating organized cellular alignment and subsequent oriented bone-like matrix (collagen/apatite) production. The oriented collagen substrates with three anisotropic levels were prepared by a hydrodynamic method. Primary osteoblasts were cultured on the fabricated substrates until mineralized matrix formation is confirmed. Osteoblast alignment was successfully regulated by the level of substrate collagen orientation, with preferential alignment along the direction of the collagen fibers. Notably, both fibrous orientation of newly synthesized collagen matrix and c-axis of produced apatite crystals showed preferential orientation along the cell direction. Because the degree of anisotropy of the deposited apatite crystals showed dependency on the directional distribution of osteoblasts cultured on the oriented collagen substrates, the cell orientation determines the crystallographic anisotropy of produced apatite crystals. To the best of our knowledge, this is the first report demonstrating that bone tissue anisotropy, even the alignment of apatite crystals, is controllable by varying the degree of osteoblast alignment via regulating the level of substrate orientation. © 2014 Wiley Periodicals, Inc.

Clinical observation of biomimetic mineralized collagen artificial bone putty for bone reconstruction of calcaneus fracture

PubMed Central

Pan, Yong-Xiong; Yang, Guang-Gang; Li, Zhong-Wan; Shi, Zhong-Min; Sun, Zhan-Dong

2018-01-01

Abstract This study investigated clinical outcomes of biomimetic mineralized collagen artificial bone putty for bone reconstruction in the treatment of calcaneus fracture. Sixty cases of calcaneal fractures surgically treated with open reduction and internal fixation in our hospital from June 2014–2015 were chosen and randomly divided into two groups, including 30 cases treated with biomimetic mineralized collagen artificial bone putty as treatment group, and 30 cases treated with autogenous ilia as control group. The average follow-up time was 17.2 ± 3.0 months. The results showed that the surgery duration and postoperative drainage volume of treatment group were significantly lower than control group; there were no statistically significant differences in the fracture healing time, American Orthopaedic Foot and Ankle Society scores at 3 and 12 months after surgery, Böhler’s angle, Gissane’s angle and height of calcaneus between the two groups. There were no significant differences in wound complication and reject reaction between the two groups, while significant difference in donor site complication. As a conclusion, the implantation of biomimetic mineralized collagen artificial bone putty in the open reduction of calcaneal fracture resulted in reliable effect and less complications, which is suitable for clinical applications in the treatment of bone defect in calcaneal fractures. PMID:29644087

Collagen Fingerprinting: A New Screening Technique for Radiocarbon Dating Ancient Bone.

PubMed

Harvey, Virginia L; Egerton, Victoria M; Chamberlain, Andrew T; Manning, Phillip L; Buckley, Michael

2016-01-01

Collagen is the dominant organic component of bone and is intimately locked within the hydroxyapatite structure of this ubiquitous biomaterial that dominates archaeological and palaeontological assemblages. Radiocarbon analysis of extracted collagen is one of the most common approaches to dating bone from late Pleistocene or Holocene deposits, but dating is relatively expensive compared to other biochemical techniques. Numerous analytical methods have previously been investigated for the purpose of screening out samples that are unlikely to yield reliable dates including histological analysis, UV-stimulated fluorescence and, most commonly, the measurement of percentage nitrogen (%N) and ratio of carbon to nitrogen (C:N). Here we propose the use of collagen fingerprinting (also known as Zooarchaeology by Mass Spectrometry, or ZooMS, when applied to species identification) as an alternative screening method for radiocarbon dating, due to its ability to provide information on collagen presence and quality, alongside species identification. The method was tested on a series of sub-fossil bone specimens from cave systems on Cayman Brac (Cayman Islands), chosen due to the observable range in diagenetic alteration, and in particular, the extent of mineralisation. Six (14)C dates, of 18 initial attempts, were obtained from remains of extinct hutia, Capromys sp. (Rodentia; Capromyidae), recovered from five distinct caves on Cayman Brac, and ranging from 393 ± 25 to 1588 ± 26 radiocarbon years before present (yr BP). All of the bone samples that yielded radiocarbon dates generated excellent collagen fingerprints, and conversely those that gave poor fingerprints also failed dating. Additionally, two successfully fingerprinted bone samples were screened out from a set of 81. Both subsequently generated (14)C dates, demonstrating successful utilisation of ZooMS as an alternative screening mechanism to identify bone samples that are suitable for 1(4)C analysis.

Collagen Fingerprinting: A New Screening Technique for Radiocarbon Dating Ancient Bone

PubMed Central

Harvey, Virginia L.; Egerton, Victoria M.; Chamberlain, Andrew T.; Manning, Phillip L.; Buckley, Michael

2016-01-01

Collagen is the dominant organic component of bone and is intimately locked within the hydroxyapatite structure of this ubiquitous biomaterial that dominates archaeological and palaeontological assemblages. Radiocarbon analysis of extracted collagen is one of the most common approaches to dating bone from late Pleistocene or Holocene deposits, but dating is relatively expensive compared to other biochemical techniques. Numerous analytical methods have previously been investigated for the purpose of screening out samples that are unlikely to yield reliable dates including histological analysis, UV-stimulated fluorescence and, most commonly, the measurement of percentage nitrogen (%N) and ratio of carbon to nitrogen (C:N). Here we propose the use of collagen fingerprinting (also known as Zooarchaeology by Mass Spectrometry, or ZooMS, when applied to species identification) as an alternative screening method for radiocarbon dating, due to its ability to provide information on collagen presence and quality, alongside species identification. The method was tested on a series of sub-fossil bone specimens from cave systems on Cayman Brac (Cayman Islands), chosen due to the observable range in diagenetic alteration, and in particular, the extent of mineralisation. Six 14C dates, of 18 initial attempts, were obtained from remains of extinct hutia, Capromys sp. (Rodentia; Capromyidae), recovered from five distinct caves on Cayman Brac, and ranging from 393 ± 25 to 1588 ± 26 radiocarbon years before present (yr BP). All of the bone samples that yielded radiocarbon dates generated excellent collagen fingerprints, and conversely those that gave poor fingerprints also failed dating. Additionally, two successfully fingerprinted bone samples were screened out from a set of 81. Both subsequently generated 14C dates, demonstrating successful utilisation of ZooMS as an alternative screening mechanism to identify bone samples that are suitable for 14C analysis. PMID:26938469

Thermal and electron stimulated luminescence of natural bones, commercial hydroxyapatite and collagen.

PubMed

Roman-Lopez, J; Correcher, V; Garcia-Guinea, J; Rivera, T; Lozano, I B

2014-01-01

The luminescence (cathodoluminescence and thermoluminescence) properties of natural bones (Siberian mammoth and adult elephant), commercial hydroxyapatite and collagen were analyzed. Chemical analyses of the natural bones were determined using by Electron Probe Micro-Analysis (EMPA). Structural, molecular and thermal characteristics were determined by X-ray Diffraction (XRD), Raman spectroscopy and Differential Thermal and Thermogravimetric analysis (DTA-TG). Cathodoluminescence (CL) spectra of natural bones and collagen showed similar intense broad bands at 440 and 490 nm related to luminescence of the tetrahedral anion [Formula: see text] or structural defects. A weaker luminescence exhibited at 310 nm could be attributed to small amount of rare earth elements (REEs). Four luminescent bands at 378, 424, 468 and 576 nm were observed in the commercial hydroxyapatite (HAP). Both natural bones and collagen samples exhibited natural thermoluminescence (NTL) with well-defined glow curves whereas that the induced thermoluminescence (ITL) only appears in the samples of commercial hydroxyapatite and collagen. Additional explanations for the TL anomalous fading of apatite, as a crucial difficulty performing dosimetry and dating, are also considered. Copyright © 2013 Elsevier B.V. All rights reserved.

Relationships between human cortical bone toughness and collagen cross-links on paired anatomical locations.

PubMed

Gauthier, Rémy; Follet, Hélène; Langer, Max; Gineyts, Evelyne; Rongiéras, Frédéric; Peyrin, Françoise; Mitton, David

2018-07-01

Human cortical bone fracture processes depend on the internal porosity network down to the lacunar length scale. Recent results show that at the collagen scale, the maturation of collagen cross-links may have a negative influence on bone mechanical behavior. While the effect of pentosidine on human cortical bone toughness has been studied, the influence of mature and immature enzymatic cross-links has only been studied in relation to strength and work of fracture. Moreover, these relationships have not been studied on different paired anatomical locations. Thus, the aim of the current study was to assess the relationships between both enzymatic and non-enzymatic collagen cross-links and human cortical bone toughness, on four human paired anatomical locations. Single Edge Notched Bending toughness tests were performed for two loading conditions: a quasi-static standard condition, and a condition representative of a fall. These tests were done with 32 paired femoral diaphyses, femoral necks and radial diaphyses (18 women, age 81 ± 12 y.o.; 14 men, age 79 ± 8 y.o.). Collagen enzymatic and non-enzymatic crosslinks were measured on the same bones. Maturation of collagen was defined as the ratio between immature and mature cross-links (CX). The results show that there was a significant correlation between collagen cross-link maturation and bone toughness when gathering femoral and radial diaphyses, but not when considering each anatomical location individually. These results show that the influence of collagen enzymatic and non-enzymatic cross-links is minor when considering human cortical bone crack propagation mechanisms. Copyright © 2018 Elsevier Inc. All rights reserved.

Techniques to assess bone ultrastructure organization: orientation and arrangement of mineralized collagen fibrils

PubMed Central

Georgiadis, Marios; Müller, Ralph; Schneider, Philipp

2016-01-01

Bone's remarkable mechanical properties are a result of its hierarchical structure. The mineralized collagen fibrils, made up of collagen fibrils and crystal platelets, are bone's building blocks at an ultrastructural level. The organization of bone's ultrastructure with respect to the orientation and arrangement of mineralized collagen fibrils has been the matter of numerous studies based on a variety of imaging techniques in the past decades. These techniques either exploit physical principles, such as polarization, diffraction or scattering to examine bone ultrastructure orientation and arrangement, or directly image the fibrils at the sub-micrometre scale. They make use of diverse probes such as visible light, X-rays and electrons at different scales, from centimetres down to nanometres. They allow imaging of bone sections or surfaces in two dimensions or investigating bone tissue truly in three dimensions, in vivo or ex vivo, and sometimes in combination with in situ mechanical experiments. The purpose of this review is to summarize and discuss this broad range of imaging techniques and the different modalities of their use, in order to discuss their advantages and limitations for the assessment of bone ultrastructure organization with respect to the orientation and arrangement of mineralized collagen fibrils. PMID:27335222

Microfibrous β-TCP/collagen scaffolds mimic woven bone in structure and composition.

PubMed

Zhang, Shen; Zhang, Xin; Cai, Qing; Wang, Bo; Deng, Xuliang; Yang, Xiaoping

2010-12-01

Woven bone, as the initial form of bone tissue, is always found in developing and repairing bone. It is thought of as a temporary scaffold for the deposition of osteogenic cells and the laying down of lamellar bone. Thus, we hypothesize that a matrix which resembles the architecture and components of woven bone can provide an osteoblastic microenvironment for bone cell growth and new bone formation. In this study, woven-bone-like beta-tricalcium phosphate (β-TCP)/collagen scaffolds were fabricated by sol-gel electrospinning and impregnating methods. Optimization studies on sol-gel synthesis and electrospinning process were conducted respectively to prepare pure β-TCP fibers with dimensions close to mineralized collagen fibrils in woven bone. The collagen-coating layer prepared by impregnation had an adhesive role that held the β-TCP fibers together, and resulted in rapid degradation and matrix mineralization in in vitro tests. MG63 osteoblast-like cells seeded on the resultant scaffolds showed three-dimensional (3D) morphologies, and merged into multicellular layers after 7 days culture. Cytotoxicity test further revealed that extracts from the resultant scaffolds could promote the proliferation of MG63 cells. Therefore, the woven-bone-like matrix that we constructed favored the attachment and proliferation of MG63 cells in three dimensions. It has great potential ability to shorten the time of formation of new bone.

Collagen Membrane and Immune Response in Guided Bone Regeneration: Recent Progress and Perspectives.

PubMed

Chu, Chenyu; Deng, Jia; Sun, Xianchang; Qu, Yili; Man, Yi

2017-10-01

Collagen is one of the important components of collagen membranes as well as the extracellular matrix (ECM). Most previous studies have focused on combining collagen membranes with various cross-linking agents, grafting materials, and cytokines to enhance their mechanical properties and bioactivities. Moreover, collagen membranes are often designed to minimize foreign body reactions involving macrophages. However, macrophages were recently found to play a pivotal role during bone regeneration based on their polarization into both proinflammatory and anti-inflammatory phenotypes. Because of the abilities to modulate macrophage polarization and mediate the balance of proinflammatory and anti-inflammatory microenvironments, immune-responsive collagen membranes may be an innovative strategy for promoting bone regeneration. Herein, following a brief review of collagen membranes and the background of macrophages, recent modulations and studies of immune-responsive collagen are described to express the potential of collagen interacting with macrophages and the necessity of further studies in the field of immune-responsive collagen membranes.

Hydroxyapatite/collagen bone-like nanocomposite.

PubMed

Kikuchi, Masanori

2013-01-01

Our group has succeeded to synthesize material with bone-like nanostructure and bone-like inorganic and organic composition via self-organization mechanism between them using simultaneous titration method under controlled pH and temperature. The hydroxyapatite/collagen (HAp/Col) bone-like nanocomposite completely incorporated into bone remodeling process to be substituted by new bone. Cells cultured on the HAp/Col revealed very interesting reactions. Osteoblast-like MG63 cells showed upregulation of alkaline phosphatase >3 times greater than MG63 cells cultured on tissue culture polystyrene (TCPS). MG63 cells 3-dimensionally cultured in a "HAp/Col sponge," a porous HAp/Col having sponge-like viscoelasticity, accumulated calcium phosphate nodules on extracellular matrices they secreted. Bone marrow cells co-cultured with osteoblasts on HAp/Col differentiated to osteoclasts without differentiation supplements. This phenomenon is not found in cells cultured on hydroxyapatite ceramics and TCPS, and rarely in cells cultured on dentin. These results suggest that HAp/Col is a good candidate for tissue engineering of bone as well as bone filler. In a clinical test as a bone filler, the HAp/Col sponge was significantly better than porous β-tricalcium phosphate. The HAp/Col sponge has been approved by the Japanese government and will be used as greatly needed bone filler in patients. In addition to the above, HAp/Col coating on titanium revealed higher osteo-conductivity than HAp-coated titanium and bare titanium and improved direct bonding between titanium and newly formed bone. The HAp/Col coating may be used for metal devices requiring osseointegration.

The Structure and Function of Non-Collagenous Bone Proteins

NASA Technical Reports Server (NTRS)

Hook, Magnus; McQuillan, David J.

1997-01-01

The research done under the cooperative research agreement for the project titled 'The structure and function of non-collagenous bone proteins' represented the first phase of an ongoing program to define the structural and functional relationships of the principal noncollagenous proteins in bone. An ultimate goal of this research is to enable design and execution of useful pharmacological compounds that will have a beneficial effect in treatment of osteoporosis, both land-based and induced by long-duration space travel. The goals of the now complete first phase were as follows: 1. Establish and/or develop powerful recombinant protein expression systems; 2. Develop and refine isolation and purification of recombinant proteins; 3. Express wild-type non-collagenous bone proteins; 4. Express site-specific mutant proteins and domains of wild-type proteins to enhance likelihood of crystal formation for subsequent solution of structure.

Development of a nanofiltration method for bone collagen 14C AMS dating

NASA Astrophysics Data System (ADS)

Boudin, Mathieu; Boeckx, Pascal; Buekenhoudt, Anita; Vandenabeele, Peter; Van Strydonck, Mark

2013-01-01

Radiocarbon dating of bones is usually performed on the collagen fraction. However, this collagen can contain exogenous molecules, including humic substances (HSs) and/or other soil components that may have a different age than the bone. Incomplete removal can result in biased 14C dates. Ultrafiltration of collagen, dissolved as gelatin (molecular weight (MW) ∼100,000 Dalton), has received considerable attention to obtain more reliable dates. Ultrafiltration is an effective method of removal of low-molecular weight contaminants from bone collagen but it does not remove high-molecular weight contaminants, such as cross-linked humic collagen complexes. However, comparative dating studies have raised the question whether this cleaning step itself may introduce contamination with carbon from the filters used. In this study, a nanofiltration method was developed using a ceramic filter to avoid a possible extraneous carbon contamination introduced by the filter. This method should be applicable to various protein materials e.g. collagen, silk, wool, leather and should be able to remove low-molecular and high molecular weight HSs. In this study bone collagen was hot acid hydrolyzed to amino acids and nanofiltrated. A filter with a molecular weight cutoff (MWCO) of 450 Dalton was chosen in order to collect the amino acids in the permeate and the HSs in the retentate. Two pilot studies were set up. Two nanofiltration types were tested in pilot study 1: dead end and cross flow filtration. Humic substance (HS)-solutions with fossil carbon and modern hydrolyzed collagen contaminated with HSs were filtrated and analyzed with spectrofluorescence to determine the HS removal. Cross flow nanofiltration showed the most efficient HS removal. A second pilot study based upon these results was set up wherein only cross flow filtration was performed. 14C measurements of the permeates of hydrolyzed modern collagen contaminated with fossil HSs demonstrate a significant but incomplete

Influence of Nano-HA Coated Bone Collagen to Acrylic (Polymethylmethacrylate) Bone Cement on Mechanical Properties and Bioactivity

PubMed Central

Li, Tao; Weng, Xisheng; Bian, Yanyan; Zhou, Lei; Cui, Fuzhai; Qiu, Zhiye

2015-01-01

Objective This research investigated the mechanical properties and bioactivity of polymethylmethacrylate (PMMA) bone cement after addition of the nano-hydroxyapatite(HA) coated bone collagen (mineralized collagen, MC). Materials & Methods The MC in different proportions were added to the PMMA bone cement to detect the compressive strength, compression modulus, coagulation properties and biosafety. The MC-PMMA was embedded into rabbits and co-cultured with MG 63 cells to exam bone tissue compatibility and gene expression of osteogenesis. Results 15.0%(wt) impregnated MC-PMMA significantly lowered compressive modulus while little affected compressive strength and solidification. MC-PMMA bone cement was biologically safe and indicated excellent bone tissue compatibility. The bone-cement interface crosslinking was significantly higher in MC-PMMA than control after 6 months implantation in the femur of rabbits. The genes of osteogenesis exhibited significantly higher expression level in MC-PMMA. Conclusions MC-PMMA presented perfect mechanical properties, good biosafety and excellent biocompatibility with bone tissues, which has profoundly clinical values. PMID:26039750

Influence of Nano-HA Coated Bone Collagen to Acrylic (Polymethylmethacrylate) Bone Cement on Mechanical Properties and Bioactivity.

PubMed

Li, Tao; Weng, Xisheng; Bian, Yanyan; Zhou, Lei; Cui, Fuzhai; Qiu, Zhiye

2015-01-01

This research investigated the mechanical properties and bioactivity of polymethylmethacrylate (PMMA) bone cement after addition of the nano-hydroxyapatite(HA) coated bone collagen (mineralized collagen, MC). The MC in different proportions were added to the PMMA bone cement to detect the compressive strength, compression modulus, coagulation properties and biosafety. The MC-PMMA was embedded into rabbits and co-cultured with MG 63 cells to exam bone tissue compatibility and gene expression of osteogenesis. 15.0%(wt) impregnated MC-PMMA significantly lowered compressive modulus while little affected compressive strength and solidification. MC-PMMA bone cement was biologically safe and indicated excellent bone tissue compatibility. The bone-cement interface crosslinking was significantly higher in MC-PMMA than control after 6 months implantation in the femur of rabbits. The genes of osteogenesis exhibited significantly higher expression level in MC-PMMA. MC-PMMA presented perfect mechanical properties, good biosafety and excellent biocompatibility with bone tissues, which has profoundly clinical values.

Blueberry consumption prevents loss of collagen in bone matrix and inhibits senescence pathways in osteoblastic cells

USDA-ARS?s Scientific Manuscript database

Ovariectomy (OVX)-induced bone loss has been linked to increased bone turnover and higher bone matrix collagen degradation as the result of osteoclast activation. However, the role of degraded collagen matrix in the fate of resident bone-forming cells is unclear. In this report, we show that OVX-i...

A Direct Role of Collagen Glycation in Bone Fracture

PubMed Central

Poundarik, Atharva A.; Wu, Ping-Cheng; Evis, Zafer; Sroga, Grazyna E.; Ural, Ani; Rubin, Mishaela; Vashishth, Deepak

2015-01-01

Non-enzymatic glycation (NEG) is an age-related process accelerated by diseases like diabetes, and causes the accumulation of advanced glycation end-products (AGEs). NEG-mediated modification of bone’s organic matrix, principally collagen type-I, has been implicated in impairing skeletal physiology and mechanics. Here, we present evidence, from in vitro and in vivo models, and establish a causal relationship between collagen glycation and alterations in bone fracture at multiple length scales. Through atomic force spectroscopy, we established that NEG impairs collagen’s ability to dissipate energy. Mechanical testing of in vitro glycated human bone specimen revealed that AGE accumulation due to NEG dramatically reduces the capacity of organic and mineralized matrix to creep and caused bone to fracture under impact at low levels of strain (3000–5000 μstrain) typically associated with fall. Fracture mechanics tests of NEG modified human cortical bone of varying ages, and their age-matched controls revealed that NEG disrupted microcracking based toughening mechanisms and reduced bone propagation and initiation fracture toughness across all age groups. A comprehensive mechanistic model, based on experimental and modeling data, was developed to explain how NEG and AGEs are causal to, and predictive of bone fragility. Furthermore, fracture mechanics and indentation testing on diabetic mice bones revealed that diabetes mediated NEG severely disrupts bone matrix quality in vivo. Finally, we show that AGEs are predictive of bone quality in aging humans and have diagnostic applications in fracture risk. PMID:26530231

Environmental, trophic, and ecological factors influencing bone collagen δ2H

NASA Astrophysics Data System (ADS)

Topalov, Katarina; Schimmelmann, Arndt; David Polly, P.; Sauer, Peter E.; Lowry, Mark

2013-06-01

Organic deuterium/hydrogen stable isotope ratios (i.e., 2H/1H, expressed as δ2H value in ‰) in animal tissues are related to the 2H/1H in diet and ingested water. Bone collagen preserves the biochemical 2H/1H isotopic signal in the δ2H value of collagen's non-exchangeable hydrogen. Therefore, δ2H preserved in bone collagen has the potential to constrain environmental and trophic conditions, which is of interest to researchers studying of both living and fossil vertebrates. Our data examine the relationship of δ2H values of collagen with geographic variation in δ2H of meteoric waters, with local variations in the ecology and trophic level of species, and with the transition from mother's milk to adult diet. Based on 97 individuals from 22 marine and terrestrial vertebrates (predominately mammals), we found the relationships of collagen δ2H to both geographic variation in meteoric water δ2H (R2 = 0.55) and to δ15N in bone collagen (R2 = 0.17) statistically significant but weaker than previously reported. The second strongest control on collagen δ2H in our data is dietary, with nearly 50 percent of the variance in δ2H explained by trophic level (R2 = 0.47). Trophic level effects potentially confound the local meteoric signal if not held constant: herbivores tend to have the lowest δ2H values, omnivores have intermediate ones, and carnivores have the highest values. Body size (most likely related to mass-specific metabolic rates) has a strong influence on collagen δ2H (R2 = 0.30), by causing greater sensitivity in smaller animals to seasonal climate variations and/or high evapotranspiration leading to 2H-enrichment in tissues. In marine mammals weaning produces a dramatic effect on collagen δ2H with adult values being universally higher than pup values (R2 = 0.79). Interestingly, the shift in δ15N at weaning is downward, even though normally hydrogen and nitrogen isotope ratios are positively correlated with one another in respect to trophic level. Our

Collagen fibril organization within rat vertebral bone modified with metastatic involvement.

PubMed

Burke, Mikhail; Golaraei, Ahmad; Atkins, Ayelet; Akens, Margarete; Barzda, Virginijus; Whyne, Cari

2017-08-01

Metastatic involvement diminishes the mechanical integrity of vertebral bone, however its specific impact on the structural characteristics of a primary constituent of bone tissue, the collagen-I fibril matrix, has not been adequately characterized. Female athymic rats were inoculated with HeLa or Ace-1 cancer cells lines producing osteolytic or mixed (osteolytic & osteoblastic) metastases respectively. A maximum of 21days was allowed between inoculation and rat sacrifice for vertebrae extraction. Linear polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and transmission electron microscopy (TEM) imaging was utilized to assess the impact of metastatic involvement on collagen fibril organization. Increased observations of deviations in the typical plywood motif or a parallel packing structure and an increased average measured susceptibility ratio (related to relative degree of in-plane vs. out-plane fibrils in the analyzed tissue area) in bone adjacent to metastatic involvement was indicative of change in fibrilar organization compared to healthy controls. In particular, collagen-I fibrils in tumour-induced osteoblastic bone growth showed no adherence to the plywood motif or parallel packing structure seen in healthy lamellar bone, exhibiting a much higher susceptibility ratio and degree of fibril disorder. Negative correlations were established between measured susceptibility ratios and the hardness and modulus of metastatic bone tissue assessed in a previous study. Characterizing modifications in tissue level properties is key in defining bone quality in the presence of metastatic disease and their potential impact on material behaviour. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessment of tricalcium phosphate/collagen (TCP/collagene)nanocomposite scaffold compared with hydroxyapatite (HA) on healing of segmental femur bone defect in rabbits.

PubMed

Mohseni, Mahmoud; Jahandideh, Alireza; Abedi, Gholamreza; Akbarzadeh, Abolfazl; Hesaraki, Saeed

2018-03-01

Bone regeneration is an important objective in clinical practice and has been used for different applications. The aim of this study was to evaluate the effectiveness of nanocomposite tricalcium phosphate (TCP)/collagen scaffolds combined with hydroxyapatite scaffold for bone healing in surgery of femoral defects in rabbits. In this study, 45 mature male New Zealand white rabbits between 6 and 8 months old and weighting between 3 and 3.5 kg were examined. Rabbits were divided into three groups. Surgical procedures were performed after intramuscular injection of Ketamine 10% (ketamine hydrochloride, 50 mg/kg) and Rompun 5% (xylazine, 5 mg/kg). Then an approximately 6 mm diameter-5 mm cylinder bone defect was created in the femur of one of the hind limbs. After inducing the surgical wound, all rabbits were coloured and randomly divided into three experimental groups of 15 animals each. Group 1 received pure medical nanocomposite TCP/collagen granules, group 2 received hydroxyapatite, and third group was a control group which received no treatment. Histopathological evaluation was performed on days 15, 30, and 45 after surgery. On days 15, 30, and 45 after surgery, the quantity and the velocity of stages of bone formation at the healing site in nanocomposite TCP/collagen group were better than HA and control groups and the quantity of newly formed lamellar bone at the healing site in nanocomposite TCP/collagen group were better than onward compared with HA and control groups. In conclusion, it seems that TCP/collagen nanocomposite has a significant role in the reconstruction of bone defects and can be used as scaffold in bone fractures.

Extracellular post-translational modifications of collagen are major determinants of biomechanical properties of fetal bovine cortical bone.

PubMed

Garnero, Patrick; Borel, Olivier; Gineyts, Evelyne; Duboeuf, Francois; Solberg, Helene; Bouxsein, Mary L; Christiansen, Claus; Delmas, Pierre D

2006-03-01

Mechanical behavior of bone depends on its mass and architecture, and on the material properties of the matrix, which is composed of a mineral phase and an organic component mainly constituted of type I collagen. Mineral accounts largely for the stiffness of bone, whereas type I collagen provides bone its ductility and toughness, i.e., its ability to undergo deformation and absorb energy after it begins to yield. The molecular mechanisms underlying the effect of alterations in type I collagen on bone mechanical properties are unclear. We used an in vitro model of fetal bovine cortical bone specimens (n = 44), where the extent of type I collagen cross-linking was modified by incubation at 37 degrees C for 0, 60, 90 and 120 days, keeping constant the architecture and the mineral content. At each incubation time, the following parameters were determined: (1) the bone concentration of enzymatic (pyridinoline; PYD and deoxypyridinoline, DPD) and non-enzymatic (pentosidine) crosslinks by HPLC, (2) the extent of aspartic acid isomerization of the type I collagen C-telopeptide (CTX) by ELISA of native (alpha CTX) and isomerized (beta CTX) forms, (3) the mineral density by DXA, (4) the porosity by micro-computed tomography and (5) the bending and compressive mechanical properties. Incubation of bone specimens at 37 degrees C for 60 days increased the level (per molecule of collagen) of PYD (+98%, P = 0.005), DPD (+42%, P = 0.013), pentosidine (+55-fold, P = 0.005), and the degree of type I collagen C-telopeptide isomerization (+4.9-fold, P = 0.005). These biochemical changes of collagen were associated with a 30% decrease in bending and compressive yield stress and a 2.5-fold increase in compressive post-yield energy absorption (P < 0.02 for all), with no significant change of bone stiffness. In multivariate analyses, the level of collagen cross-linking was associated with yield stress and post-yield energy absorption independently of bone mineral density, explaining up to

[Bio-Oss collagen bone grafting in the treatment of endodontic-periodontic lesion].

PubMed

Sun, Jiang; Liu, Qiong

2009-09-01

To study the effect of Bio-Oss collagen for bone grafting in the treatment of endodontic-periodontic lesion. Thirty patients (37 teeth) with endodontic-periodontic lesions received root canal therapy and periodontal treatment. Four weeks after the treatment, Bio-Oss collagen was grafted in these teeth. Clinical examinations were performed 1, 3, 6, 9, and 12 months after the surgery. The mobility of these teeth improved 1 month after the surgery. At 3, 6, 9, and 12 months after the surgery, the pocket depth reduced obviously good attachment and further improvement of the tooth mobility. Bio-Oss collagen for bone grafting can achieve good therapeutic effect for endodontic-periodontic lesions.

Fabrication and Characterization of Biomimetic Collagen-Apatite Scaffolds with Tunable Structures for Bone Tissue Engineering

PubMed Central

Xia, Zengmin; Yu, Xiaohua; Jiang, Xi; Brody, Harold D; Rowe, David W; Wei, Mei

2013-01-01

The objective of the current study is to prepare a biomimetic collagen-apatite (Col-Ap) scaffold for improved bone repair and regeneration. A novel bottom-up approach has been developed, which combines a biomimetic self-assembly method with a controllable freeze casting technology. In this study, the mineralized collagen fibers were generated using a simple one-step co-precipitation method which involved collagen self-assembly and in situ apatite precipitation in a collagen-containing modified simulated body fluid (m-SBF). The precipitates were subjected to controllable freeze casting, forming scaffolds with either an isotropic equiaxed structure or a unidirectional lamellar structure. These scaffolds were comprised of collagen fibers and poorly crystalline bone-like carbonated apatite nanoparticles. The mineral content in the scaffold could be tailored in a range 0–54 wt% by simply adjusting the collagen content in the m-SBF. Further, the mechanisms of the formation of both the equiaxed and the lamellar scaffolds were investigated, and freezing regimes for equiaxed and lamellar solidification were established. Finally, bone forming capability of such prepared scaffolds was evaluated in vivo in a mouse calvarial defect model. It was confirmed that the scaffolds well support new bone formation. PMID:23567944

The temporal degradation of bone collagen: A histochemical approach.

PubMed

Boaks, Amelia; Siwek, Donald; Mortazavi, Farzad

2014-07-01

As forensic anthropologists are currently unable to estimate reliably and quantitatively the postmortem interval (PMI) of skeletonized remains, the current study was conducted to determine if degradation of bone collagen over time could be quantified using sirius red/fast green staining, and whether the degradation would occur at a predictive rate such that it may be used to estimate the PMI of skeletonized individuals. Resin embedded 200-300μm cross-sections of pig (Sus scrofa) long bones with known provenience and PMIs ranging from fresh to 12 months were stained using a histochemical reaction which differentially stains collagenous (Co) and non-collagenous (NCo) proteins. Spectrophotometry was used to determine the concentration of Co and NCo proteins in each bone section, after which the ratio of these proteins was calculated. The results of this study revealed a significant decline in the ratios of Co/NCo protein concentrations over the time period studied (p<0.001). Furthermore, a significant negative correlation between the ratios of Co/NCo protein concentrations and time (r=-0.563, p<0.0001) was observed. Despite a significant correlation, the moderate r-value obtained suggests that, at present, this method is useful primarily for detecting and quantifying the degradation of Co and NCo proteins in bones. Future studies that include shorter time intervals and environmental factors, such as soil pH, temperature, and hydrology may prove to be critical for using this method for PMI estimation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Effects of the bilayer nano-hydroxyapatite/mineralized collagen-guided bone regeneration membrane on site preservation in dogs.

PubMed

Sun, Yi; Wang, Chengyue; Chen, Qixin; Liu, Hai; Deng, Chao; Ling, Peixue; Cui, Fu-Zhai

2017-08-01

This study was aimed at assessing the effects of the porous mineralized collagen plug with or without the bilayer mineralized collagen-guided bone regeneration membrane on alveolar ridge preservation in dogs. The third premolars in the bilateral maxilla of mongrel dogs ( N = 12) were extracted. Twenty-four alveolar sockets were thus randomly divided into three groups: membrane + collagen plug (MP, n = 8), nonmembrane + collagen plug (NP, n = 8) and blank group without any implantation (BG, n = 8). Radiographic assessment was carried out immediately and in the 2nd, 6th, and 12th week after surgery. The bone-repairing effects of the two grafts were respectively evaluated by clinical observation, X-ray micro-computed tomography examination, and histological analysis in the 8th and 12th week after surgery. Three groups presented excellent osseointegration without any inflammation or dehiscence. X-ray micro-computed tomography and histological assessment indicated that the ratios of new bone formation of MP group were significantly higher than those of NP group and BG group in the 8th and 12th week after surgery ( P < 0.05). As a result, the porous mineralized collagen plug with or without the bilayer mineralized collagen-guided bone regeneration membrane could reduce the absorption of alveolar ridge compared to BG group, and the combined use of porous mineralized collagen plug and bilayer mineralized collagen-guided bone regeneration could further improve the activity of bone regeneration.

Collagen and mineral deposition in rabbit cortical bone during maturation and growth: effects on tissue properties.

PubMed

Isaksson, Hanna; Harjula, Terhi; Koistinen, Arto; Iivarinen, Jarkko; Seppänen, Kari; Arokoski, Jari P A; Brama, Pieter A; Jurvelin, Jukka S; Helminen, Heikki J

2010-12-01

We characterized the composition and mechanical properties of cortical bone during maturation and growth and in adult life in the rabbit. We hypothesized that the collagen network develops earlier than the mineralized matrix. Growth was monitored, and the rabbits were euthanized at birth (newborn), and at 1, 3, 6, 9, and 18 months of age. The collagen network was assessed biochemically (collagen content, enzymatic and non-enzymatic cross-links) in specimens from the mid-diaphysis of the tibia and femur and biomechanically (tensile testing) from decalcified whole tibia specimens. The mineralized matrix was analyzed using pQCT and 3-point bend tests from intact femur specimens. The collagen content and the Young's modulus of the collagen matrix increased significantly until the rabbits were 3 months old, and thereafter remained stable. The amount of HP and LP collagen cross-links increased continuously from newborn to 18 months of age, whereas PEN cross-links increased after 6 months of age. Bone mineral density and the Young's modulus of the mineralized bone increased until the rabbits were at least 6 months old. We concluded that substantial changes take place during the normal process of development in both the biochemical and biomechanical properties of rabbit cortical bone. In cortical bone, the collagen network reaches its mature composition and mechanical strength prior to the mineralized matrix. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Bone Healing in Extraction Sockets Covered With Collagen Membrane Alone or Associated With Porcine-Derived Bone Graft: a Comparative Histological and Histomorphometric Analysis.

PubMed

Guarnieri, Renzo; Testarelli, Luca; Stefanelli, Luigi; De Angelis, Francesca; Mencio, Francesca; Pompa, Giorgio; Di Carlo, Stefano

2017-01-01

The present paper reports data of a randomized study aimed to analyse and compare the histologic and histomorphometric aspects of bone healing in extraction sites covered with collagen membrane alone or associated with porcine-derived bone graft. Thirty patients, with single extraction sockets without severe bone wall defects in the premolar/molar region, were included. Ten extraction sockets were grafted with porcine-derived bone and covered with collagen membrane (group 1), 10 sites were covered with collagen membrane alone (group 2), and 10 sites healed spontaneously (group 3). After 4 months of healing, 26 (8 in group 1, 9 in group 2, and 9 in group 3) bone core specimens were harvested for histologic evaluation, then dental implants were placed. Sites in the group 1 and in the group 2 showed similar histologic and histomorphometric results without significantly differences in the percentage of vital bone (57.43% [SD 4.8] vs. 60.01% [SD 3.2]), and non-mineralized connective tissue 22.99% (SD 5.3) vs. 18.53% (SD 6.2). In group 1 a 16.57% (SD 3.8) of residual material was found. Results showed that the use of collagen membrane alone or associated to porcine-derived bone improves the healing bone process compared to that of extraction sites spontaneously healed. Moreover, histomorphometric data related to bone quality, indicated that extraction sites without severe walls defects and with a vestibular bone thickness > 1.5 mm, treated with a low resorbtion rate collagen membrane alone, do not need more than 4 months for dental implant insertion.

Hydrolyzed collagen improves bone metabolism and biomechanical parameters in ovariectomized mice: an in vitro and in vivo study.

PubMed

Guillerminet, Fanny; Beaupied, Hélène; Fabien-Soulé, Véronique; Tomé, Daniel; Benhamou, Claude-Laurent; Roux, Christian; Blais, Anne

2010-03-01

Collagen has an important structural function in several organs of the body, especially in bone and cartilage. The aim of this study was to investigate the effect of hydrolyzed collagen on bone metabolism, especially in the perspective of osteoporosis treatment and understanding of its mechanism of action. An in vivo study was carried out in 12-week-old female C3H/HeN mice. These were either ovariectomized (OVX) or sham-operated (SHAM) and fed for 12 weeks with a diet containing 10 or 25 g/kg of hydrolyzed collagen. We measured bone mineral density (BMD) using dual-energy X-ray absorptiometry (DXA). C-terminal telopeptide of type I collagen (CTX), marker of bone resorption, and alkaline phosphatase (ALP), marker of bone formation, were assayed after 4 and 12 weeks. Femur biomechanical properties were studied by a 3-point bending test and bone architecture by microtomography. The BMD for OVX mice fed the diet including 25 g/kg of hydrolyzed collagen was significantly higher as compared to OVX mice. The blood CTX level significantly decreased when mice were fed with either of the diets containing hydrolyzed collagen. Finally, we have shown a significant increase in bone strength correlated to geometrical changes for the OVX mice fed the 25 g/kg hydrolyzed collagen diet. Primary cultures of murine bone cells were established from the tibia and femur marrow of BALB/c mice. The growth and differentiation of osteoclasts and osteoblasts cultured with different concentrations (from 0.2 to 1.0 mg/mL) of bovine, porcine or fish hydrolyzed collagens (2 or 5 kDa) were measured. Hydrolyzed collagens (2 or 5 kDa) in the tissue culture medium did not have any significant effects on cell growth as compared to controls. However, there was a significant and dose-dependent increase in ALP activity, a well-known marker of osteogenesis, and a decrease in octeoclast activity in primary culture of bone cells cultured with hydrolyzed collagens (2 kDa only) as compared to the control. It

Effects of fixation and demineralization on bone collagen D-spacing as analyzed by atomic force microscopy.

PubMed

Wallace, Joseph M

2015-04-01

Collagen's role in bone is often considered secondary. As increased attention is paid to collagen, understanding the impact of tissue preservation is important in interpreting experimental results. The goal of this study was to test the hypothesis that bone fixation prior to demineralization would maintain its collagen ultrastructure in an undisturbed state when analyzed using Atomic Force Microscopy (AFM). The anterior diaphysis of a pig femur was cut into 6 mm pieces along its length. Samples were mounted, polished and randomly assigned to control or fixation groups (n = 5/group). Fixation samples were fixed for 24 h prior to demineralization. All samples were briefly demineralized to expose collagen, and imaged using AFM. Mouse tail tendons were also analyzed to explore effects of dehydration and fixation. Measurements from each bone sample were averaged and compared using a Mann-Whitney U-test. Tendon sample means were compared using RMANOVA. To investigate differences in D-spacing distributions, Kolmogorov-Smirnov tests were used. Fixation decreased D-spacing variability within and between bone samples and induced or maintained a higher average D-spacing versus control by shifting the D-spacing population upward. Tendon data indicate that fixing and drying samples leaves collagen near its undisturbed and hydrated native state. Fixation in bone prior to demineralization decreased D-spacing variability. D-spacing was shifted upward in fixed samples, indicating that collagen is stretched with mineral present and relaxes upon its removal. The ability to decrease variability in bone suggests that fixation might increase the power to detect changes in collagen due to disease or other pressures.

Experimental Evaluation of the Effectiveness of Demineralized Bone Matrix and Collagenated Heterologous Bone Grafts Used Alone or in Combination with Platelet-Rich Fibrin on Bone Healing in Sinus Floor Augmentation.

PubMed

Peker, Elif; Karaca, Inci Rana; Yildirim, Benay

2016-01-01

The aim of this study was an experimental evaluation of the effectiveness of demineralized bone matrix (DBM) and collagenated heterologous bone graft (CHBG) used alone or in combination with platelet-rich fibrin on bone healing in sinus floor augmentation procedures. In this study, 36 New Zealand rabbits were used. The bilateral sinus elevation was performed, and 72 defects were obtained. The rabbit maxillary sinuses were divided into four groups according to the augmentation biomaterials obtained: demineralized bone matrix (Grafton DBM Putty, Osteotech; DBM group), DBM combined with platelet-rich fibrin (PRF; DBM + PRF group), collagenated heterologous bone graft (CHBG; Apatos Mix, OsteoBiol, Tecnoss; CHBG group), CHBG combined with PRF (CHBG + PRF group). All groups were sacrificed at 2, 4, and 8 weeks after surgery for histologic, histomorphometric, and immunohistochemical analyses. The inflammatory reaction was moderate to intense at the second week in all groups and declined from 2 to 8 weeks. New bone formation was started at the second week and increased from 2 to 8 weeks in all groups. There was no significant difference in bone formation between the experimental groups that used PRF mixed graft material and control groups that used only graft material. The percentage of new bone formation showed a significant difference in DBM groups and DBM + PRF groups compared with other groups. There were osteoclasts around all the bone graft materials used, but the percentage of residual graft particles was significantly higher in CHBG groups and CHBG + PRF groups at the eighth week. There is no beneficial effect of the application of PRF in combination with demineralized bone matrix or collagenated heterologous bone graft on bone formation in sinus floor augmentation. The results of this study showed that both collagenated heterologous bone graft and demineralized bone matrix have osteoconductive properties, but demineralized bone matrix showed more bone formation

Fabrication and characterization of biomimetic collagen-apatite scaffolds with tunable structures for bone tissue engineering.

PubMed

Xia, Zengmin; Yu, Xiaohua; Jiang, Xi; Brody, Harold D; Rowe, David W; Wei, Mei

2013-07-01

The objective of the current study is to prepare a biomimetic collagen-apatite scaffold for improved bone repair and regeneration. A novel bottom-up approach has been developed, which combines a biomimetic self-assembly method with a controllable freeze-casting technology. In this study, the mineralized collagen fibers were generated using a simple one-step co-precipitation method which involved collagen self-assembly and in situ apatite precipitation in a collagen-containing modified simulated body fluid (m-SBF). The precipitates were then subjected to controllable freeze casting, forming scaffolds with either an isotropic equiaxed structure or a unidirectional lamellar structure. These scaffolds were comprised of collagen fibers and poorly crystalline bone-like carbonated apatite nanoparticles. The mineral content in the scaffold could be tailored in the range 0-54wt.% by simply adjusting the collagen content in the m-SBF. Further, the mechanisms of the formation of both the equiaxed and the lamellar scaffolds were investigated, and freezing regimes for equiaxed and lamellar solidification were established. Finally, the bone-forming capability of such prepared scaffolds was evaluated in vivo in a mouse calvarial defect model. It was confirmed that the scaffolds well support new bone formation. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Accelerated craniofacial bone regeneration through dense collagen gel scaffolds seeded with dental pulp stem cells

NASA Astrophysics Data System (ADS)

Chamieh, Frédéric; Collignon, Anne-Margaux; Coyac, Benjamin R.; Lesieur, Julie; Ribes, Sandy; Sadoine, Jérémy; Llorens, Annie; Nicoletti, Antonino; Letourneur, Didier; Colombier, Marie-Laure; Nazhat, Showan N.; Bouchard, Philippe; Chaussain, Catherine; Rochefort, Gael Y.

2016-12-01

Therapies using mesenchymal stem cell (MSC) seeded scaffolds may be applicable to various fields of regenerative medicine, including craniomaxillofacial surgery. Plastic compression of collagen scaffolds seeded with MSC has been shown to enhance the osteogenic differentiation of MSC as it increases the collagen fibrillary density. The aim of the present study was to evaluate the osteogenic effects of dense collagen gel scaffolds seeded with mesenchymal dental pulp stem cells (DPSC) on bone regeneration in a rat critical-size calvarial defect model. Two symmetrical full-thickness defects were created (5 mm diameter) and filled with either a rat DPSC-containing dense collagen gel scaffold (n = 15), or an acellular scaffold (n = 15). Animals were imaged in vivo by microcomputer tomography (Micro-CT) once a week during 5 weeks, whereas some animals were sacrificed each week for histology and histomorphometry analysis. Bone mineral density and bone micro-architectural parameters were significantly increased when DPSC-seeded scaffolds were used. Histological and histomorphometrical data also revealed significant increases in fibrous connective and mineralized tissue volume when DPSC-seeded scaffolds were used, associated with expression of type I collagen, osteoblast-associated alkaline phosphatase and osteoclastic-related tartrate-resistant acid phosphatase. Results demonstrate the potential of DPSC-loaded-dense collagen gel scaffolds to benefit of bone healing process.

Pyridinium cross-links in bone of patients with osteogenesis imperfecta: evidence of a normal intrafibrillar collagen packing.

PubMed

Bank, R A; Tekoppele, J M; Janus, G J; Wassen, M H; Pruijs, H E; Van der Sluijs, H A; Sakkers, R J

2000-07-01

The brittleness of bone in patients with osteogenesis imperfecta (OI) has been attributed to an aberrant collagen network. However, the role of collagen in the loss of tissue integrity has not been well established. To gain an insight into the biochemistry and structure of the collagen network, the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) and the level of triple helical hydroxylysine (Hyl) were determined in bone of OI patients (types I, III, and IV) as well as controls. The amount of triple helical Hyl was increased in all patients. LP levels in OI were not significantly different; in contrast, the amount of HP (and as a consequence the HP/LP ratio and the total pyridinoline level) was significantly increased. There was no relationship between the sum of pyridinolines and the amount of triple helical Hyl, indicating that lysyl hydroxylation of the triple helix and the telopeptides are under separate control. Cross-linking is the result of a specific three-dimensional arrangement of collagens within the fibril; only molecules that are correctly aligned are able to form cross-links. Inasmuch as the total amount of pyridinoline cross-links in OI bone is similar to control bone, the packing geometry of intrafibrillar collagen molecules is not disturbed in OI. Consequently, the brittleness of bone is not caused by a disorganized intrafibrillar collagen packing and/or loss of cross-links. This is an unexpected finding, because mutant collagen molecules with a random distribution within the fibril are expected to result in disruptions of the alignment of neighboring collagen molecules. Pepsin digestion of OI bone revealed that collagen located at the surface of the fibril had lower cross-link levels compared with collagen located at the inside of the fibril, indicating that mutant molecules are not distributed randomly within the fibril but are located preferentially at the surface of the fibril.

Greener synthesis of electrospun collagen/hydroxyapatite composite fibers with an excellent microstructure for bone tissue engineering

PubMed Central

Zhou, Yuanyuan; Yao, Hongchang; Wang, Jianshe; Wang, Dalu; Liu, Qian; Li, Zhongjun

2015-01-01

In bone tissue engineering, collagen/hydroxyapatite (HAP) fibrous composite obtained via electrospinning method has been demonstrated to support the cells’ adhesion and bone regeneration. However, electrospinning of natural collagen often requires the use of cytotoxic organic solvents, and the HAP crystals were usually aggregated and randomly distributed within a fibrous matrix of collagen, limiting their clinical potential. Here, an effective and greener method for the preparation of collagen/HAP composite fibers was developed for the first time, and this green product not only had 40 times higher mechanical properties than that previously reported, but also had an excellent microstructure similar to that of natural bone. By dissolving type I collagen in environmentally friendly phosphate buffered saline/ethanol solution instead of the frequently-used cytotoxic organic solvents, followed with the key step of desalination, co-electrospinning the collagen solution with the HAP sol, generates a collagen/HAP composite with a uniform and continuous fibrous morphology. Interestingly, the nano-HAP needles were found to preferentially orient along the longitudinal direction of the collagen fibers, which mimicked the nanostructure of natural bones. Based on the characterization of the related products, the formation mechanism for this novel phenomenon was proposed. After cross-linking with 1-ethyl-3-(3-dimethyl-aminopropyl)-1-carbodiimide hydrochloride/N-hydroxysuccinimide, the obtained composite exhibited a significant enhancement in mechanical properties. In addition, the biocompatibility of the obtained composite fibers was evaluated by in vitro culture of the human myeloma cells (U2-OS). Taken together, the process outlined herein provides an effective, non-toxic approach for the fabrication of collagen/HAP composite nanofibers that could be good candidates for bone tissue engineering. PMID:25995630

Greener synthesis of electrospun collagen/hydroxyapatite composite fibers with an excellent microstructure for bone tissue engineering.

PubMed

Zhou, Yuanyuan; Yao, Hongchang; Wang, Jianshe; Wang, Dalu; Liu, Qian; Li, Zhongjun

2015-01-01

In bone tissue engineering, collagen/hydroxyapatite (HAP) fibrous composite obtained via electrospinning method has been demonstrated to support the cells' adhesion and bone regeneration. However, electrospinning of natural collagen often requires the use of cytotoxic organic solvents, and the HAP crystals were usually aggregated and randomly distributed within a fibrous matrix of collagen, limiting their clinical potential. Here, an effective and greener method for the preparation of collagen/HAP composite fibers was developed for the first time, and this green product not only had 40 times higher mechanical properties than that previously reported, but also had an excellent microstructure similar to that of natural bone. By dissolving type I collagen in environmentally friendly phosphate buffered saline/ethanol solution instead of the frequently-used cytotoxic organic solvents, followed with the key step of desalination, co-electrospinning the collagen solution with the HAP sol, generates a collagen/HAP composite with a uniform and continuous fibrous morphology. Interestingly, the nano-HAP needles were found to preferentially orient along the longitudinal direction of the collagen fibers, which mimicked the nanostructure of natural bones. Based on the characterization of the related products, the formation mechanism for this novel phenomenon was proposed. After cross-linking with 1-ethyl-3-(3-dimethyl-aminopropyl)-1-carbodiimide hydrochloride/N-hydroxysuccinimide, the obtained composite exhibited a significant enhancement in mechanical properties. In addition, the biocompatibility of the obtained composite fibers was evaluated by in vitro culture of the human myeloma cells (U2-OS). Taken together, the process outlined herein provides an effective, non-toxic approach for the fabrication of collagen/HAP composite nanofibers that could be good candidates for bone tissue engineering.

Micro-Nanostructures of Cellulose-Collagen for Critical Sized Bone Defect Healing.

PubMed

Aravamudhan, Aja; Ramos, Daisy M; Nip, Jonathan; Kalajzic, Ivo; Kumbar, Sangamesh G

2018-02-01

Bone tissue engineering strategies utilize biodegradable polymeric matrices alone or in combination with cells and factors to provide mechanical support to bone, while promoting cell proliferation, differentiation, and tissue ingrowth. The performance of mechanically competent, micro-nanostructured polymeric matrices, in combination with bone marrow stromal cells (BMSCs), is evaluated in a critical sized bone defect. Cellulose acetate (CA) is used to fabricate a porous microstructured matrix. Type I collagen is then allowed to self-assemble on these microstructures to create a natural polymer-based, micro-nanostructured matrix (CAc). Poly (lactic-co-glycolic acid) matrices with identical microstructures serve as controls. Significantly higher number of implanted host cells are distributed in the natural polymer based micro-nanostructures with greater bone density and more uniform cell distribution. Additionally, a twofold increase in collagen content is observed with natural polymer based scaffolds. This study establishes the benefits of natural polymer derived micro-nanostructures in combination with donor derived BMSCs to repair and regenerate critical sized bone defects. Natural polymer based materials with mechanically competent micro-nanostructures may serve as an alternative material platform for bone regeneration. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probabilistic failure analysis of bone using a finite element model of mineral-collagen composites.

PubMed

Dong, X Neil; Guda, Teja; Millwater, Harry R; Wang, Xiaodu

2009-02-09

Microdamage accumulation is a major pathway for energy dissipation during the post-yield deformation of bone. In this study, a two-dimensional probabilistic finite element model of a mineral-collagen composite was developed to investigate the influence of the tissue and ultrastructural properties of bone on the evolution of microdamage from an initial defect in tension. The probabilistic failure analyses indicated that the microdamage progression would be along the plane of the initial defect when the debonding at mineral-collagen interfaces was either absent or limited in the vicinity of the defect. In this case, the formation of a linear microcrack would be facilitated. However, the microdamage progression would be scattered away from the initial defect plane if interfacial debonding takes place at a large scale. This would suggest the possible formation of diffuse damage. In addition to interfacial debonding, the sensitivity analyses indicated that the microdamage progression was also dependent on the other material and ultrastructural properties of bone. The intensity of stress concentration accompanied with microdamage progression was more sensitive to the elastic modulus of the mineral phase and the nonlinearity of the collagen phase, whereas the scattering of failure location was largely dependent on the mineral to collagen ratio and the nonlinearity of the collagen phase. The findings of this study may help understanding the post-yield behavior of bone at the ultrastructural level and shed light on the underlying mechanism of bone fractures.

Probabilistic Failure Analysis of Bone Using a Finite Element Model of Mineral-Collagen Composites

PubMed Central

Dong, X. Neil; Guda, Teja; Millwater, Harry R.; Wang, Xiaodu

2009-01-01

Microdamage accumulation is a major pathway for energy dissipation during the post-yield deformation of bone. In this study, a two-dimensional probabilistic finite element model of a mineral-collagen composite was developed to investigate the influence of the tissue and ultrastructural properties of bone on the evolution of microdamage from an initial defect in tension. The probabilistic failure analyses indicated that the microdamage progression would be along the plane of the initial defect when the debonding at mineral-collagen interfaces was either absent or limited in the vicinity of the defect. In this case, the formation of a linear microcrack would be facilitated. However, the microdamage progression would be scattered away from the initial defect plane if interfacial debonding takes place at a large scale. This would suggest the possible formation of diffuse damage. In addition to interfacial debonding, the sensitivity analyses indicated that the microdamage progression was also dependent on the other material and ultrastructural properties of bone. The intensity of stress concentration accompanied with microdamage progression was more sensitive to the elastic modulus of the mineral phase and the nonlinearity of the collagen phase, whereas the scattering of failure location was largely dependent on the mineral to collagen ratio and the nonlinearity of the collagen phase. The findings of this study may help understanding the post-yield behavior of bone at the ultrastructural level and shed light on the underlying mechanism of bone fractures. PMID:19058806

Osteogenesis imperfecta due to mutations in non-collagenous genes: lessons in the biology of bone formation.

PubMed

Marini, Joan C; Reich, Adi; Smith, Simone M

2014-08-01

Osteogenesis imperfecta or 'brittle bone disease' has mainly been considered a bone disorder caused by collagen mutations. Within the last decade, however, a surge of genetic discoveries has created a new paradigm for osteogenesis imperfecta as a collagen-related disorder, where most cases are due to autosomal dominant type I collagen defects, while rare, mostly recessive, forms are due to defects in genes whose protein products interact with collagen protein. This review is both timely and relevant in outlining the genesis, development, and future of this paradigm shift in the understanding of osteogenesis imperfecta. Bone-restricted interferon-induced transmembrane (IFITM)-like protein (BRIL) and pigment epithelium-derived factor (PEDF) defects cause types V and VI osteogenesis imperfecta via defective bone mineralization, while defects in cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1), and cyclophilin B (CYPB) cause types VII-IX osteogenesis imperfecta via defective collagen post-translational modification. Heat shock protein 47 (HSP47) and FK506-binding protein-65 (FKBP65) defects cause types X and XI osteogenesis imperfecta via aberrant collagen crosslinking, folding, and chaperoning, while defects in SP7 transcription factor, wingless-type MMTV integration site family member 1 (WNT1), trimeric intracellular cation channel type b (TRIC-B), and old astrocyte specifically induced substance (OASIS) disrupt osteoblast development. Finally, absence of the type I collagen C-propeptidase bone morphogenetic protein 1 (BMP1) causes type XII osteogenesis imperfecta due to altered collagen maturation/processing. Identification of these multiple causative defects has provided crucial information for accurate genetic counseling, inspired a recently proposed functional grouping of osteogenesis imperfecta types by shared mechanism to simplify current nosology, and has prodded investigations into common pathways in osteogenesis imperfecta. Such

Functional grading of mineral and collagen in the attachment of tendon to bone.

PubMed

Genin, Guy M; Kent, Alistair; Birman, Victor; Wopenka, Brigitte; Pasteris, Jill D; Marquez, Pablo J; Thomopoulos, Stavros

2009-08-19

Attachment of dissimilar materials is a major challenge because high levels of localized stress may develop at their interfaces. An effective biologic solution to this problem exists at one of nature's most extreme interfaces: the attachment of tendon (a compliant, structural "soft tissue") to bone (a stiff, structural "hard tissue"). The goal of our study was to develop biomechanical models to describe how the tendon-to-bone insertion derives its mechanical properties. We examined the tendon-to-bone insertion and found two factors that give the tendon-to-bone transition a unique grading in mechanical properties: 1), a gradation in mineral concentration, measured by Raman spectroscopy; and 2), a gradation in collagen fiber orientation, measured by polarized light microscopy. Our measurements motivate a new physiological picture of the tissue that achieves this transition, the tendon-to-bone insertion, as a continuous, functionally graded material. Our biomechanical model suggests that the experimentally observed increase in mineral accumulation within collagen fibers can provide significant stiffening of the partially mineralized fibers, but only for concentrations of mineral above a "percolation threshold" corresponding to formation of a mechanically continuous mineral network within each collagen fiber (e.g., the case of mineral connectivity extending from one end of the fiber to the other). Increasing dispersion in the orientation distribution of collagen fibers from tendon to bone is a second major determinant of tissue stiffness. The combination of these two factors may explain the nonmonotonic variation of stiffness over the length of the tendon-to-bone insertion reported previously. Our models explain how tendon-to-bone attachment is achieved through a functionally graded material composition, and provide targets for tissue engineered surgical interventions and biomimetic material interfaces.

Accelerated craniofacial bone regeneration through dense collagen gel scaffolds seeded with dental pulp stem cells

PubMed Central

Chamieh, Frédéric; Collignon, Anne-Margaux; Coyac, Benjamin R.; Lesieur, Julie; Ribes, Sandy; Sadoine, Jérémy; Llorens, Annie; Nicoletti, Antonino; Letourneur, Didier; Colombier, Marie-Laure; Nazhat, Showan N.; Bouchard, Philippe; Chaussain, Catherine; Rochefort, Gael Y.

2016-01-01

Therapies using mesenchymal stem cell (MSC) seeded scaffolds may be applicable to various fields of regenerative medicine, including craniomaxillofacial surgery. Plastic compression of collagen scaffolds seeded with MSC has been shown to enhance the osteogenic differentiation of MSC as it increases the collagen fibrillary density. The aim of the present study was to evaluate the osteogenic effects of dense collagen gel scaffolds seeded with mesenchymal dental pulp stem cells (DPSC) on bone regeneration in a rat critical-size calvarial defect model. Two symmetrical full-thickness defects were created (5 mm diameter) and filled with either a rat DPSC-containing dense collagen gel scaffold (n = 15), or an acellular scaffold (n = 15). Animals were imaged in vivo by microcomputer tomography (Micro-CT) once a week during 5 weeks, whereas some animals were sacrificed each week for histology and histomorphometry analysis. Bone mineral density and bone micro-architectural parameters were significantly increased when DPSC-seeded scaffolds were used. Histological and histomorphometrical data also revealed significant increases in fibrous connective and mineralized tissue volume when DPSC-seeded scaffolds were used, associated with expression of type I collagen, osteoblast-associated alkaline phosphatase and osteoclastic-related tartrate-resistant acid phosphatase. Results demonstrate the potential of DPSC-loaded-dense collagen gel scaffolds to benefit of bone healing process. PMID:27934940

Rheological, biocompatibility and osteogenesis assessment of fish collagen scaffold for bone tissue engineering.

PubMed

Elango, Jeevithan; Zhang, Jingyi; Bao, Bin; Palaniyandi, Krishnamoorthy; Wang, Shujun; Wenhui, Wu; Robinson, Jeya Shakila

2016-10-01

In the present investigation, an attempt was made to find an alternative to mammalian collagen with better osteogenesis ability. Three types of collagen scaffolds - collagen, collagen-chitosan (CCH), and collagen-hydroxyapatite (CHA) - were prepared from the cartilage of Blue shark and investigated for their physico-functional and mechanical properties in relation to biocompatibility and osteogenesis. CCH scaffold was superior with pH 4.5-4.9 and viscosity 9.7-10.9cP. Notably, addition of chitosan and HA (hydroxyapatite) improved the stiffness (11-23MPa) and degradation rate but lowered the water binding capacity and porosity of the scaffold. Interestingly, CCH scaffolds remained for 3days before complete in-vitro biodegradation. The decreased amount of viable T-cells and higher level of FAS/APO-1 were substantiated the biocompatibility properties of prepared collagen scaffolds. Osteogenesis study revealed that the addition of CH and HA in both fish and mammalian collagen scaffolds could efficiently promote osteoblast cell formation. The ALP activity was significantly high in CHA scaffold-treated osteoblast cells, which suggests an enhanced bone-healing process. Therefore, the present study concludes that the composite scaffolds prepared from fish collagen with higher stiffness, lower biodegradation rate, better biocompatible, and osteogenesis properties were suitable biomaterial for a bone tissue engineering application as an alternative to mammalian collagen scaffolds. Copyright © 2016 Elsevier B.V. All rights reserved.

Persistence of bone collagen cross-links in skeletons of the Nuraghi population living in Sardinia 1500-1200 B.C

NASA Technical Reports Server (NTRS)

Wojtowicz, A.; Yamauchi, M.; Montella, A.; Bandiera, P.; Sotowski, R.; Ostrowski, K.

1999-01-01

Bone collagen has a specific molecular ultrastructure which can be proved by birefringence. This protein, forming the main organic component of bone tissue, is known to survive millennia in paleontological bones and teeth. Birefringence of bone collagen obtained from the skeletons of the Nuraghi population living in Sardinia c-ca 1500 years B.C. was found previously by the use of polarizing microscopy [1]. In this paper, using high pressure liquid chromatography (HPLC) techniques, we show the existence of bone collagen cross-links preserved in Nuraghi skeletons after more than 3000 years.

Bone resorption analysis of platelet-derived growth factor type BB application on collagen for bone grafts secured by titanium mesh over a pig jaw defect model

PubMed Central

Herford, Alan Scott; Cicciù, Marco

2012-01-01

Purpose: The aim of this investigation was to evaluate whether the addition of the platelet derived growth factor type BB (PDGF-BB) to a collagen matrix applied on a titanium mesh would favor healing and resorption onto the grafted bone. A histologic and radiographic study of two different groups (test and control) was performed. Designs: A surgical procedure was performed on 8 pigs to obtain 16 bilateral mandibular alveolar defects. All the defects were then reconstructed with a mixture of autogenous bovine bone using titanium mesh positioning. Two groups, with a total of 16 defects were created: The first to study collagen sponge and PDGF-BB and the second to control collagen only. The collagen matrix was positioned directly over the mesh and soft tissue was closed without tensions onto both groups without attempting to obtain primary closure. Possible exposure of the titanium mesh as well as the height and volume of the new bone was recorded. Results: New bone formation averaged about 6.68 mm in the test group studied; the control group had less regenerated bone at 4.62 mm. Conclusion: PDGF-BB addition to the collagen matrix induced a strong increase in hard and soft tissue healing and favored bone formation, reducing bone resorption even if the mesh was exposed. PMID:23833493

Red deer bone and antler collagen are not isotopically equivalent in carbon and nitrogen.

PubMed

Stevens, Rhiannon E; O'Connell, Tamsin C

2016-09-15

Bone and antler collagen δ(13) C and δ(15) N values are often assumed to be equivalent when measured in palaeodietary, palaeoclimate and palaeocological studies. Although compositionally similar, bone grows slowly and is remodelled whereas antler growth is rapid and remodelling does not occur. These different patterns of growth could result in isotopic difference within antler and between the two tissue types. Here we test whether red deer (Cervus elaphus) bone and antler δ(13) C and δ(15) N values are equivalent, and whether intra-antler isotopic values are uniform. Bone and antler were isotopically analysed from six stags that lived in a temperate maritime climate on the Isle of Rum, Scotland. Multiple antlers from different years were sampled per individual, together with a single bone sample per individual. Up to 12 samples were taken along the length of each antler (total of 25 antlers, 259 samples) so that a chronological record of the isotopic composition during antler growth could be obtained. Collagen was extracted and its δ(13) C and δ(15) N values were measured by continuous-flow isotope ratio mass spectrometry. Intra-antler collagen isotope signatures vary, and show that not all antlers from an individual or a growth year are equivalent in carbon and nitrogen isotopic ratios. δ(15) N values typically increase with distance along antler length, but no overall trend is observed in δ(13) C values. An isotopic offset is visible between bone and antler, with bone δ(13) C and δ(15) N values being higher in most cases. Bone and antler collagen δ(13) C and δ(15) N values are not isotopically equivalent and are therefore not directly comparable in palaeodietary, palaeoclimate and palaeocological studies. Bone and antler collagen isotopic differences probably relate to differential metabolic processes during the formation of the two tissues. Intra- and inter-antler isotopic variations probably reflect the isotopic composition of an individual's diet

Osteogenesis Imperfecta due to Mutations in Non-Collagenous Genes-Lessons in the Biology of Bone Formation

PubMed Central

Marini, Joan C.; Reich, Adi; Smith, Simone M.

2014-01-01

Purpose of Review Osteogenesis imperfecta (OI), or “brittle bone disease”, has mainly been considered a bone disorder caused by collagen mutations. Within the last decade, however, a surge of genetic discoveries has created a new paradigm for OI as a collagen-related disorder, where autosomal dominant type I collagen defects cause most cases, while rare, mostly recessive forms are due to defects in genes whose protein products interact with collagen protein. This review is both timely and relevant in outlining the genesis, development and future of this paradigm shift in the understanding of OI. Recent Findings BRIL and PEDF defects cause types V and VI OI via defective bone mineralization, while defects in CRTAP, P3H1 and CyPB cause types VII-IX via defective collagen post-translational modification. Hsp47 and FKBP65 defects cause types X and XI OI via aberrant collagen crosslinking, folding and chaperoning, while defects in SP7, WNT1, TRIC-B and OASIS disrupt osteoblast development. Finally, absence of the type I collagen C-propeptidase BMP1 causes type XII OI due to altered collagen maturation/processing. Summary Identification of these multiple causative defects has provided crucial information for accurate genetic counseling, inspired a recently proposed functional grouping of OI types by shared mechanism to simplify current nosology, and should prod investigations into common pathways in OI. Such investigations could yield critical information on cellular and bone tissue mechanisms and translate to new mechanistic insight into clinical therapies for patients. PMID:25007323

Normal Collagen and Bone Production by Gene-targeted Human Osteogenesis Imperfecta iPSCs

PubMed Central

Deyle, David R; Khan, Iram F; Ren, Gaoying; Wang, Pei-Rong; Kho, Jordan; Schwarze, Ulrike; Russell, David W

2012-01-01

Osteogenesis imperfecta (OI) is caused by dominant mutations in the type I collagen genes. In principle, the skeletal abnormalities of OI could be treated by transplantation of patient-specific, bone-forming cells that no longer express the mutant gene. Here, we develop this approach by isolating mesenchymal cells from OI patients, inactivating their mutant collagen genes by adeno-associated virus (AAV)-mediated gene targeting, and deriving induced pluripotent stem cells (iPSCs) that were expanded and differentiated into mesenchymal stem cells (iMSCs). Gene-targeted iMSCs produced normal collagen and formed bone in vivo, but were less senescent and proliferated more than bone-derived MSCs. To generate iPSCs that would be more appropriate for clinical use, the reprogramming and selectable marker transgenes were removed by Cre recombinase. These results demonstrate that the combination of gene targeting and iPSC derivation can be used to produce potentially therapeutic cells from patients with genetic disease. PMID:22031238

The predominant role of collagen in the nucleation, growth, structure and orientation of bone apatite

NASA Astrophysics Data System (ADS)

Wang, Yan; Azaïs, Thierry; Robin, Marc; Vallée, Anne; Catania, Chelsea; Legriel, Patrick; Pehau-Arnaudet, Gérard; Babonneau, Florence; Giraud-Guille, Marie-Madeleine; Nassif, Nadine

2012-08-01

The involvement of collagen in bone biomineralization is commonly admitted, yet its role remains unclear. Here we show that type I collagen in vitro can initiate and orientate the growth of carbonated apatite mineral in the absence of any other vertebrate extracellular matrix molecules of calcifying tissues. We also show that the collagen matrix influences the structural characteristics on the atomic scale, and controls the size and the three-dimensional distribution of apatite at larger length scales. These results call into question recent consensus in the literature on the need for Ca-rich non-collagenous proteins for collagen mineralization to occur in vivo. Our model is based on a collagen/apatite self-assembly process that combines the ability to mimic the in vivo extracellular fluid with three major features inherent to living bone tissue, that is, high fibrillar density, monodispersed fibrils and long-range hierarchical organization.

Glyoxal Crosslinking of Cell-Seeded Chitosan/Collagen Hydrogels for Bone Regeneration

PubMed Central

Wang, Limin; Stegemann, Jan P.

2011-01-01

Chitosan and collagen are natural biomaterials that have been used extensively in tissue engineering, both separately and as composite materials. Most methods to fabricate chitosan/collagen composites use freeze drying and chemical crosslinking to create stable porous scaffolds, which subsequently can be seeded with cells. In this study, we directly embedded human bone marrow stem cells (hBMSC) in chitosan/collagen materials by initiating gelation using β-glycerophosphate at physiological temperature and pH. We further examined the use of glyoxal, a dialdehyde with relatively low toxicity, to crosslink these materials and characterized the resulting changes in matrix and cell properties. The cytocompatibility of glyoxal and the crosslinked gels were investigated in terms of hBMSC metabolic activity, viability, proliferation, and osteogenic differentiation. These studies revealed that glyoxal was cytocompatible at concentrations below about 1 mM for periods of exposure up to 15 h, though the degree of cell spreading and proliferation were dependent on matrix composition. Glyoxal-crosslinked matrices were stiffer and compacted less than uncrosslinked controls. It was further demonstrated that hBMSC can attach and proliferate in 3D matrices composed of 50/50 chitosan/collagen, and that these materials supported osteogenic differentiation in response to stimulation. Such glyoxal-crosslinked chitosan/collagen composite materials may find utility as cell delivery vehicles for enhancing the repair of bone defects. PMID:21345389

Elastic properties of woven bone: effect of mineral content and collagen fibrils orientation.

PubMed

García-Rodríguez, J; Martínez-Reina, J

2017-02-01

Woven bone is a type of tissue that forms mainly during fracture healing or fetal bone development. Its microstructure can be modeled as a composite with a matrix of mineral (hydroxyapatite) and inclusions of collagen fibrils with a more or less random orientation. In the present study, its elastic properties were estimated as a function of composition (degree of mineralization) and fibril orientation. A self-consistent homogenization scheme considering randomness of inclusions' orientation was used for this purpose. Lacuno-canalicular porosity in the form of periodically distributed void inclusions was also considered. Assuming collagen fibrils to be uniformly oriented in all directions led to an isotropic tissue with a Young's modulus [Formula: see text] GPa, which is of the same order of magnitude as that of woven bone in fracture calluses. By contrast, assuming fibrils to have a preferential orientation resulted in a Young's modulus in the preferential direction of 9-16 GPa depending on the mineral content of the tissue. These results are consistent with experimental evidence for woven bone in foetuses, where collagen fibrils are aligned to a certain extent.

Increased cartilage type II collagen degradation in patients with osteogenesis imperfecta used as a human model of bone type I collagen alterations.

PubMed

Rousseau, Jean-Charles; Chevrel, Guillaume; Schott, Anne-Marie; Garnero, Patrick

2010-04-01

We investigated whether cartilage degradation is altered in adult patients with mild osteogenesis imperfecta (OI) used as a human model of bone type I collagen-related osteoarthritis (OA). Sixty-four adult patients with OI (39% women, mean age+/-SD: 37+/-12 years) and 64 healthy age-matched controls (54% women, 39+/-7 years) were included. We also compared data in 87 patients with knee OA (73% women, 63+/-8 years, mean disease duration: 6 years) and 291 age-matched controls (80% women, 62+/-10 years). Urinary C-terminal cross-linked telopeptide of type II collagen (CTX-II), a marker of cartilage degradation, urinary helical peptide of type I collagen (Helix-I), a marker of bone resorption, and the urinary ratio between non-isomerised/isomerised (alpha/beta CTX-I) type I collagen C-telopeptide, a marker of type I collagen maturation, were measured. Patients with OI had CTX-II levels similar to those of subjects with knee OA (p=0.89; mean+/-SEM; 460+/-57 ng/mmol Cr for OI group and 547+/-32 ng/mmol Cr for OA group) and significantly higher than both young (144+/-7.8 ng/mmol Cr, p<0.0001) and old controls (247+/-7 ng/mmol Cr, p<0.0001). In patients with OI, increased Helix-I (p<0.0001) and alpha/beta CTX-I (p=0.0067) were independently associated with increased CTX-II and together explained 26% of its variance (p< 0.0001). In patients with knee OA, increased levels of alpha/beta CTX-I ratio were also associated with higher CTX-II levels. Adult patients with OI or knee OA are characterized by increased cartilage type II collagen degradation, which is associated with increased type I collagen degradation for OI and lower type I collagen maturation for both OI and OA. These data suggest that both quantitative and qualitative alterations of bone type I collagen metabolism are involved in increased cartilage degradation in patients with OI or knee OA. Copyright 2009 Elsevier Inc. All rights reserved.

Chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines formed by cross-link of bone and synovium collagen.

PubMed

Anastasia, Luigi; Rota, Paola; Anastasia, Mario; Allevi, Pietro

2013-09-21

This review focuses on the chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines (Pyds), fluorescent collagen cross-links, with a pyridinium salt structure. Pyds derive from the degradation of bone collagen and have attracted attention for their use as biochemical markers of bone resorption and to assess fracture risk prediction in persons suffering from osteoporosis, bone cancer and other bone or collagen diseases. We consider and critically discuss all reported syntheses of free and glycosylated Pyds evidencing an unrevised chemistry, original and of general utility, analysis of which allows us to also support a previously suggested non-enzymatic formation of Pyds in collagen better rationalizing and justifying the chemical events.

Bone-to-bone Fixation Enhances Functional Healing of the Porcine Anterior Cruciate Ligament Using a Collagen-Platelet Composite

PubMed Central

Murray, Martha M.; Magarian, Elise; Zurakowski, David; Fleming, Braden C.

2010-01-01

Purpose The purpose of this study was to determine if providing bony stabilization between the tibia and femur would improve the structural properties of an “enhanced” ACL repair using a collagen-platelet composite when compared to the traditional (Marshall) suture technique. Methods Twelve pigs underwent unilateral ACL transection and were treated with sutures connecting the bony femoral ACL attachment site to the distal ACL stump (LIGAMENT group), or to the tibia via a bone tunnel (TIBIA group). A collagen-platelet composite was placed around the sutures to enhance the biologic repair in both groups. Anteroposterior (AP) knee laxity and the graft structural properties were measured after 15 weeks of healing in both the ACL-repaired and contralateral ACL-intact joints. Results Enhanced ACL repair with bone-to-bone fixation significantly improved yield load and linear stiffness of the ACL repairs (p<0.05) after 15 weeks of healing. However, laxity values of the knees were similar in both groups of repaired knees (p>0.10). Conclusions Using an enhanced ACL suture repair technique that includes bone-to-bone fixation to protect the repair in the initial healing stages resulted in an ACL with improved structural properties after 15 weeks in the porcine model. Clinical Relevance The healing response of an ACL suture repair using a collagen-platelet composite can be enhanced by providing bony stabilization between the tibia and femur to protect the graft during the initial healing process in a translational model. PMID:20810092

Nanocellulose-collagen-apatite composite associated with osteogenic growth peptide for bone regeneration.

PubMed

Saska, Sybele; Teixeira, Lucas Novaes; de Castro Raucci, Larissa Moreira Spinola; Scarel-Caminaga, Raquel Mantuaneli; Franchi, Leonardo Pereira; Dos Santos, Raquel Alves; Santagneli, Silvia Helena; Capela, Marisa Veiga; de Oliveira, Paulo Tambasco; Takahashi, Catarina Satie; Gaspar, Ana Maria Minarelli; Messaddeq, Younès; Ribeiro, Sidney José Lima; Marchetto, Reinaldo

2017-10-01

Despite advances in the field of biomaterials for bone repair/regeneration, some challenges for developing an ideal bone substitute need to be overcome. Herein, this study synthesized and evaluated in vitro a nanocomposite based on bacterial cellulose (BC), collagen (COL), apatite (Ap) and osteogenic growth peptide (OGP) or its C-terminal pentapeptide [OGP(10-14)] for bone regeneration purposes. The BC-COL nanocomposites were successfully obtained by carbodiimide-mediated coupling as demonstrated by spectroscopy analysis. SEM, FTIR and 31 P NMR analyses revealed that in situ synthesis to apatite was an effective route for obtaining of bone-like apatite. The OGP-containing (BC-COL)-Ap stimulated the early development of the osteoblastic phenotype. Additionally, the association among collagen, apatite, and OGP peptides enhanced cell growth compared with OGP-containing BC-Ap. Furthermore, none of the nanocomposites showed cytotoxic, genotoxic or mutagenic effects. These promising results suggest that the (BC-COL)-Ap associated with OGP peptides might be considered a potential candidate for bone tissue engineering applications. Copyright © 2017 Elsevier B.V. All rights reserved.

The influence of collagen membrane and autogenous bone chips on bone augmentation in the anterior maxilla: a preclinical study.

PubMed

Janner, Simone F M; Bosshardt, Dieter D; Cochran, David L; Chappuis, Vivianne; Huynh-Ba, Guy; Jones, Archie A; Buser, Daniel

2017-11-01

To evaluate the effect of a resorbable collagen membrane and autogenous bone chips combined with deproteinized bovine bone mineral (DBBM) on the healing of buccal dehiscence-type defects. The second incisors and the first premolars were extracted in the maxilla of eight mongrels. Reduced diameter, bone-level implants were placed 5 weeks later. Standardized buccal dehiscence-type defects were created and grafted at implant surgery. According to an allocation algorithm, the graft composition of each of the four maxillary sites was DBBM + membrane (group D + M), autogenous bone chips + DBBM + membrane (group A + D + M), DBBM alone (group D) or autogenous bone chips + DBBM (group A + D). Four animals were sacrificed after 3 weeks of healing and four animals after 12 weeks. Histological and histomorphometric analyses were performed on oro-facial sections. The pattern of bone formation and resorption within the grafted area showed high variability among the same group and healing time. The histomorphometric analysis of the 3-week specimens showed a positive effect of autogenous bone chips on both implant osseointegration and bone formation into the grafted region (P < 0.05). The presence of the collagen membrane correlated with greater bone formation around the DBBM particles and greater bone formation in the grafted region after 12 weeks of healing (P < 0.05). The oro-facial width of the augmented region at the level of the implant shoulder was significantly reduced in cases where damage of the protection splints occurred in the first week of healing (P < 0.05). The addition of autogenous bone chips and the presence of the collagen membrane increased bone formation around DBBM particles. Wound protection from mechanical noxa during early healing may be critical for bone formation within the grafted area. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Preparation of flexible bone tissue scaffold utilizing sea urchin test and collagen.

PubMed

Manchinasetty, Naga Vijaya Lakshmi; Oshima, Sho; Kikuchi, Masanori

2017-10-13

Gonads of sea urchin are consumed in Japan and some countries as food and most parts including its tests are discarded as marine wastes. Therefore, utilization of them as functional materials would reduce the waste as well as encourage Japanese fishery. In this study, magnesium containing calcite granules collected from sea urchin tests were hydrothermally phosphatized and the obtained granules were identified as approximately 82% in mass of magnesium containing β-tricalcium phosphate and 18% in mass of nonstoichiometric hydroxyapatite, i.e., a biphasic calcium phosphate, maintaining the original porous network. Shape-controlled scaffolds were fabricated with the obtained biphasic calcium phosphate granules and collagen. The scaffolds showed good open porosity (83.84%) and adequate mechanical properties for handling during cell culture and subsequent operations. The MG-63 cells showed higher proliferation and osteogenic differentiation in comparison to a control material, the collagen sponge with the same size. Furthermore, cell viability assay proved that the scaffolds were not cytotoxic. These results suggest that scaffold prepared using sea urchin test derived calcium phosphate and collagen could be a potential candidate of bone void fillers for non-load bearing defects in bone reconstruction as well as scaffolds for bone tissue engineering.

Osteolytic and mixed cancer metastasis modulates collagen and mineral parameters within rat vertebral bone matrix.

PubMed

Burke, Mikhail V; Atkins, Ayelet; Akens, Margarete; Willett, Thomas L; Whyne, Cari M

2016-12-01

Metastatic involvement in vertebral bone diminishes the mechanical integrity of the spine; however minimal data exist on the potential impact of metastases on the intrinsic material characteristics of the bone matrix. Thirty-four (34) female athymic rats were inoculated with HeLa (N = 17) or Ace-1 (N = 17) cancer cells lines producing osteolytic or mixed (osteolytic and osteoblastic) metastases, respectively. A maximum of 21 days was allowed between inoculation and rat sacrifice for vertebrae extraction. High performance liquid chromatography (HPLC) was utilized to determine modifications in collagen-I parameters such as proline hydroxylation and the formation of specific enzymatic and non-enzymatic (pentosidine) cross-links. Raman spectroscopy was used to determine relative changes in mineral crystallinity, mineral carbonation, mineral/collagen matrix ratio, collagen quality ratio, and proline hydroxylation. HPLC results showed significant increase in the formation of pentosidine and decrease in the formation of the enzymatic cross-link deoxy-pryridinoline within osteolytic bone compared to mixed bone. Raman results showed decreased crystallinity, increased carbonation, and collagen quality (aka 1660/1690 sub-band) ratio with osteolytic bone compared to mixed bone and healthy controls along with an observed increase in proline hydroxylation with metastatic involvement. The mineral/matrix ratio decreased in both osteolytic and mixed bone compared to healthy controls. Quantifying modifications within the intrinsic characteristics of bone tissue will provide a foundation to assess the impact of current therapies on the material behavior of bone tissue in the metastatic spine and highlight targets for the development of new therapeutics and approaches for treatment. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2126-2136, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

A new procedure for extraction of collagen from modern and archaeological bones for 14C dating.

PubMed

Maspero, F; Sala, S; Fedi, M E; Martini, M; Papagni, A

2011-10-01

Bones are potentially the best age indicators in a stratigraphic study, because they are closely related to the layer in which they are found. Collagen is the most suitable fraction and is the material normally used in radiocarbon dating. Bone contaminants can strongly alter the carbon isotopic fraction values of the samples, so chemical pretreatment for (14)C dating by accelerator mass spectrometry (AMS) is essential. The most widespread method for collagen extraction is based on the Longin procedure, which consists in HCl demineralization to dissolve the inorganic phase of the samples, followed by dissolution of collagen in a weak acid solution. In this work the possible side effects of this procedure on a modern bone are presented; the extracted collagen was analyzed by ATR-IR spectroscopy. An alternative procedure, based on use of HF instead of HCl, to minimize unwanted degradation of the organic fraction, is also given. A study by ATR-IR spectroscopic analysis of collagen collected after different demineralization times and with different acid volumes, and a study of an archaeological sample, are also presented.

In vitro deposition of hydroxyapatite on cortical bone collagen stimulated by deformation-induced piezoelectricity.

PubMed

Noris-Suárez, Karem; Lira-Olivares, Joaquin; Ferreira, Ana Marina; Feijoo, José Luis; Suárez, Nery; Hernández, Maria C; Barrios, Esteban

2007-03-01

In the present work, we have studied the effect of the piezoelectricity of elastically deformed cortical bone collagen on surface using a biomimetic approach. The mineralization process induced as a consequence of the piezoelectricity effect was evaluated using scanning electron microscopy (SEM), thermally stimulated depolarization current (TSDC), and differential scanning calorimetry (DSC). SEM micrographs showed that mineralization occurred predominantly over the compressed side of bone collagen, due to the effect of piezoelectricity, when the sample was immersed in the simulated body fluid (SBF) in a cell-free system. The TSDC method was used to examine the complex collagen dielectric response. The dielectric spectra of deformed and undeformed collagen samples with different hydration levels were compared and correlated with the mineralization process followed by SEM. The dielectric measurements showed that the mineralization induced significant changes in the dielectric spectra of the deformed sample. DSC and TSDC results demonstrated a reduction of the collagen glass transition as the mineralization process advanced. The combined use of SEM, TSDC, and DSC showed that, even without osteoblasts present, the piezoelectric dipoles produced by deformed collagen can produce the precipitation of hydroxyapatite by electrochemical means, without a catalytic converter as occurs in classical biomimetic deposition.

Bone conditioned media (BCM) improves osteoblast adhesion and differentiation on collagen barrier membranes.

PubMed

Fujioka-Kobayashi, Masako; Caballé-Serrano, Jordi; Bosshardt, Dieter D; Gruber, Reinhard; Buser, Daniel; Miron, Richard J

2016-07-04

The use of autogenous bone chips during guided bone regeneration procedures has remained the gold standard for bone grafting due to its excellent combination of osteoconduction, osteoinduction and osteogenesis. Recent protocols established by our group have characterized specific growth factors and cytokines released from autogenous bone that have the potential to be harvested and isolated into bone conditioned media (BCM). Due to the advantageous osteo-promotive properties of BCM, the aims of the present study was to pre-coat collagen barrier membranes with BCM and investigate its effect on osteoblast adhesion, proliferation and differentiation for possible future clinical use. Scanning electron microscopy (SEM) was first used to qualitative assess BCM protein accumulation on the surface of collagen membranes. Thereafter, undifferentiated mouse ST2 stromal bone marrow cells were seeded onto BioGide porcine derived collagen barrier membranes (control) or barrier membranes pre-coated with BCM (test group). Control and BCM samples were compared for cell adhesion at 8 h, cell proliferation at 1, 3 and 5 days and real-time PCR at 5 days for osteoblast differentiation markers including Runx2, alkaline phosphatase (ALP), osteocalcin (OCN) and bone sialoprotein (BSP). Mineralization was further assessed with alizarin red staining at 14 days post seeding. SEM images demonstrated evidence of accumulated proteins found on the surface of collagen membranes following coating with BCM. Analysis of total cell numbers revealed that the additional pre-coating with BCM markedly increased cell attachment over 4 fold when compared to cells seeded on barrier membranes alone. No significant difference could be observed for cell proliferation at all time points. BCM significantly increased mRNA levels of osteoblast differentiation markers including ALP, OCN and BSP at 5 days post seeding. Furthermore, barrier membranes pre-coated with BCM demonstrated a 5-fold increase in alizarin

Effect of nicotinamide on amino acids content in bone collagen depending on biological availability of vitamins in diabetic rats.

PubMed

Guzyk, M M; Sergiichuk, Iu T; Dyakun, K O; Yanitska, L V; Kuchmerovska, T M

2014-01-01

Connective tissue is highly susceptible to imbalances induced by diabetes. Diabetes-related osteopenia, decreased bone strength etc. may be associated with altered metabolism of various collagens: Although it is assumed that alterations in collagen amino acids (AA) may strongly affect protein properties andphysiological functions, however, very limited evidences are present at the moment regarding AA composition of bone type I collagen and its relevance to abnormal availability of vitamins which are necessary for collagen synthesis in diabetes. We have tested whether nicotinamide (NAm) can influence type Icollagen formation and AA composition as well as vitamins availability in diabetes. After 4 weeks of STZ-induced diabetes (60 mg/ kg) male Wistar rats were injected for 2 weeks with/without NAm (200 mg/kg b. w). Acid extraction of type I collagen from the bones was performed with following stepwise salting out. The content of type I collagen after its acid extraction from the bones was estimated by the amounts of hydroxyproline. Amino acids were assayed by cation exchange chromatography Diabetes-associated changes in AA composition of type I collagen mainly affect those amino acids which are known to be involved in helix formation and cross-linking of the molecules. Diabetes was found to significantly reduce bone collagen contents of o-Pro, Gly, Ala, o-Lys and Pro, whereas Lys, His, Arg, Glu, Thr, Leu, Phe contents were elevated (P < 0.05). NAm treatment was able to partially normalise AA contents. In diabetes, blood serum and hepatic vitamin C and B3 contents were shown to be significantly lowered, whereas a-tocopherol was slightly increased compared with control (P < 0.05). Restoration of circulatory and liver vitamin C and B3 was observed. The data demonstrate the close relationship between the diabetes-associated decrease in type I collagen deposition, altered amino acids metabolism and impaired availability of vitamins, which are necessary for collagen

Collagen type I from bovine bone. Effect of animal age, bone anatomy and drying methodology on extraction yield, self-assembly, thermal behaviour and electrokinetic potential.

PubMed

Ferraro, Vincenza; Gaillard-Martinie, Brigitte; Sayd, Thierry; Chambon, Christophe; Anton, Marc; Santé-Lhoutellier, Véronique

2017-04-01

Natural collagen is easily available from animal tissues such as bones. Main limitations reported in the use of natural collagen are heterogeneity and loss of integrity during recovery. However, its natural complexity, functionality and bioactivity still remain to be achieved through synthetic and recombinant ways. Variability of physicochemical properties of collagen extracted from bovine bone by acetic acid was then investigated taking into account endogenous and exogenous factors. Endogenous: bovine's bones age (4 and 7 years) and anatomy (femur and tibia); exogenous: thermal treatments (spray-drying and lyophilisation). Scanning electron microscopy, spectroscopy (EDS, FTIR, UV/Vis and CD), differential scanning calorimetry (DSC), centesimal composition, mass spectrometry, amino acids and zeta-potential analysis were used for the purpose. Age correlated negatively with yield of recovery and positively with minerals and proteoglycans content. Comparing the anatomy, higher yields were found for tibias, and higher stability of tibias collagen in solution was noticed. Whatever the age and the anatomy, collagens were able to renature and to self-assemble into tri-dimensional structures. Nonetheless thermal stability and kinetics of renaturation were different. Variability of natural collagen with bone age and anatomy, and drying methodology, may be a crucial advantage to conceive tailor-made applications in either the biological or technical sector. Copyright © 2016 Elsevier B.V. All rights reserved.

Disruption of collagen/apatite alignment impairs bone mechanical function in osteoblastic metastasis induced by prostate cancer.

PubMed

Sekita, Aiko; Matsugaki, Aira; Nakano, Takayoshi

2017-04-01

Prostate cancer (PCa) frequently metastasizes to the bone, generally inducing osteoblastic alterations that increase bone brittleness. Although there is growing interest in the management of the physical capability of patients with bone metastasis, the mechanism underlying the impairment of bone mechanical function remains unclear. The alignment of both collagen fibrils and biological apatite (BAp) c-axis, together with bone mineral density, is one of the strongest contributors to bone mechanical function. In this study, we analyzed the bone microstructure of the mouse femurs with and without PCa cell inoculation. Histological assessment revealed that the bone-forming pattern in the PCa-bearing bone was non-directional, resulting in a spongious structure, whereas that in the control bone was unidirectional and layer-by-layer, resulting in a compact lamellar structure. The degree of preferential alignment of collagen fibrils and BAp, which was evaluated by quantitative polarized microscopy and microbeam X-ray diffraction, respectively, were significantly lower in the PCa-bearing bone than in the control bone. Material parameters including Young's modulus and toughness, measured by the three-point bending test, were simultaneously decreased in the PCa-bearing bone. Specifically, there was a significant positive correlation between the degree of BAp c-axis orientation and Young's modulus. In conclusion, the impairment of mechanical function in the PCa-bearing bone is attributable to disruption of the anisotropic microstructure of bone in multiple phases. This is the first report demonstrating that cancer bone metastasis induces disruption of the collagen/BAp alignment in long bones, thereby impairing their mechanical function. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Thermogelling chitosan and collagen composite hydrogels initiated with β-glycerophosphate for bone tissue engineering

PubMed Central

Wang, Limin; Stegemann, Jan P.

2010-01-01

Chitosan and collagen type I are naturally-derived materials used as cell carriers because of their ability to mimic the extracellular environment and direct cell function. In this study beta-glycerophosphate (beta-GP), an osteogenic medium supplement and a weak base, was used to simultaneously initiate gelation of pure chitosan, pure collagen, and chitosan-collagen composite materials at physiological pH and temperature. Adult human bone marrow-derived stem cells (hBMSC) encapsulated in such hydrogels at chitosan/collagen ratios of 100/0, 65/35, 25/75, and 0/100 wt% exhibited high viability at day 1 after encapsulation, but DNA content dropped by about half over 12 days in pure chitosan materials while it increased two-fold in materials containing collagen. Collagen-containing materials compacted more strongly and were significantly stiffer than pure chitosan gels. In monolayer culture, exposure of hBMSC to beta-GP resulted in decreased cell metabolic activity that varied with concentration and exposure time, but washing effectively removed excess beta-GP from hydrogels. The presence of chitosan in materials resulted in higher expression of osterix and bone sialoprotein genes in medium with and without osteogenic supplements. Chitosan also increased alkaline phosphatase activity and calcium deposition in osteogenic medium. Chitosan-collagen composite materials have potential as matrices for cell encapsulation and delivery, or as in situ gel-forming materials for tissue repair. PMID:20170955

Cross-linking connectivity in bone collagen fibrils: the COOH-terminal locus of free aldehyde

NASA Technical Reports Server (NTRS)

Otsubo, K.; Katz, E. P.; Mechanic, G. L.; Yamauchi, M.

1992-01-01

Quantitative analyses of the chemical state of the 16c residue of the alpha 1 chain of bone collagen were performed on samples from fetal (4-6-month embryo) and mature (2-3 year old) bovine animals. All of this residue could be accounted for in terms of three chemical states, in relative amounts which depended upon the age of the animal. Most of the residue was incorporated into either bifunctional or trifunctional cross-links. Some of it, however, was present as free aldehyde, and the content increased with maturation. This was established by isolating and characterizing the aldehyde-containing peptides generated by tryptic digestion of NaB3H4-reduced mature bone collagen. We have concluded that the connectivity of COOH-terminal cross-linking in bone collagen fibrils changes with maturation in the following way: at first, each 16c residue in each of the two alpha 1 chains of the collagen molecule is incorporated into a sheet-like pattern of intermolecular iminium cross-links, which stabilizes the young, nonmineralized fibril as a whole. In time, some of these labile cross-links maturate into pyridinoline while others dissociate back to their precursor form. The latter is likely due to changes in the molecular packing brought about by the mineralization of the collagen fibrils. The resultant reduction in cross-linking connectivity may provide a mechanism for enhancing certain mechanical characteristics of the skeleton of a mature animal.

Ionic liquid as a potential solvent for preparation of collagen-alginate-hydroxyapatite beads as bone filler.

PubMed

Iqbal, Bushra; Sarfaraz, Zenab; Muhammad, Nawshad; Ahmad, Pervaiz; Iqbal, Jibran; Khan, Zia Ul Haq; Gonfa, Girma; Iqbal, Farasat; Jamal, Arshad; Rahim, Abdur

2018-07-01

In this study, collagen/alginate/hydroxyapatite beads having different proportions were prepared as bone fillers for the restoration of osteological defects. Ionic liquid was used to dissolve the collagen and subsequently the solution was mixed with sodium alginate solution. Hydroxyapatite was added in different proportions, with the rationale to enhance mechanical as well as biological properties. The prepared solutions were given characteristic bead shapes by dropwise addition into calcium chloride solution. The prepared beads were characterized using FTIR, XRD, TGA and SEM analysis. Microhardness testing was used to evaluate the mechanical properties. The prepared beads were investigated for water adsorption behavior to ascertain its ability for body fluid uptake and adjusted accordingly to the bone cavity. Drug loading and subsequently the antibacterial activity was investigated for the prepared beads. The biocompatibility was assessed using the hemolysis testing and cell proliferation assay. The prepared collagen-alginate-HA beads, having biocompatibility and good mechanical properties, have showed an option of promising biologically active bone fillers for bone regeneration.

Pigment epithelium-derived factor upregulates collagen I and downregulates matrix metalloproteinase 2 in osteosarcoma cells, and colocalises to collagen I and heat shock protein 47 in fetal and adult bone.

PubMed

Alcantara, Marice B; Nemazannikova, Natalie; Elahy, Mina; Dass, Crispin R

2014-11-01

Pigment epithelium-derived factor (PEDF) has proven anti-osteosarcoma activity. However, the mechanism(s) underpinning its ability to reduce primary bone tumour (osteosarcoma) metastasis is unknown. Adult and fetal murine bone were immunostained for PEDF, collagen I (major protein in bone) and its processing proteins, heat shock protein 47 (HSP47, a chaperone protein for collagen I), membrane type I matrix metalloproteinase (MT1-MMP, a collagenase), and matrix metalloproteinase 2 (MMP-2, which is activated by MT1-MMP). Immunoblotting and immunocytochemistry were used to observe levels of the above biomarkers when human osteosarcoma cells were treated with PEDF. Immunohistochemical staining in adult and fetal bone mirrors collagen I. PEDF localised to ridges of trabecular bone in tibial cortex and to megakaryocytes within bone marrow. Second, we observed that PEDF upregulates collagen I, HSP47 and MT1-MMP, while downregulating MMP-2 in osteosarcoma cells in vitro. PEDF is a promising antagonist to osteosarcoma cell metastasis via downregulation of MMP-2, and can induce tumour cells to further adopt differentiative properties, thereby possibly reducing their aggressive growth in vitro and in vivo. © 2014 Royal Pharmaceutical Society.

Differential Actions of the Endocytic Collagen Receptor uPARAP/Endo180 and the Collagenase MMP-2 in Bone Homeostasis

PubMed Central

Madsen, Daniel H.; Jürgensen, Henrik J.; Ingvarsen, Signe; Melander, Maria C.; Albrechtsen, Reidar; Hald, Andreas; Holmbeck, Kenn; Bugge, Thomas H.; Behrendt, Niels; Engelholm, Lars H.

2013-01-01

A well-coordinated remodeling of uncalcified collagen matrices is a pre-requisite for bone development and homeostasis. Collagen turnover proceeds through different pathways, either involving extracellular reactions exclusively, or being dependent on endocytic processes. Extracellular collagen degradation requires the action of secreted or membrane attached collagenolytic proteases, whereas the alternative collagen degradation pathway proceeds intracellularly after receptor-mediated uptake and delivery to the lysosomes. In this study we have examined the functional interplay between the extracellular collagenase, MMP-2, and the endocytic collagen receptor, uPARAP, by generating mice with combined deficiency of both components. In both uPARAP-deficient and MMP-2-deficient adult mice the length of the tibia and femur was decreased, along with a reduced bone mineral density and trabecular bone quality. An additional decrease in bone length was observed when combining the two deficiencies, pointing to both components being important for the remodeling processes in long bone growth. In agreement with results found by others, a different effect of MMP-2 deficiency was observed in the distinct bone structures of the calvaria. These membranous bones were found to be thickened in MMP-2-deficient mice, an effect likely to be related to an accompanying defect in the canalicular system. Surprisingly, both of the latter defects in MMP-2-deficient mice were counteracted by concurrent uPARAP deficiency, demonstrating that the collagen receptor does not support the same matrix remodeling processes as the MMP in the growth of the skull. We conclude that both uPARAP and MMP-2 take part in matrix turnover processes important for bone growth. However, in some physiological situations, these two components do not support the same step in the growth process. PMID:23940733

Guided bone regeneration with asymmetric collagen-chitosan membranes containing aspirin-loaded chitosan nanoparticles.

PubMed

Zhang, Jiayu; Ma, Shiqing; Liu, Zihao; Geng, Hongjuan; Lu, Xin; Zhang, Xi; Li, Hongjie; Gao, Chenyuan; Zhang, Xu; Gao, Ping

2017-01-01

Membranes allowing the sustained release of drugs that can achieve cell adhesion are very promising for guided bone regeneration. Previous studies have suggested that aspirin has the potential to promote bone regeneration. The purpose of this study was to prepare a local drug delivery system with aspirin-loaded chitosan nanoparticles (ACS) contained in an asymmetric collagen-chitosan membrane (CCM). In this study, the ACS were fabricated using different concentrations of aspirin (5 mg, 25 mg, 50 mg, and 75 mg). The drug release behavior of ACS was studied. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to examine the micromorphology of ACS and aspirin-loaded chitosan nanoparticles contained in chitosan-collagen membranes (ACS-CCM). In vitro bone mesenchymal stem cells (BMSCs) were cultured and critical-sized cranial defects on Sprague-Dawley rats were made to evaluate the effect of the ACS-CCM on bone regeneration. Drug release behavior results of ACS showed that the nanoparticles fabricated in this study could successfully sustain the release of the drug. TEM showed the morphology of the nanoparticles. SEM images indicated that the asymmetric membrane comprised a loose collagen layer and a dense chitosan layer. In vitro studies showed that ACS-CCM could promote the proliferation of BMSCs, and that the degree of differentiated BMSCs seeded on CCMs containing 50 mg of ACS was higher than that of other membranes. Micro-computed tomography showed that 50 mg of ACS-CCM resulted in enhanced bone regeneration compared with the control group. This study shows that the ACS-CCM would allow the sustained release of aspirin and have further osteogenic potential. This membrane is a promising therapeutic approach to guiding bone regeneration.

Injectable calcium sulfate/mineralized collagen-based bone repair materials with regulable self-setting properties.

PubMed

Chen, Zonggang; Liu, Huanye; Liu, Xi; Cui, Fu-Zhai

2011-12-15

An injectable and self-setting bone repair materials (nano-hydroxyapatite/collagen/calcium sulfate hemihydrate, nHAC/CSH) was developed in this study. The nano-hydroxyapatite/collagen (nHAC) composite, which is the mineralized fibril by self-assembly of nano-hydrocyapatite and collagen, has the same features as natural bone in both main hierarchical microstructure and composition. It is a bioactive osteoconductor due to its high level of biocompatibility and appropriate degradation rate. However, this material lacks handling characteristics because of its particle or solid-preformed block shape. Herein, calcium sulfate hemihydrate (CSH) was introduced into nHAC to prepare an injectable and self-setting in situ bone repair materials. The morphology of materials was observed using SEM. Most important and interesting of all, calcium sulfate dihydrate (CSD), which is not only the reactant of preparing CSH but also the final solidified product of CSH, was introduced into nHAC as setting accelerator to regulate self-setting properties of injectable nHAC/CSH composite, and thus the self-setting time of nHAC/CSH composite can be regulated from more than 100 min to about 30 min and even less than 20 min by adding various amount of setting accelerator. The compressive properties of bone graft substitute after final setting are similar to those of cancellous bone. CSD as an excellent setting accelerator has no significant effect on the mechanical property and degradability of bone repair materials. In vitro biocompatibility and in vivo histology studies demonstrated that the nHAC/CSH composite could provide more adequate stimulus for cell adhesion and proliferation, embodying favorable cell biocompatibility and a strong ability to accelerate bone formation. It can offer a satisfactory biological environment for growing new bone in the implants and for stimulating bone formation. Copyright © 2011 Wiley Periodicals, Inc.

Beneath the minerals, a layer of round lipid particles was identified to mediate collagen calcification in compact bone formation.

PubMed

Xu, Shaohua; Yu, Jianqing J

2006-12-01

Astronauts lose 1-2% of their bone minerals per month during space flights. A systematic search for a countermeasure relies on a good understanding of the mechanism of bone formation at the molecular level. How collagen fibers, the dominant matrix protein in bones, are mineralized remains mysterious. Atomic force microscopy was carried out, in combination with immunostaining and Western blotting, on bovine tibia to identify unrecognized building blocks involved in bone formation and for an elucidation of the process of collagen calcification in bone formation. Before demineralization, tiles of hydroxyapatite crystals were found stacked along bundles of collagen fibers. These tiles were homogeneous in size and shape with dimensions 0.69 x 0.77 x 0.2 micro m(3). Demineralization dissolved these tiles and revealed small spheres with an apparent diameter around 145 nm. These spheres appeared to be lipid particles since organic solvents dissolved them. The parallel collagen bundles had widths mostly <2 micro m. Composition analysis of compact bones indicated a high content of apolar lipids, including triglycerides and cholesterol esters. Apolar lipids are known to form lipid droplets or lipoproteins, and these spheres are unlikely to be matrix vesicles as reported for collagen calcification in epiphyseal cartilages. Results from this study suggest that the layer of round lipid particles on collagen fibers mediates the mineral deposition onto the fibers. The homogeneous size of these lipid particles and the presence of apolipoprotein in demineralized bone tissue suggest the possibility that these particles might be of lipoprotein origin. More studies are needed to verify the last claim and to exclude the possibility that they are secreted lipid droplets.

Ductile sliding between mineral crystals followed by rupture of collagen crosslinks: experimentally supported micromechanical explanation of bone strength.

PubMed

Fritsch, Andreas; Hellmich, Christian; Dormieux, Luc

2009-09-21

There is an ongoing discussion on how bone strength could be explained from its internal structure and composition. Reviewing recent experimental and molecular dynamics studies, we here propose a new vision on bone material failure: mutual ductile sliding of hydroxyapatite mineral crystals along layered water films is followed by rupture of collagen crosslinks. In order to cast this vision into a mathematical form, a multiscale continuum micromechanics theory for upscaling of elastoplastic properties is developed, based on the concept of concentration and influence tensors for eigenstressed microheterogeneous materials. The model reflects bone's hierarchical organization, in terms of representative volume elements for cortical bone, for extravascular and extracellular bone material, for mineralized fibrils and the extrafibrillar space, and for wet collagen. In order to get access to the stress states at the interfaces between crystals, the extrafibrillar mineral is resolved into an infinite amount of cylindrical material phases oriented in all directions in space. The multiscale micromechanics model is shown to be able to satisfactorily predict the strength characteristics of different bones from different species, on the basis of their mineral/collagen content, their intercrystalline, intermolecular, lacunar, and vascular porosities, and the elastic and strength properties of hydroxyapatite and (molecular) collagen.

Mineralization Induction Effects of Osteopontin, Bone Sialoprotein, and Dentin Phosphoprotein on a Biomimetic Collagen Substrate

PubMed Central

Zurick, Kevin M.; Qin, Chunlin; Bernards, Matthew T.

2012-01-01

Native bone tissue is composed of a matrix of collagen, non-collagenous proteins, and calcium phosphate minerals, which are primarily hydroxyapatite (HA). The SIBLING (small integrin-binding ligand, N-linked glycoprotein) family of proteins is the primary non-collagenous protein group found in mineralized tissues. In this work, the mineralization induction capabilities of three of the SIBLING members, bone sialoprotein (BSP), osteopontin (OPN), and the calcium binding subdomain of dentin sialophosphoprotein, dentin phosphoprotein (DPP), are directly compared on a biomimetic collagen substrate. A self-assembled, loosely aligned collagen fibril substrate was prepared and then 125I radiolabeled adsorption isotherms were developed for BSP, OPN, and DPP. The results showed that BSP exhibited the highest binding capacity for collagen at lower concentrations, followed by DPP and OPN. However, at the highest concentrations all three proteins had similar adsorption levels. The adsorption isotherms were then used to identify conditions that resulted in identical amounts of adsorbed protein. These substrates were prepared and placed in simulated body fluid for 5 hours, 10 hours, and 24 hours at 37°C. The resulting mineral morphology was assessed by atomic force microscopy and the composition was determined using photochemical assays. Mineralization was seen in the presence of all of the proteins. However, DPP was seen to be the only protein that formed individual mineral nodules similar to those seen in developing bone. This suggests that DPP plays a significant role in the biomineralization process and that the incorporation of DPP into tissue engineering constructs may facilitate the induction of biomimetic mineral formation. PMID:23161527

Lipids and collagen matrix restrict the hydraulic permeability within the porous compartment of adult cortical bone

PubMed Central

Wen, Demin; Androjna, Caroline; Vasanji, Amit; Belovich, Joanne; Midura, Ronald J.

2010-01-01

In vivo the hydraulic permeability of cortical bone influences the transport of nutrients, waste products and signaling molecules, thus influencing the metabolic functions of osteocytes and osteoblasts. In the current study two hypotheses were tested: the presence of (1) lipids and (2) collagen matrix in the porous compartment of cortical bone restricts its permeability. Our approach was to measure the radial permeability of adult canine cortical bone before and after extracting lipids with acetone-methanol, and before and after digesting collagen with bacterial collagenase. Our results showed that the permeability of adult canine cortical bone was below 4.0 × 10−17 m2, a value consistent with prior knowledge. After extracting lipids, permeability increased to a median value of 8.6 × 10−16 m2. After further digesting with collagenase, permeability increased to a median value of 1.4 × 10−14 m2. We conclude that the presence of both lipids and collagen matrix within the porous compartment of cortical bone restricts its radial permeability. These novel findings suggest that the chemical composition of the tissue matrix within the porous compartment of cortical bone influences the transport and exchange of nutrients and waste products, and possibly influences the metabolic functions of osteocytes and osteoblasts. PMID:19967451

Thermogelling chitosan and collagen composite hydrogels initiated with beta-glycerophosphate for bone tissue engineering.

PubMed

Wang, Limin; Stegemann, Jan P

2010-05-01

Chitosan and collagen type I are naturally derived materials used as cell carriers because of their ability to mimic the extracellular environment and direct cell function. In this study beta-glycerophosphate (beta-GP), an osteogenic medium supplement and a weak base, was used to simultaneously initiate gelation of pure chitosan, pure collagen, and chitosan-collagen composite materials at physiological pH and temperature. Adult human bone marrow-derived stem cells (hBMSC) encapsulated in such hydrogels at chitosan/collagen ratios of 100/0, 65/35, 25/75, and 0/100 wt% exhibited high viability at day 1 after encapsulation, but DNA content dropped by about half over 12 days in pure chitosan materials while it increased twofold in materials containing collagen. Collagen-containing materials compacted more strongly and were significantly stiffer than pure chitosan gels. In monolayer culture, exposure of hBMSC to beta-GP resulted in decreased cell metabolic activity that varied with concentration and exposure time, but washing effectively removed excess beta-GP from hydrogels. The presence of chitosan in materials resulted in higher expression of osterix and bone sialoprotein genes in medium with and without osteogenic supplements. Chitosan also increased alkaline phosphatase activity and calcium deposition in osteogenic medium. Chitosan-collagen composite materials have potential as matrices for cell encapsulation and delivery, or as in situ gel-forming materials for tissue repair. Copyright 2010 Elsevier Ltd. All rights reserved.

Adhesion of MC3T3-E1 cells to bone sialoprotein and bone osteopontin specifically bound to collagen I.

PubMed

Bernards, Matthew T; Qin, Chunlin; Ratner, Buddy D; Jiang, Shaoyi

2008-09-01

Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of proteins commonly found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen. In this work, the orientation of BSP under similar circumstances is examined and compared with OPN. Radiolabeled adsorption isotherms were obtained for BSP bound to both tissue culture polystyrene (TCPS) and collagen-coated TCPS. The results show that collagen has the capacity to bind almost twice as much OPN under identical conditions. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of BSP on either TCPS or collagen-coated TCPS with identical amounts of adsorbed protein. It was found that there is no significant difference in the cell binding ability of BSP on either of the substrates. For cell binding studies on collagen-coated TCPS, it was shown that there are a greater number of cells bound to substrates with adsorbed OPN as compared with BSP. The preferable orientation of OPN for cell binding coupled with the higher binding capability of collagen for OPN indicates that OPN is more important than BSP for osteoblast adhesion to the collagen matrix. In addition, a cell inhibition assay was performed to show that all of the cell binding that occurred throughout these studies was dependent upon integrin interactions with the RGD cell binding moiety.

Adhesion of MC3T3-E1 cells to bone sialoprotein and bone osteopontin specifically bound to collagen I

PubMed Central

Bernards, Matthew T.; Qin, Chunlin; Ratner, Buddy D.; Jiang, Shaoyi

2009-01-01

Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of proteins commonly found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen. In this work, the orientation of BSP under similar circumstances is examined and compared with OPN. Radiolabeled adsorption isotherms were obtained for BSP bound to both tissue culture polystyrene (TCPS) and collagen-coated TCPS. The results show that collagen has the capacity to bind almost twice as much OPN under identical conditions. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of BSP on either TCPS or collagen-coated TCPS with identical amounts of adsorbed protein. It was found that there is no significant difference in the cell binding ability of BSP on either of the substrates. For cell binding studies on collagen-coated TCPS, it was shown that there are a greater number of cells bound to substrates with adsorbed OPN as compared with BSP. The preferable orientation of OPN for cell binding coupled with the higher binding capability of collagen for OPN indicates that OPN is more important than BSP for osteoblast adhesion to the collagen matrix. In addition, a cell inhibition assay was performed to show that all of the cell binding that occurred throughout these studies was dependent upon integrin interactions with the RGD cell binding moiety. PMID:18041732

Guided bone regeneration with asymmetric collagen-chitosan membranes containing aspirin-loaded chitosan nanoparticles

PubMed Central

Zhang, Jiayu; Ma, Shiqing; Liu, Zihao; Geng, Hongjuan; Lu, Xin; Zhang, Xi; Li, Hongjie; Gao, Chenyuan; Zhang, Xu; Gao, Ping

2017-01-01

Introduction Membranes allowing the sustained release of drugs that can achieve cell adhesion are very promising for guided bone regeneration. Previous studies have suggested that aspirin has the potential to promote bone regeneration. The purpose of this study was to prepare a local drug delivery system with aspirin-loaded chitosan nanoparticles (ACS) contained in an asymmetric collagen-chitosan membrane (CCM). Methods In this study, the ACS were fabricated using different concentrations of aspirin (5 mg, 25 mg, 50 mg, and 75 mg). The drug release behavior of ACS was studied. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to examine the micromorphology of ACS and aspirin-loaded chitosan nanoparticles contained in chitosan-collagen membranes (ACS-CCM). In vitro bone mesenchymal stem cells (BMSCs) were cultured and critical-sized cranial defects on Sprague-Dawley rats were made to evaluate the effect of the ACS-CCM on bone regeneration. Results Drug release behavior results of ACS showed that the nanoparticles fabricated in this study could successfully sustain the release of the drug. TEM showed the morphology of the nanoparticles. SEM images indicated that the asymmetric membrane comprised a loose collagen layer and a dense chitosan layer. In vitro studies showed that ACS-CCM could promote the proliferation of BMSCs, and that the degree of differentiated BMSCs seeded on CCMs containing 50 mg of ACS was higher than that of other membranes. Micro-computed tomography showed that 50 mg of ACS-CCM resulted in enhanced bone regeneration compared with the control group. Conclusion This study shows that the ACS-CCM would allow the sustained release of aspirin and have further osteogenic potential. This membrane is a promising therapeutic approach to guiding bone regeneration. PMID:29276386

Alveolar ridge preservation with deproteinized bovine bone graft and collagen membrane and delayed implants.

PubMed

Pang, Chaoyuan; Ding, Yuxiang; Zhou, Hongzhi; Qin, Ruifeng; Hou, Rui; Zhang, Guoliang; Hu, Kaijin

2014-09-01

To evaluate clinically and radiographically an alveolar ridge, preservation technique with deproteinized bovine bone graft and absorbable collagen membrane and then restoration with delayed implants were done. The study included 30 patients. The trial group's sockets were filled with deproteinized bovine bone graft (Bio-Oss) and covered with absorbable collagen membrane (Bio-Gide). The control group's sockets healed without any treatment. Panoramic radiograph and computed tomography were taken immediately after graft and 3 and 6 months later to evaluate the height, width, and volume change of the alveolar ridge bone. Dental implants were inserted in all sockets at 6 months, and osseointegration condition was evaluated in the following 12 months. All sockets healed uneventfully. In the trial group, the mean (SD) height reduction of the alveolar ridge bone was 1.05 (0.24) mm at 3 months and 1.54 (0.25) mm at 6 months. The width reduction was 1.11 (0.13) mm at 3 months and 1.84 (0.35) mm at 6 months. Bone volume reduction was 193.79 (21.47) mm at 3 months and 262.06 (33.08) mm at 6 months. At the same trend, in the control group, the bone height reduction was 2.12 (0.15) mm at 3 months and 3.26 (0.29) mm at 6 months. The width reduction was 2.72 (0.19) mm at 3 months and 3.56 (0.28) mm at 6 months. Bone volume reduction was 252.19 (37.21) mm at 3 months and 342.32 (36.41) mm at 6 months. There was a significant difference in alveolar ridge bone height, width, and volume reduction in the 2 groups. The osseointegration condition had no significant difference between the 2 groups. This study suggested that the deproteinized bovine bone graft and absorbable collagen membrane were beneficial to preserve the alveolar ridge bone and had no influence on the osseointegration of delayed implant.

Effect of Resorbable Collagen Plug on Bone Regeneration in Rat Critical-Size Defect Model.

PubMed

Liu, Weiqing; Kang, Ning; Dong, Yuliang; Guo, Yuchen; Zhao, Dan; Zhang, Shiwen; Zhou, Liyan; Seriwatanachai, Dutmanee; Liang, Xing; Yuan, Quan

2016-04-01

The purpose of this investigation was to examine the effect of resorbable collagen plug (RCP) on bone regeneration in rat calvarial critical-size defects. About 5-mm-diameter calvarial defects were created in forty 12-week-old male Sprague-Dawley rats and implanted with or without RCP. Animals were killed at 1, 2, 4, and 8 weeks postoperatively. After being killed, specimens were collected and subjected to micro-computed tomography (μCT) and histological analysis. The μCT showed a significant increase of newly formed bone volume/tissue volume in RCP-implanted defect compared with controls at all designated time points. After 8 weeks, the defects implanted with RCP displayed almost complete closure. Hematoxylin and eosin staining of the decalcified sections confirmed these observations and evidenced active bone regeneration in the RCP group. In addition, Masson's trichrome staining demonstrated that RCP implantation accelerated the process of collagen maturation. The RCP enhances bone regeneration in rat critical-size cranial defects, which suggest it might be a desired material for bone defect repair.

Ectopic bone formation in nude rats using human osteoblasts seeded poly(3)hydroxybutyrate embroidery and hydroxyapatite-collagen tapes constructs.

PubMed

Mai, Ronald; Hagedorn, Manolo Gunnar; Gelinsky, Michael; Werner, Carsten; Turhani, Dritan; Späth, Heike; Gedrange, Tomas; Lauer, Günter

2006-09-01

The aim of this study was to evaluate the ectopic bone formation using tissue engineered cell-seeded constructs with two different scaffolds and primary human maxillary osteoblasts in nude rats over an implantation period of up to 96 days. Collagen I-coated Poly(3)hydroxybutyrate (PHB) embroidery and hydroxyapatite (HAP) collagen tapes were seeded with primary human maxillary osteoblasts (hOB) and implanted into athymic rnu/run rats. A total of 72 implants were placed into the back muscles of 18 rats. 24, 48 and 96 days after implantation, histological and histomorphometric analyses were made. The osteoblastic character of the cells was confirmed by immunocytochemistry and RT-PCR for osteocalcin. Histological analysis demonstrated that all cell-seeded constructs induced ectopic bone formation after 24, 48 and 96 days of implantation. There was more mineralized tissue in PHB constructs than in HAP-collagen tapes (at day 24; p < 0.05). Bone formation decreased with the increasing length of the implantation period. Osteocalcin expression verified the osteoblastic character of the cell-seeded constructs after implantation time. No bone formation and no osteocalcin expression were found in the control groups. Cell-seeded constructs either with PHB embroidery or HAP-collagen tapes can induce ectopic bone formation. However, the amount of bone formed decreased with increasing length of implantation.

Deposition of collagen type I onto skeletal endothelium reveals a new role for blood vessels in regulating bone morphology

PubMed Central

Ben Shoham, Adi; Rot, Chagai; Stern, Tomer; Krief, Sharon; Akiva, Anat; Dadosh, Tali; Sabany, Helena; Lu, Yinhui; Kadler, Karl E.

2016-01-01

Recently, blood vessels have been implicated in the morphogenesis of various organs. The vasculature is also known to be essential for endochondral bone development, yet the underlying mechanism has remained elusive. We show that a unique composition of blood vessels facilitates the role of the endothelium in bone mineralization and morphogenesis. Immunostaining and electron microscopy showed that the endothelium in developing bones lacks basement membrane, which normally isolates the blood vessel from its surroundings. Further analysis revealed the presence of collagen type I on the endothelial wall of these vessels. Because collagen type I is the main component of the osteoid, we hypothesized that the bone vasculature guides the formation of the collagenous template and consequently of the mature bone. Indeed, some of the bone vessels were found to undergo mineralization. Moreover, the vascular pattern at each embryonic stage prefigured the mineral distribution pattern observed one day later. Finally, perturbation of vascular patterning by overexpressing Vegf in osteoblasts resulted in abnormal bone morphology, supporting a role for blood vessels in bone morphogenesis. These data reveal the unique composition of the endothelium in developing bones and indicate that vascular patterning plays a role in determining bone shape by forming a template for deposition of bone matrix. PMID:27621060

Defective collagen crosslinking in bone, but not in ligament or cartilage, in Bruck syndrome: Indications for a bone-specific telopeptide lysyl hydroxylase on chromosome 17

PubMed Central

Bank, Ruud A.; Robins, Simon P.; Wijmenga, Cisca; Breslau-Siderius, Liesbeth J.; Bardoel, Alfons F. J.; Van der Sluijs, Hans A.; Pruijs, Hans E. H.; TeKoppele, Johan M.

1999-01-01

Bruck syndrome is characterized by the presence of osteoporosis, joint contractures, fragile bones, and short stature. We report that lysine residues within the telopeptides of collagen type I in bone are underhydroxylated, leading to aberrant crosslinking, but that the lysine residues in the triple helix are normally modified. In contrast to bone, cartilage and ligament show unaltered telopeptide hydroxylation as evidenced by normal patterns of crosslinking. The results provide compelling evidence that collagen crosslinking is regulated primarily by tissue-specific enzymes that hydroxylate only telopeptide lysine residues and not those destined for the helical portion of the molecule. This new family of enzymes appears to provide the primary regulation for controlling the different pathways of collagen crosslinking and explains why crosslink patterns are tissue specific and not related to a genetic collagen type. A genome screen identified only a single region on chromosome 17p12 where all affected sibs shared a cluster of haplotypes identical by descent; this might be the BS (Bruck syndrome) locus and consequently the region where bone telopeptidyl lysyl hydroxylase is located. Further knowledge of this enzyme has important implications for conditions where aberrant expression of telopeptide lysyl hydroxylase occurs, such as fibrosis and scar formation. PMID:9927692

Role of Collagen and Inorganic Components in Electrical Polarizability of Bone

PubMed Central

HIRATAI, Rumi; NAKAMURA, Miho; YAMASHITA, Kimihiro

2013-01-01

ABSTRACT Hydroxyapatite (HA) has polarization capability and is able to accumulate electrical storage in bone. Experiments were conducted to measure the polarization capability of rabbit femurs. After preparing and polarizing bone samples using 2% KOH treatment (denoted 2% koh), 2% KOH and baking (2% koh+bake) and decalcification (decalcification) as well as untreated bone (untreated), stored charges were quantitatively determined using thermally stimulated depolarization current (TSDC) measurements. In TSDC spectra, untreated and 2% koh samples showed peaks at 100 and 500°C, while 2% koh+bake showed one peak at 580°C and decalcification one peak around 100°C. These evidences indicated that collagen and inorganic components play a major role in polarization of the bone at different temperature conditions. PMID:24141277

Content and distribution of noncollagenous matrix proteins in bone and cementum: relationship to speed of formation and collagen packing density.

PubMed

Nanci, A

1999-06-30

The organic matrix of collagen-based calcified tissues consists of a supporting collagen meshwork and various noncollagenous matrix proteins (NCPs). Together, they contribute to determining the structure and biomechanical properties of the tissue. Their respective organization and interrelation can advantageously be examined by immunocytochemistry, an approach which allows correlation of composition with structure. The aim of this article is to review postembedding immuno- and lectin-gold-labeling data on the characterization of the noncollagenous compartment in rat and human bone and cementum, and on its relationship to collagen. The two major NCPs, bone sialoprotein and osteopontin, generally codistribute and accumulate in cement lines and in the spaces among the mineralized collagen fibrils. However, there are variations in their distribution and density of labeling throughout the tissue. Indeed, bone and cementum can form in environments that are either poor or enriched in NCPs. The amount of NCPs generally correlates with bone and cementum types and with speed of formation of the tissue and packing density of collagen fibrils. Taken together, the data suggest that production of both collagenous and noncollagenous constituents can be "modulated" during formation of collagen-based calcified tissues. It is concluded that, in addition to structural and compositional parameters, tissue dynamics must be taken into consideration in order to understand the significance of the apparent accumulation of NCPs at some sites and to determine the mechanisms of normal and pathological calcified tissue formation. Copyright 1999 Academic Press.

Deamidation of collagen.

PubMed

Hurtado, Pilar Perez; O'Connor, Peter B

2012-03-20

Collagen is the major component of skin, tendons, ligaments, teeth, and bones, it provides the framework that holds most multicellular animals together, and collagen type I constitutes the major fibrillar collagen of bone. Because of the complexity of collagen's structure, the study of post-translational modifications such as deamidation for this protein is challenging. Although there is no evidence of this protein being used for age assessment, it has been shown that deamidation of collagen is remarkably increased in old bones from mammals. Nonspectrometric methodologies have been used for the determination of the extent of deamidation as a measure of the amount of amide nitrogen released in ammonia as well as constant rates for deamidation of asparagine in collagen. In general, these methodologies required more sample and separation processes. To understand if collagen plays a significant role in the aging process of fossil materials, a simpler and more accurate method is needed to determine the extent of deamidation at the whole protein level. The present work shows a method to determine the extent of deamidation in collagen using Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) along with collisionally activated dissociation (CAD) and electron capture dissociation (ECD). The measured deamidation half-life for three different tryptic peptides from collagen (I) ranged from 2000 to 6000 s under high temperature conditions (∼62 °C) and pH 7.5.

Sika Deer Antler Collagen Type I-Accelerated Osteogenesis in Bone Marrow Mesenchymal Stem Cells via the Smad Pathway

PubMed Central

Li, Na; Zhang, Min; Drummen, Gregor P. C.; Zhao, Yu; Tan, Yin Fen; Luo, Su; Qu, Xiao Bo

2016-01-01

Deer antler preparations have been used to strengthen bones for centuries. It is particularly rich in collagen type I. This study aimed to unravel part of the purported bioremedial effect of Sika deer antler collagen type I (SDA-Col I) on bone marrow mesenchymal stem cells. The results suggest that SDA-Col I might be used to promote and regulate osteoblast proliferation and differentiation. SDA-Col I might potentially provide the basis for novel therapeutic strategies in the treatment of bone injury and/or in scaffolds for bone replacement strategies. Finally, isolation of SDA-Col I from deer antler represents a renewable, green, and uncomplicated way to obtain a biomedically valuable therapeutic. PMID:27066099

Scaffolds for bone regeneration made of hydroxyapatite microspheres in a collagen matrix.

PubMed

Cholas, Rahmatullah; Kunjalukkal Padmanabhan, Sanosh; Gervaso, Francesca; Udayan, Gayatri; Monaco, Graziana; Sannino, Alessandro; Licciulli, Antonio

2016-06-01

Biomimetic scaffolds with a structural and chemical composition similar to native bone tissue may be promising for bone tissue regeneration. In the present work hydroxyapatite mesoporous microspheres (mHA) were incorporated into collagen scaffolds containing an ordered interconnected macroporosity. The mHA were obtained by spray drying of a nano hydroxyapatite slurry prepared by the precipitation technique. X-ray diffraction (XRD) analysis revealed that the microspheres were composed only of hydroxyapatite (HA) phase, and energy-dispersive x-ray spectroscopy (EDS) analysis revealed the Ca/P ratio to be 1.69 which is near the value for pure HA. The obtained microspheres had an average diameter of 6 μm, a specific surface area of 40 m(2)/g as measured by Brunauer-Emmett-Teller (BET) analysis, and Barrett-Joyner-Halenda (BJH) analysis showed a mesoporous structure with an average pore diameter of 16 nm. Collagen/HA-microsphere (Col/mHA) composite scaffolds were prepared by freeze-drying followed by dehydrothermal crosslinking. SEM observations of Col/mHA scaffolds revealed HA microspheres embedded within a porous collagen matrix with a pore size ranging from a few microns up to 200 μm, which was also confirmed by histological staining of sections of paraffin embedded scaffolds. The compressive modulus of the composite scaffold at low and high strain values was 1.7 and 2.8 times, respectively, that of pure collagen scaffolds. Cell proliferation measured by the MTT assay showed more than a 3-fold increase in cell number within the scaffolds after 15 days of culture for both pure collagen scaffolds and Col/mHA composite scaffolds. Attractive properties of this composite scaffold include the potential to load the microspheres for drug delivery and the controllability of the pore structure at various length scales. Copyright © 2016 Elsevier B.V. All rights reserved.

Radiographic and histological evaluation of deproteinized bovine bone mineral vs. deproteinized bovine bone mineral with 10% collagen in ridge preservation. A randomized controlled clinical trial.

PubMed

Nart, Jose; Barallat, Lucía; Jimenez, Daniel; Mestres, Jaume; Gómez, Alberto; Carrasco, Miguel Angel; Violant, Deborah; Ruíz-Magaz, Vanessa

2017-07-01

The aims of this randomized clinical trial were to compare the dimensional changes and the histological composition after using deproteinized bovine bone mineral (DBBM) or deproteinized bovine bone mineral with 10% collagen (DBBM-C) and a collagen membrane in ridge preservation procedures. Patients who required an extraction and a subsequent implant-supported rehabilitation at a non-molar site were recruited. After extraction, a cone beam computed tomography (CBCT) was performed and sites were randomly treated either with DBBM or DBBM-C plus a collagen membrane. At 5 months, before implant placement, a second CBCT was performed and a biopsy of the area was obtained. A blinded investigator superimposed the CBCTs and performed measurements to determine bone volume changes between the two time points. Additionally, a histomorphometric analysis of the biopsies was performed in a blinded manner. Eleven sites belonged to the DBBM group and eleven to the DBBM-C group. All together, a significant reduction in height and width was observed at 5 months of healing, but no statistically significant differences were observed between the DBBM and the DBBM-C group. The histomorphometric analysis revealed a similar composition in terms of newly formed bone, connective tissue and residual graft particles in both groups. Deproteinized bovine bone mineral with 10% collagen showed a similar behaviour as DBBM not only in its capacity to minimize ridge contraction but also from a histological point of view. Thus, both graft materials seem to be suitable for ridge preservation procedures. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

When size matters: differences in demineralized bone matrix particles affect collagen structure, mesenchymal stem cell behavior, and osteogenic potential.

PubMed

Dozza, B; Lesci, I G; Duchi, S; Della Bella, E; Martini, L; Salamanna, F; Falconi, M; Cinotti, S; Fini, M; Lucarelli, E; Donati, D

2017-04-01

Demineralized bone matrix (DBM) is a natural, collagen-based, osteoinductive biomaterial. Nevertheless, there are conflicting reports on the efficacy of this product. The purpose of this study was to evaluate whether DBM collagen structure is affected by particle size and can influence DBM cytocompatibility and osteoinductivity. Sheep cortical bone was ground and particles were divided in three fractions with different sizes, defined as large (L, 1-2 mm), medium (M, 0.5-1 mm), and small (S, <0.5 mm). After demineralization, the chemical-physical analysis clearly showed a particle size-dependent alteration in collagen structure, with DBM-M being altered but not as much as DBM-S. DBM-M displayed a preferable trend in almost all biological characteristics tested, although all DBM particles revealed an optimal cytocompatibility. Subcutaneous implantation of DBM particles into immunocompromised mice resulted in bone induction only for DBM-M. When sheep MSC were seeded onto particles before implantation, all DBM particles were able to induce new bone formation with the best incidence for DBM-M and DBM-S. In conclusion, the collagen alteration in DBM-M is likely the best condition to promote bone induction in vivo. Furthermore, the choice of 0.5-1 mm particles may enable to obtain more efficient and consistent results among different research groups in bone tissue-engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1019-1033, 2017. © 2017 Wiley Periodicals, Inc.

Periodontal regeneration with stem cells-seeded collagen-hydroxyapatite scaffold.

PubMed

Liu, Zeping; Yin, Xing; Ye, Qingsong; He, Wulin; Ge, Mengke; Zhou, Xiaofu; Hu, Jing; Zou, Shujuan

2016-07-01

Re-establishing compromised periodontium to its original structure, properties and function is demanding, but also challenging, for successful orthodontic treatment. In this study, the periodontal regeneration capability of collagen-hydroxyapatite scaffolds, seeded with bone marrow stem cells, was investigated in a canine labial alveolar bone defect model. Bone marrow stem cells were isolated, expanded and characterized. Porous collagen-hydroxyapatite scaffold and cross-linked collagen-hydroxyapatite scaffold were prepared. Attachment, migration, proliferation and morphology of bone marrow stem cells, co-cultured with porous collagen-hydroxyapatite or cross-linked collagen-hydroxyapatite, were evaluated in vitro. The periodontal regeneration capability of collagen-hydroxyapatite scaffold with or without bone marrow stem cells was tested in six beagle dogs, with each dog carrying one sham-operated site as healthy control, and three labial alveolar bone defects untreated to allow natural healing, treated with bone marrow stem cells - collagen-hydroxyapatite scaffold implant or collagen-hydroxyapatite scaffold implant, respectively. Animals were euthanized at 3 and 6 months (3 animals per group) after implantation and the resected maxillary and mandibular segments were examined using micro-computed tomography scan, H&E staining, Masson's staining and histometric evaluation. Bone marrow stem cells were successfully isolated and demonstrated self-renewal and multi-potency in vitro. The porous collagen-hydroxyapatite and cross-linked collagen-hydroxyapatite had average pore sizes of 415 ± 20 µm and 203 ± 18 µm and porosity of 69 ± 0.5% and 50 ± 0.2%, respectively. The attachment, proliferation and migration of bone marrow stem cells were satisfactory on both porous collagen-hydroxyapatite and cross-linked collagen-hydroxyapatite scaffolds. Implantation of bone marrow stem cells - collagen-hydroxyapatite or collagen-hydroxyapatite scaffold in

Effects of mangosteen peel extract combined with demineralized freeze-dried bovine bone xenograft on osteocalcin, collagen 1, and osteoblast as alveolar bone regeneration in socket preservation.

PubMed

Kresnoadi, Utari; Raharjo, Tika; Rostiny, Rostiny

2018-01-01

Tooth extraction will provoke changes in alveolar bone morphology and dimensions. Postextraction bone resorption can lead to significant problems for restorative dentistry. Therefore, the extracted tooth socket needs to be preserved to reduce alveolar ridge bone resorption. This research aimed to analyze the expression and levels of osteocalcin, collagen 1, and osteoblasts in extracted tooth sockets filled with a combination of mangosteen peel extract and demineralized freeze-dried bovine bone xenograft (DFDBBX). Fifty-six Cavia cobaya , whose lower left incisors had been extracted, were divided into eight groups according to the substance used to fill their sockets on days 7 and 30, Poly ethylene glycol, DFDBBX, mangosteen peel extract, or a combination of mangosteen peel extract and DFDBBX. This research was conducted in several stages; the application of mangosteen peel extract combined with graft material was performed as the form of tooth extraction socket preservation. The C. cobaya rats were subsequently examined by immunohistochemical methods to measure osteocalcin and collagen 1 expressions, whereas histological examination was conducted to calculate the number of osteoblasts in accordance with the duration of the research. On days 7 and 30, the group treated with a combination of DFDBBX and mangosteen peel extract which had the highest expression and levels of osteocalcin, collagen 1, and osteoblasts. The administration of mangosteen peel extract combined with DFDBBX as a means of tooth extraction socket preservation can increase osteocalcin and collagen 1 expression. Consequently, osteoblasts as a means of alveolar bone regeneration will increase in number.

Anti-epileptic drugs and bone loss: Phenytoin reduces pro-collagen I and alters the electrophoretic mobility of osteonectin in cultured bone cells.

PubMed

Wilson, Emma L; Garton, Mark; Fuller, Heidi R

2016-05-01

Phenytoin is an antiepileptic drug used in the management of partial and tonic-clonic seizures. In previous studies we have shown that valproate, another antiepileptic drug, reduced the amount of two key bone proteins, pro-collagen I and osteonectin (SPARC, BM-40), in both skin fibroblasts and cultured osteoblast-like cells. Here we show that phenytoin also reduces pro-collagen I production in osteoblast-like cells, but does not appear to cause a decrease in osteonectin message or protein production. Instead, a 24h exposure to a clinically relevant concentration of phenytoin resulted in a dose-dependent change in electrophoretic mobility of osteonectin, which was suggestive of a change in post-translational modification status. The perturbation of these important bone proteins could be one of the mechanisms to explain the bone loss that has been reported following long-term treatment with phenytoin. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of 3D printed PCL/PLGA/β-TCP versus collagen membranes for guided bone regeneration in a beagle implant model.

PubMed

Won, J-Y; Park, C-Y; Bae, J-H; Ahn, G; Kim, C; Lim, D-H; Cho, D-W; Yun, W-S; Shim, J-H; Huh, J-B

2016-10-07

Here, we compared 3D-printed polycaprolactone/poly(lactic-co-glycolic acid)/β-tricalcium phosphate (PCL/PLGA/β-TCP) membranes with the widely used collagen membranes for guided bone regeneration (GBR) in beagle implant models. For mechanical property comparison in dry and wet conditions and cytocompatibility determination, we analyzed the rate and pattern of cell proliferation of seeded fibroblasts and preosteoblasts using the cell counting kit-8 assay and scanning electron microscopy. Osteogenic differentiation was verified using alizarin red S staining. At 8 weeks following implantation in vivo using beagle dogs, computed tomography and histological analyses were performed after sacrifice. Cell proliferation rates in vitro indicated that early cell attachment was higher in collagen than in PCL/PLGA/β-TCP membranes; however, the difference subsided by day 7. Similar outcomes were found for osteogenic differentiation, with approximately 2.5 times greater staining in collagen than PCL/PLGA/β-TCP, but without significant difference by day 14. In vivo, bone regeneration in the defect area, represented by new bone formation and bone-to-implant contact, paralleled those associated with collagen membranes. However, tensile testing revealed that whereas the PCL/PLGA/β-TCP membrane mechanical properties were conserved in both wet and dry states, the tensile property of collagen was reduced by 99% under wet conditions. Our results demonstrate in vitro and in vivo that PCL/PLGA/β-TCP membranes have similar levels of biocompatibility and bone regeneration as collagen membranes. In particular, considering that GBR is always applied to a wet environment (e.g. blood, saliva), we demonstrated that PCL/PLGA/β-TCP membranes maintained their form more reliably than collagen membranes in a wet setting, confirming their appropriateness as a GBR membrane.

Radiocarbon dating of charcoal and bone collagen associated with early pottery at Yuchanyan Cave, Hunan Province, China

PubMed Central

Boaretto, Elisabetta; Wu, Xiaohong; Yuan, Jiarong; Bar-Yosef, Ofer; Chu, Vikki; Pan, Yan; Liu, Kexin; Cohen, David; Jiao, Tianlong; Li, Shuicheng; Gu, Haibin; Goldberg, Paul; Weiner, Steve

2009-01-01

Yuchanyan Cave in Daoxian County, Hunan Province (People's Republic of China), yielded fragmentary remains of 2 or more ceramic vessels, in addition to large amounts of ash, a rich animal bone assemblage, cobble and flake artifacts, bone tools, and shell tools. The artifacts indicate that the cave was a Late Paleolithic foragers' camp. Here we report on the radiocarbon ages of the sediments based on analyses of charcoal and bone collagen. The best-preserved charcoal and bone samples were identified by prescreening in the field and laboratory. The dates range from around 21,000 to 13,800 cal BP. We show that the age of the ancient pottery ranges between 18,300 and 15,430 cal BP. Charcoal and bone collagen samples located above and below one of the fragments produced dates of around 18,000. These ceramic potsherds therefore provide some of the earliest evidence for pottery making in China. PMID:19487667

Collagen hydrolysate based collagen/hydroxyapatite composite materials

NASA Astrophysics Data System (ADS)

Ficai, Anton; Albu, Madalina Georgiana; Birsan, Mihaela; Sonmez, Maria; Ficai, Denisa; Trandafir, Viorica; Andronescu, Ecaterina

2013-04-01

The aim of this study was to study the influence of collagen hydrolysate (HAS) on the formation of ternary collagen-hydrolysate/hydroxyapatite composite materials (COLL-HAS/HA). During the precipitation process of HA, a large amount of brushite is resulted at pH = 7 but, practically pure HA is obtained at pH ⩾ 8. The FTIR data reveal the duplication of the most important collagen absorption bands due to the presence of the collagen hydrolysate. The presence of collagen hydrolysate is beneficial for the management of bone and joint disorders such as osteoarthritis and osteoporosis.

Collagen implants equipped with 'fish scale'-like nanoreservoirs of growth factors for bone regeneration.

PubMed

Eap, Sandy; Ferrand, Alice; Schiavi, Jessica; Keller, Laetitia; Kokten, Tunay; Fioretti, Florence; Mainard, Didier; Ladam, Guy; Benkirane-Jessel, Nadia

2014-01-01

Implants triggering rapid, robust and durable tissue regeneration are needed to shorten recovery times and decrease risks of postoperative complications for patients. Here, we describe active living collagen implants with highly promising bone regenerative properties. Bioactivity of the implants is obtained through the protective and stabilizing layer-by-layer immobilization of a protein growth factor in association with a polysaccharide (chitosan), within the form of nanocontainers decorating the collagen nanofibers. All components of the implants are US FDA approved. From both in vitro and in vivo evaluations, the sophisticated strategy described here should enhance, at a reduced cost, the safety and efficacy of the therapeutic implants in terms of large bone defects repair compared with current simplistic approaches based on the soaking of the implants with protein growth factor.

Alveolar socket preservation with demineralised bovine bone mineral and a collagen matrix

PubMed Central

2017-01-01

Purpose The aim of the present study was to evaluate the healing of post-extraction sockets following alveolar ridge preservation clinically, radiologically, and histologically. Methods Overall, 7 extraction sockets in 7 patients were grafted with demineralised bovine bone mineral and covered with a porcine-derived non-crosslinked collagen matrix (CM). Soft tissue healing was clinically evaluated on the basis of a specific healing index. Horizontal and vertical ridge dimensional changes were assessed clinically and radiographically at baseline and 6 months after implant placement. For histological and histomorphometric analysis, bone biopsies were harvested from the augmented sites during implant surgery 6 months after the socket preservation procedure. Results Clinically, healing proceeded uneventfully in all the sockets. A trend towards reduced horizontal and vertical socket dimensions was observed from baseline to the final examination. The mean width and height of resorption were 1.21 mm (P=0.005) and 0.46 mm (P=0.004), respectively. Histologically, residual xenograft particles (31.97%±3.52%) were surrounded by either newly formed bone (16.02%±7.06%) or connective tissue (50.67%±8.42%) without fibrous encapsulation. The CM underwent a physiological substitution process in favour of well-vascularised collagen-rich connective tissue. Conclusions Socket preservation using demineralised bovine bone mineral in combination with CM provided stable dimensional changes of the alveolar ridge associated with good re-epithelialisation of the soft tissues during a 6-month healing period. PMID:28861284

Mineralization induction effects of osteopontin, bone sialoprotein, and dentin phosphoprotein on a biomimetic collagen substrate.

PubMed

Zurick, Kevin M; Qin, Chunlin; Bernards, Matthew T

2013-06-01

Native bone tissue is composed of a matrix of collagen, noncollagenous proteins, and calcium phosphate minerals, which are primarily hydroxyapatite. The SIBLING (small integrin-binding ligand, N-linked glycoprotein) family of proteins is the primary noncollagenous protein group found in mineralized tissues. In this work, the mineralization induction capabilities of three of the SIBLING members, bone sialoprotein (BSP), osteopontin (OPN), and the calcium-binding subdomain of dentin sialophosphoprotein, dentin phosphoprotein (DPP), are directly compared on a biomimetic collagen substrate. A self-assembled, loosely aligned collagen fibril substrate was prepared, and then (125) I-radiolabeled adsorption isotherms were developed for BSP, OPN, and DPP. The results showed that BSP exhibited the highest binding capacity for collagen at lower concentrations, followed by DPP and OPN. However, at the highest concentrations, all three proteins had similar adsorption levels. The adsorption isotherms were then used to identify conditions that resulted in identical amounts of adsorbed protein. These substrates were prepared and placed in simulated body fluid for 5, 10, and 24 h at 37°C. The resulting mineral morphology was assessed by atomic force microscopy, and the composition was determined using photochemical assays. Mineralization was seen in the presence of all the proteins. However, DPP was seen to be the only protein that formed individual mineral nodules similar to those seen in developing bone. This suggests that DPP plays a significant role in the biomineralization process and that the incorporation of DPP into tissue engineering constructs may facilitate the induction of biomimetic mineral formation. Copyright © 2012 Wiley Periodicals, Inc.

Bacterial challenge of NISSHO ultrafilter ETF 609: results of in vitro testing.

PubMed

Krautzig, S; Lonnemann, G; Shaldon, S; Koch, K M

1996-07-01

In hemodialysis, a certain degree of bacterial contamination on the dialysate side is a regular finding. Concern has been growing that this contamination may lead to a chronic inflammatory response in the patient. Ultrafiltration of dialysate can be used to reduce bacterial content and levels of cytokine-inducing substances upstream of the patient's dialyzer. The aim of this study was to test in vitro the rejection capacity of a polysulfone hollow-fiber ultrafilter (ETF 609, NISSHO Co., Osaka, Japan) challenged with bacterial filtrates derived from Pseudomonas aeruginosa PA103. Results showed a reduction of interleukin-1 beta-inducing activity (measured on peripheral blood mononuclear cells) from 5,035 +/- 394 pg/ml prefilter to nondetectable levels postfilter and endotoxin levels (limulus amebocyte lysate assay) of 4,167 +/- 1,079 versus 12 +/- 2 pg/ml, respectively. In conclusion, ultrafiltration of dialysate with the polysulfone ultrafilter ETF 609 leads to a potent reduction of cytokine-inducing activity.

Osteoinduction and proliferation of bone-marrow stromal cells in three-dimensional poly (ε-caprolactone)/ hydroxyapatite/collagen scaffolds.

PubMed

Wang, Ting; Yang, Xiaoyan; Qi, Xin; Jiang, Chaoyin

2015-05-08

Osteoinduction and proliferation of bone-marrow stromal cells (BMSCs) in three-dimensional (3D) poly(ε-caprolactone) (PCL) scaffolds have not been studied throughly and are technically challenging. This study aimed to optimize nanocomposites of 3D PCL scaffolds to provide superior adhesion, proliferation and differentiation environment for BMSCs in this scenario. BMSCs were isolated and cultured in a novel 3D tissue culture poly(ε-caprolactone) (PCL) scaffold coated with poly-lysine, hydroxyapatite (HAp), collagen and HAp/collagen. Cell morphology was observed and BMSC biomarkers for osteogenesis, osteoblast differentiation and activation were analyzed. Scanning Electron Microscope (SEM) micrographs showed that coating materials were uniformly deposited on the surface of PCL scaffolds and BMSCs grew and aggregated to form clusters during 3D culture. Both mRNA and protein levels of the key players of osteogenesis and osteoblast differentiation and activation, including runt-related transcription factor 2 (Runx2), alkaline phosphates (ALP), osterix, osteocalcin, and RANKL, were significantly higher in BMSCs seeded in PCL scaffolds coated with HAp or HAp/collagen than those seeded in uncoated PCL scaffolds, whereas the expression levels were not significantly different in collagen or poly-lysine coated PCL scaffolds. In addition, poly-lysine, collagen, HAp/collagen, and HAp coated PCL scaffolds had significantly more viable cells than uncoated PCL scaffolds, especially scaffolds with HAp/collagen and collagen-alone coatings. That BMSCs in HAp or HAp/collagen PCL scaffolds had remarkably higher ALP activities than those in collagen-coated alone or uncoated PCL scaffolds indicating higher osteogenic differentiation levels of BMSCs in HAp or HAp/collagen PCL scaffolds. Moreover, morphological changes of BMSCs after four-week of 3D culture confirmed that BMSCs successfully differentiated into osteoblast with spread-out phenotype in HAp/collagen coated PCL scaffolds

An effective delivery vehicle of demineralized bone matrix incorporated with engineered collagen-binding human bone morphogenetic protein-2 to accelerate spinal fusion at low dose.

PubMed

Zhu, Weiguo; Qiu, Yong; Sheng, Fei; Yuan, Xinxin; Xu, Leilei; Bao, Hongda; Dai, Jianwu; Zhu, Zezhang

2017-12-01

The aim of this study was to investigate the feasibility and efficacy of a new delivery matrix using demineralized bone matrix (DBM) incorporated with collagen-binding bone morphogenetic protein-2 (CBD-BMP-2) in the rat inter-transverse spinal fusion model. Sixty rats undergoing posterolateral (inter-transverse) spinal fusion were divided into 3 groups according to the fusion materials containing different components (n = 20 per group). Group A were implanted with DBM, Group B with combination of DBM and BMP-2 and Group C with combination of DBM and CBD-BMP-2. After surgery, the spinal fusion of all the rats was assessed by plain radiography, CT + 3D reconstruction, manual palpation and histological evaluation. Significant difference was found in terms of solid fusion rate among the three groups, with 95% in Group C, 65% in Group B and 0% in Group A (P < 0.001). Compared with Groups B and A, new bone formation was observed earlier and was obvious larger, trabecular bone microarchitecture assessment was better and bone mineral density was statistically larger in Group C. In addition, more newly woven bone and osteocytes were shown by histological evaluation in Group C at 4 weeks post-operation. The present study showed CBD domain could help BMP-2 to improve the efficiency of posterolateral spinal fusion. DBM scaffold activated by collagen-binding BMP-2 was a feasible and promising bone repair vehicle. The present study showed better results in terms of plain radiography, CT + 3D reconstruction, manual palpation and histological evaluation in the rat inter-transverse spinal fusion model using DBM+CBD-BMP-2, compared with DBM+BMP-2 and DBM alone, indicating DBM scaffold activated by collagen-binding BMP-2 was a feasible and promising bone repair vehicle.

Characterization of bone collagen organization defects in murine hypophosphatasia using a Zernike model of optical aberrations

NASA Astrophysics Data System (ADS)

Tehrani, Kayvan Forouhesh; Pendleton, Emily G.; Leitmann, Bobby; Barrow, Ruth; Mortensen, Luke J.

2018-02-01

Bone growth and strength is severely impacted by Hypophosphatasia (HPP). It is a genetic disease that affects the mineralization of the bone. We hypothesize that it impacts overall organization, density, and porosity of collagen fibers. Lower density of fibers and higher porosity cause less absorption and scattering of light, and therefore a different regime of transport mean free path. To find a cure for this disease, a metric for the evaluation of bone is required. Here we present an evaluation method based on our Phase Accumulation Ray Tracing (PART) method. This method uses second harmonic generation (SHG) in bone collagen fiber to model bone indices of refraction, which is used to calculate phase retardation on the propagation path of light in bone. The calculated phase is then expanded using Zernike polynomials up to 15th order, to make a quantitative analysis of tissue anomalies. Because the Zernike modes are a complete set of orthogonal polynomials, we can compare low and high order modes in HPP, compare them with healthy wild type mice, to identify the differences between their geometry and structure. Larger coefficients of low order modes show more uniform fiber density and less porosity, whereas the opposite is shown with larger coefficients of higher order modes. Our analyses show significant difference between Zernike modes in different types of bone evidenced by Principal Components Analysis (PCA).

Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography

NASA Technical Reports Server (NTRS)

Landis, W. J.; Hodgens, K. J.; Arena, J.; Song, M. J.; McEwen, B. F.

1996-01-01

Aspects of the ultrastructural interaction between collagen and mineral crystals in embryonic chick bone have been examined by the novel technique of high voltage electron microscopic tomography to obtain three-dimensional information concerning extracellular calcification in this tissue. Newly mineralizing osteoid along periosteal surfaces of mid-diaphyseal regions from normal chick tibiae was embedded, cut into 0.25 microns thick sections, and documented at 1.0 MV in the Albany AEI-EM7 high voltage electron microscope. The areas of the tissue studied contained electron dense mineral crystals associated with collagen fibrils, some marked by crystals disposed along their cylindrically shaped lengths. Tomographic reconstructions of one site with two mineralizing fibrils were computed from a 5 degrees tilt series of micrographs over a +/- 60 degrees range. Reconstructions showed that the mineral crystals were platelets of irregular shape. Their sizes were variable, measured here up to 80 x 30 x 8 nm in length, width, and thickness, respectively. The longest crystal dimension, corresponding to the c-axis crystallographically, was generally parallel to the collagen fibril long axis. Individual crystals were oriented parallel to one another in each fibril examined. They were also parallel in the neighboring but apparently spatially separate fibrils. Crystals were periodically (approximately 67 nm repeat distance) arranged along the fibrils and their location appeared to correspond to collagen hole and overlap zones defined by geometrical imaging techniques. The crystals appeared to be continuously distributed along a fibril, their size and number increasing in a tapered fashion from a relatively narrow tip containing smaller and infrequent crystals to wider regions having more densely packed and larger crystals. Defined for the first time by direct visual 3D imaging, these data describe the size, shape, location, orientation, and development of early crystals in normal

Photopolymerizable chitosan-collagen hydrogels for bone tissue engineering.

PubMed

Arakawa, Christopher; Ng, Ronald; Tan, Steven; Kim, Soyon; Wu, Benjamin; Lee, Min

2017-01-01

Photopolymerizable hydrogels derived from naturally occurring polymers have attracted significant interest in tissue-engineering applications due to their excellent biocompatibility, hydrophilic nature favourable for cell ingrowth and ability to be cured in situ through a minimally invasive procedure. In this study, we developed a composite hydrogel consisting of photocrosslinkable methacrylated glycol chitosan (MeGC) and semi-interpenetrating collagen (Col) with a riboflavin photoinitiator under blue light. The incorporation of Col in MeGC hydrogels enhanced the compressive modulus and slowed the degradation rate of the hydrogels. MeGC-Col composite hydrogels significantly enhanced cellular attachment, spreading, proliferation and osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) seeded on the hydrogels compared with pure MeGC hydrogels, as observed by upregulated alkaline phosphatase (ALP) activity as well as increased mineralization. Similarly, when cells were encapsulated within hydrogels, BMSCs exhibited greater proliferation, ALP activity and mineral deposits in the presence of Col. These findings demonstrate that MeGC-Col composite hydrogels may be useful in promoting bone regeneration. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

Reconstruction of irradiated bone segmental defects with a biomaterial associating MBCP+(R), microstructured collagen membrane and total bone marrow grafting: an experimental study in rabbits.

PubMed

Jégoux, Franck; Goyenvalle, Eric; Cognet, Ronan; Malard, Olivier; Moreau, Francoise; Daculsi, Guy; Aguado, Eric

2009-12-15

The bone tissue engineering models used today are still a long way from any oncologic application as immediate postimplantation irradiation would decrease their osteoinductive potential. The aim of this study was to reconstruct a segmental critical size defect in a weight-bearing bone irradiated after implantation. Six white New Zealand rabbits were immediately implanted with a biomaterial associating resorbable collagen membrane EZ(R) filled and micro-macroporous biphasic calcium phosphate granules (MBCP+(R)). After a daily schedule of radiation delivery, and within 4 weeks, a total autologous bone marrow (BM) graft was injected percutaneously into the center of the implant. All the animals were sacrificed at 16 weeks. Successful osseous colonization was found to have bridged the entire length of the defects. Identical distribution of bone ingrowth and residual ceramics at the different levels of the implant suggests that the BM graft plays an osteoinductive role in the center of the defect. Periosteum-like formation was observed at the periphery, with the collagen membrane most likely playing a role. This model succeeded in bridging a large segmental defect in weight-bearing bone with immediate postimplantation fractionated radiation delivery. This has significant implications for the bone tissue engineering approach to patients with cancer-related bone defects.

Zebrafish Collagen Type I: Molecular and Biochemical Characterization of the Major Structural Protein in Bone and Skin

PubMed Central

Gistelinck, C.; Gioia, R.; Gagliardi, A.; Tonelli, F.; Marchese, L.; Bianchi, L.; Landi, C.; Bini, L.; Huysseune, A.; Witten, P. E.; Staes, A.; Gevaert, K.; De Rocker, N.; Menten, B.; Malfait, F.; Leikin, S.; Carra, S.; Tenni, R.; Rossi, A.; De Paepe, A.; Coucke, P.; Willaert, A.; Forlino, A.

2016-01-01

Over the last years the zebrafish imposed itself as a powerful model to study skeletal diseases, but a limit to its use is the poor characterization of collagen type I, the most abundant protein in bone and skin. In tetrapods collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. In contrast, in zebrafish three type I collagen genes exist, col1a1a, col1a1b and col1a2 coding for α1(I), α3(I) and α2(I) chains. During embryonic and larval development the three collagen type I genes showed a similar spatio-temporal expression pattern, indicating their co-regulation and interdependence at these stages. In both embryonic and adult tissues, the presence of the three α(I) chains was demonstrated, although in embryos α1(I) was present in two distinct glycosylated states, suggesting a developmental-specific collagen composition. Even though in adult bone, skin and scales equal amounts of α1(I), α3(I) and α2(I) chains are present, the presented data suggest a tissue-specific stoichiometry and/or post-translational modification status for collagen type I. In conclusion, this data will be useful to properly interpret results and insights gained from zebrafish models of skeletal diseases. PMID:26876635

RAMAN SPECTRAL CLASSIFICATION OF MINERAL- AND COLLAGEN-BOUND WATER’S ASSOCIATIONS TO ELASTIC AND POST-YIELD MECHANICAL PROPERTIES OF CORTICAL BONE

PubMed Central

Unal, Mustafa; Akkus, Ozan

2015-01-01

Water that is bound to bone’s matrix is implied as a predictor of fracture resistance; however, bound water is an elusive variable to be measured nondestructively. To date, the only nondestructive method used for studying bone hydration status is magnetic resonance variants (NMR or MRI). For the first time, bone hydration status was studied by short-wave infrared (SWIR) Raman spectroscopy to investigate associations of mineral-bound and collagen-bound water compartments with mechanical properties. Thirty cortical bone samples were used for quantitative Raman-based water analysis, gravimetric analysis, porosity measurement, and biomechanical testing. A sequential dehydration protocol was developed to replace unbound (heat drying) and bound (ethanol treatment) water in bone. Raman spectra were collected serially to track the OH-stretch band during dehydration. Four previously identified peaks were investigated: I3220/I2949, I3325/I2949 and I3453/I2949 reflect status of organic-matrix related water (mostly collagen-related water) compartments and collagen portion of bone while I3584/I2949 reflects status of mineral-related water compartments and mineral portion of bone. These spectroscopic biomarkers were correlated with elastic and post-yield mechanical properties of bone. Collagen-water related biomarkers (I3220/I2949 and I3325/I2949) correlated significantly and positively with toughness (R2=0.81 and R2=0.79; p<0.001) and post-yield toughness (R2=0.65 and R2=0.73; p<0.001). Mineral-water related biomarker correlated significantly and negatively with elastic modulus (R2=0.78; p<0.001) and positively with strength (R2=0.46; p < 0.001). While MR-based techniques have been useful in measuring unbound and bound water, this is the first study which probed bound-water compartments differentially for collagen and mineral-bound water. For the first time, we showed an evidence for contributions of different bound-water compartments to mechanical properties of wet bone and

[Development of a novel absorbable nanofiber chitosan-collagen membrane by electrospinning and the preliminary study on guided bone regeneration].

PubMed

Gao, B; Li, X J; Lin, M; Li, Y Y; Dong, Y

2018-02-09

Objective: To evaluate the application effect of nanofiber chitosan-collagen membrane (NCM) on guided bone regeneration (GBR). Methods: The mixture of collagen, chitosan, polyethylene oxide was used to make up the NCM by electrospinning, then the NCM was crosslinked by glutaraldehyde vapor. The physical property of the NCM was measured by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). MC3T3-E1 osteoblasts were cultured on NCM to characterize the biocompatibility. The effectiveness of four groups [contrast group, Bio-gide membrane (BGM), compressed chitosan-collagen menbrane (CCM), NCM/CCM] on bone regeneration were evaluated in critical-sized defects (diameter = 5 mm) in SD rats. Results: When the mixed solution consists of 4.0% collagen, 1.0% chitosan and 3.5% polyethylene oxide, the NCM could be validly fabricated by electrospinning. After cross-linking by glutaraldehyde vapor, the tensile strength and the stability of NCM in damp was enhanced. No cytotoxicity of the NCM was detected on MC3T3-E1 osteoblasts. In vivo study showed that the new bone regeneration ratio of NCM/CCM group was [(43.10±1.49)%], and this was similar to that of the group of BGM [(41.36±2.60)%] ( P> 0.05), but higher than that of the CCM group [(33.10±1.41)%] and the contrast group [(7.22±2.46)%] ( P< 0.05). Conclusions: The NCM can promote new bone regeneration effectively in GBR procedure.

Determination of osteogenic or adipogenic lineages in muscle-derived stem cells (MDSCs) by a collagen-binding peptide (CBP) derived from bone sialoprotein (BSP).

PubMed

Choi, Yoon Jung; Lee, Jue Yeon; Lee, Seung Jin; Chung, Chong-Pyoung; Park, Yoon Jeong

2012-03-09

Bone sialoprotein (BSP) is a mineralized, tissue-specific, non-collagenous protein that is normally expressed only in mineralized tissues such as bone, dentin, cementum, and calcified cartilage, and at sites of new mineral formation. The binding of BSP to collagen is thought to be important for initiating bone mineralization and bone cell adhesion to the mineralized matrix. Several recent studies have isolated stem cells from muscle tissue, but their functional properties are still unclear. In this study, we examined the effects of a synthetic collagen-binding peptide (CBP) on the differentiation efficiency of muscle-derived stem cells (MDSCs). The CBP sequence (NGVFKYRPRYYLYKHAYFYPHLKRFPVQ) corresponds to residues 35-62 of bone sialoprotein (BSP), which are located within the collagen-binding domain in BSP. Interestingly, this synthetic CBP inhibited adipogenic differentiation but increased osteogenic differentiation in MDSCs. The CBP also induced expression of osteoblastic marker proteins, including alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx2), and osteocalcin; prevented adipogenic differentiation in MDSCs; and down-regulated adipose-specific mRNAs, such as adipocyte protein 2 (aP2) and peroxisome proliferator-activated receptor γ. The CBP increased Extracellular signal-regulated kinases (ERK) 1/2 protein phosphorylation, which is important in lineage determination. These observations suggest that this CBP determines the osteogenic or adipogenic lineage in MDSCs by activating ERK1/2. Taken together, a novel CBP could be a useful candidate for regenerating bone and treating osteoporosis, which result from an imbalance in osteogenesis and adipogenesis differentiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging.

PubMed

Schmidt, F N; Zimmermann, E A; Campbell, G M; Sroga, G E; Püschel, K; Amling, M; Tang, S Y; Vashishth, D; Busse, B

2017-04-01

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic cross-links, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692cm -1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p<0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high-performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging

PubMed Central

Schmidt, F.N.; Zimmermann, E.A.; Campbell, G.M.; Sroga, G.E.; Püschel, K.; Amling, M.; Tang, S. Y.; Vashishth, D.; Busse, B.

2017-01-01

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic crosslinks, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692 cm−1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p< 0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture. PMID:28109917

Low-level laser therapy induces an upregulation of collagen gene expression during the initial process of bone healing: a microarray analysis

NASA Astrophysics Data System (ADS)

Tim, Carla Roberta; Bossini, Paulo Sérgio; Kido, Hueliton Wilian; Malavazi, Iran; von Zeska Kress, Marcia Regina; Carazzolle, Marcelo Falsarella; Rennó, Ana Cláudia; Parizotto, Nivaldo Antonio

2016-08-01

This study investigates the histological modifications produced by low level laser therapy (LLLT) on the first day of bone repair, as well as evaluates the LLLT effects on collagen expression on the site of a fracture. Twenty Wistar rats were distributed into a control group (CG) and a laser group (LG). Laser irradiation of Ga-Al-As laser 830 nm, 30 mW, 94 s, 2.8 J was performed in five sessions. Animals were euthanized on day 5 postsurgery. Histopathological analysis showed that LLLT was able to increase deposition of granulation tissue and newly formed bone at the site of the injury. In addition, picrosirius analysis showed that collagen fiber organization in the LG was enhanced compared to CG. Microarray analysis demonstrated that LLLT produced an upregulation type I collagen (COL-I). Immunohistochemical analysis revealed that the subjects that were treated presented a higher immunoexpression of COL-I. Our findings indicated that LLLT improves bone healing by producing a significant increase in the expression of collagen genes.

Comparative Efficacies of Collagen-Based 3D Printed PCL/PLGA/β-TCP Composite Block Bone Grafts and Biphasic Calcium Phosphate Bone Substitute for Bone Regeneration.

PubMed

Hwang, Kyoung-Sub; Choi, Jae-Won; Kim, Jae-Hun; Chung, Ho Yun; Jin, Songwan; Shim, Jin-Hyung; Yun, Won-Soo; Jeong, Chang-Mo; Huh, Jung-Bo

2017-04-17

The purpose of this study was to compare bone regeneration and space maintaining ability of three-dimensional (3D) printed bone grafts with conventional biphasic calcium phosphate (BCP). After mixing polycaprolactone (PCL), poly (lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) in a 4:4:2 ratio, PCL/PLGA/β-TCP particulate bone grafts were fabricated using 3D printing technology. Fabricated particulate bone grafts were mixed with atelocollagen to produce collagen-based PCL/PLGA/β-TCP composite block bone grafts. After formation of calvarial defects 8 mm in diameter, PCL/PLGA/β-TCP composite block bone grafts and BCP were implanted into bone defects of 32 rats. Although PCL/PLGA/β-TCP composite block bone grafts were not superior in bone regeneration ability compared to BCP, the results showed relatively similar performance. Furthermore, PCL/PLGA/β-TCP composite block bone grafts showed better ability to maintain bone defects and to support barrier membranes than BCP. Therefore, within the limitations of this study, PCL/PLGA/β-TCP composite block bone grafts could be considered as an alternative to synthetic bone grafts available for clinical use.

In vivo bone regeneration with injectable chitosan/hydroxyapatite/collagen composites and mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Huang, Zhi; Chen, Yan; Feng, Qing-Ling; Zhao, Wei; Yu, Bo; Tian, Jing; Li, Song-Jian; Lin, Bo-Miao

2011-09-01

For reconstruction of irregular bone defects, injectable biomaterials are more appropriate than the preformed biomaterials. We herein develop a biomimetic in situ-forming composite consisting of chitosan (CS) and mineralized collagen fibrils (nHAC), which has a complex hierarchical structure similar to natural bone. The CS/nHAC composites with or without mesenchymal stem cells (MSCs) are injected into cancellous bone defects at the distal end of rabbit femurs. Defects are assessed by radiographic, histological diagnosis and Raman microscopy until 12 weeks. The results show that MSCs improve the biocompatibility of CS/nHAC composites and enhance new bone formation in vivo at 12 weeks. It can be concluded that the injectable CS/nHAC composites combined with MSCs may be a novel method for reconstruction of irregular bone defects.

A single topical dose of erythropoietin applied on a collagen carrier enhances calvarial bone healing in pigs

PubMed Central

2014-01-01

Background and purpose The osteogenic potency of erythropoietin (EPO) has been documented. However, its efficacy in a large-animal model has not yet been investigated; nor has a clinically safe dosage. The purpose of this study was to overcome such limitations of previous studies and thereby pave the way for possible clinical application. Our hypothesis was that EPO increases calvarial bone healing compared to a saline control in the same subject. Methods We used a porcine calvarial defect model. In each of 18 pigs, 6 cylindrical defects (diameter: 1 cm; height: 1 cm) were drilled, allowing 3 pairwise comparisons. Treatment consisted of either 900 IU/mL EPO or an equal volume of saline in combination with either autograft, a collagen carrier, or a polycaprolactone (PCL) scaffold. After an observation time of 5 weeks, the primary outcome (bone volume fraction (BV/TV)) was assessed with high-resolution quantitative computed tomography. Secondary outcome measures were histomorphometry and blood samples. Results The median BV/TV ratio of the EPO-treated collagen group was 1.06 (CI: 1.02–1.11) relative to the saline-treated collagen group. Histomorphometry showed a similar median effect size, but it did not reach statistical significance. Autograft treatment had excellent healing potential and was able to completely regenerate the bone defect independently of EPO treatment. Bony ingrowth into the PCL scaffold was sparse, both with and without EPO. Neither a substantial systemic effect nor adverse events were observed. The number of blood vessels was similar in EPO-treated defects and saline-treated defects. Interpretation Topical administration of EPO on a collagen carrier moderately increased bone healing. The dosing regime was safe, and could have possible application in the clinical setting. However, in order to increase the clinical relevance, a more potent but still clinically safe dose should be investigated. PMID:24564750

Carnivore specific bone bioapatite and collagen carbon isotope fractionations: Case studies of modern and fossil grey wolf populations

NASA Astrophysics Data System (ADS)

Fox-Dobbs, K.; Wheatley, P. V.; Koch, P. L.

2006-12-01

Stable isotope analyses of modern and fossil biogenic tissues are routinely used to reconstruct present and past vertebrate foodwebs. Accurate isotopic dietary reconstructions require a consumer and tissue specific understanding of how isotopes are sorted, or fractionated, between trophic levels. In this project we address the need for carnivore specific isotope variables derived from populations that are ecologically well- characterized. Specifically, we investigate the trophic difference in carbon isotope values between mammalian carnivore (wolf) bone bioapatite and herbivore (prey) bone bioapatite. We also compare bone bioapatite and collagen carbon isotope values collected from the same individuals. We analyzed bone specimens from two modern North American grey wolf (Canis lupus) populations (Isle Royale National Park, Michigan and Yellowstone National Park, Wyoming), and the ungulate herbivores that are their primary prey (moose and elk, respectively). Because the diets of both wolf populations are essentially restricted to a single prey species, there were no confounding effects due to carnivore diet variability. We measured a trophic difference of approximately -1.3 permil between carnivore (lower value) and herbivore (higher value) bone bioapatite carbon isotope values, and an average inter-tissue difference of 5.1 permil between carnivore bone collagen (lower value) and bioapatite (higher value) carbon isotope values. Both of these isotopic differences differ from previous estimates derived from a suite of African carnivores; our carnivore-herbivore bone bioapatite carbon isotope spacing is smaller (-1.3 vs. -4.0 permil), and our carnivore collagen-bioapatite carbon difference is larger (5.1 vs. 3.0 permil). These discrepancies likely result from comparing values measured from a single hypercarnivore (wolf) to average values calculated from several carnivore species, some of which are insectivorous or partly omnivorous. The trophic and inter

Induction and quantification of collagen fiber alignment in a three-dimensional hydroxyapatite-collagen composite scaffold.

PubMed

Banglmaier, Richard F; Sander, Edward A; VandeVord, Pamela J

2015-04-01

Hydroxyapatite-collagen composite scaffolds are designed to serve as a regenerative load bearing replacement that mimics bone. However, the material properties of these scaffolds are at least an order of magnitude less than that of bone and subject to fail under physiological loading conditions. These scaffolds compositionally resemble bone but they do not possess important structural attributes such as an ordered arrangement of collagen fibers, which is a correlate to the mechanical properties in bone. Furthermore, it is unclear how much ordering of structure is satisfactory to mimic bone. Therefore, quantitative methods are needed to characterize collagen fiber alignment in these scaffolds for better correlation between the scaffold structure and the mechanical properties. A combination of extrusion and compaction was used to induce collagen fiber alignment in composite scaffolds. Collagen fiber alignment, due to extrusion and compaction, was quantified from polarized light microscopy images with a Fourier transform image processing algorithm. The Fourier transform method was capable of resolving the degree of collagen alignment from polarized light images. Anisotropy indices of the image planes ranged from 0.08 to 0.45. Increases in the degree of fiber alignment induced solely by extrusion (0.08-0.25) or compaction (0.25-0.44) were not as great as those by the combination of extrusion and compaction (0.35-0.45). Additional measures of randomness and fiber direction corroborate these anisotropy findings. This increased degree of collagen fiber alignment was induced in a preferred direction that is consistent with the extrusion direction and parallel with the compacted plane. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Effect of bone sialoprotein and collagen coating on cell attachment to TICER and pure titanium implant surfaces.

PubMed

Graf, H-L; Stoeva, S; Armbruster, F P; Neuhaus, J; Hilbig, H

2008-07-01

To improve integration between implants and biological tissues, this study compared bone sialoprotein (BSP) as a surface-coating material against the major organic and inorganic components of bone, collagen type I and hydroxyapatite (TICER). The expression of osteocalcin, osteonectin and transforming growth factor ss was evaluated using immunohistochemical staining procedures. The distribution patterns of osteoblasts on the surface of pure titanium with a smooth machined surface and a rough surface (TICER) were determined by image processing using confocal laser scanning microscopy. The results compared to uncoated control materials showed that, at all times investigated, the number of cells on the surface of the TICER and pure titanium samples differed significantly (P<0.1), demonstrating the superiority of TICER over pure titanium in this respect. For pure titanium implants, collagen-precoated surfaces were not beneficial for the attachment of bone-derived cells with the exception of day 3 in vitro (P<0.01). BSP-precoated implant surfaces displayed non-significantly higher numbers of settled cells. BSP-precoated implant surfaces were beneficial for osteoinduction as revealed by osteocalcin and osteonectin expression. BSP precoating of the rough TICER implant surface enhanced the osteoinductive effect much more than did collagen precoating. These results contribute to the consideration of at least two distinct pathways of osseointegration.

A novel 3D bone-mimetic scaffold composed of collagen/MTA/MWCNT modulates cell migration and osteogenesis.

PubMed

Valverde, Thalita M; Castro, Elisandra G; Cardoso, Maíssa H S; Martins-Júnior, Paulo A; Souza, Lívia M O; Silva, Patrícia P; Ladeira, Luiz O; Kitten, Gregory T

2016-10-01

This study characterized a three-dimensional (3D) biocomposite scaffolds produced using type I collagen, mineral trioxide aggregate (MTA) and multi-walled carbon nanotubes (MWCNT) to be used in bone tissue regeneration. The scaffolds were analyzed via scanning (SEM) and transmission (TEM) electron microscopy, as well as the viability and migration of osteoblasts and mineralization of the scaffolds. SEM and TEM analyses showed that MTA and MWCNT were distributed as both large agglomerates entrapped within the collagen network and as smaller accumulations or individual molecules dispersed throughout the scaffold. Ultrastructural analysis revealed that osteoblastic MC3T3-E1 cells grown in the biocomposite endocytosed MWCNT, which were localized in the cytoplasm and in vesicles. Analysis of cells grown in the 3D scaffolds demonstrated that >95% of the cells remained viable in all tested combinations and concentrations of the biocomposite. MC3T3-E1 osteoblasts migrated into scaffolds formed with concentrations of type I collagen between 1.75 and 3.0mg/mL. Cells displayed increased migration into scaffolds formed with collagen and a range of low to high concentrations of MTA. In contrast, the presence of MWCNT in the biocomposite had a slight negative effect on migration. Collagen gels containing specific concentrations of MTA, or MWCNT, or combinations of MTA/MWCNT, caused an increase in mineralization of scaffolds. Scaffolds composed of defined concentrations of type I collagen, MTA and MWCNT are biocompatible, promote migration and mineralization of osteoblasts, and hence may be useful as bone tissue mimetics. Copyright © 2016 Elsevier Inc. All rights reserved.

The osteogenic differentiation of dog bone marrow mesenchymal stem cells in a thermo-sensitive injectable chitosan/collagen/β-glycerophosphate hydrogel: in vitro and in vivo.

PubMed

Sun, Bin; Ma, Wei; Su, Fang; Wang, Yi; Liu, Jiaqiang; Wang, Dongshen; Liu, Hongchen

2011-09-01

Type I collagen was added to the composite chitosan solution in a ratio of 1:2 to build a physical cross-linked self-forming chitosan/collagen/β-GP hydrogel. Osteogenic properties of this novel injectable hydrogel were evaluated. Gelation time was about 8 min which offered enough time for handling a mixture containing cells and the subsequent injection. Scanning electronic microscopy (SEM) observations indicated good spreading of bone marrow mesenchymal stem cells (BMSCs) in this hydrogel scaffold. Mineral nodules were found in the dog-BMSCs inoculated hydrogel by SEM after 28 days. After subcutaneous injection into nude mouse dorsum for 4 weeks, partial bone formation was observed in the chitosan/collagen/β-GP hydrogel loaded with pre-osteodifferentiated dog-BMSCs, which indicated that chitosan/collagen/β-GP hydrogel composite could induce osteodifferentiation in BMSCs without exposure to a continual supply of external osteogenic factors. In conclusion, the novel chitosan/collagen/β-GP hydrogel composite should prove useful as a bone regeneration scaffold.

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs.

PubMed

Lu, Yongbo; Kamel-El Sayed, Suzan A; Wang, Kun; Tiede-Lewis, LeAnn M; Grillo, Michael A; Veno, Patricia A; Dusevich, Vladimir; Phillips, Charlotte L; Bonewald, Lynda F; Dallas, Sarah L

2018-06-01

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel

Design and Use of Chimeric Proteins Containing a Collagen-Binding Domain for Wound Healing and Bone Regeneration.

PubMed

Addi, Cyril; Murschel, Frederic; De Crescenzo, Gregory

2017-04-01

Collagen-based biomaterials are widely used in the field of tissue engineering; they can be loaded with biomolecules such as growth factors (GFs) to modulate the biological response of the host and thus improve its potential for regeneration. Recombinant chimeric GFs fused to a collagen-binding domain (CBD) have been reported to improve their bioavailability and the host response, especially when combined with an appropriate collagen-based biomaterial. This review first provides an extensive description of the various CBDs that have been fused to proteins, with a focus on the need for accurate characterization of their interaction with collagen. The second part of the review highlights the benefits of various CBD/GF fusion proteins that have been designed for wound healing and bone regeneration.

Determination of osteogenic or adipogenic lineages in muscle-derived stem cells (MDSCs) by a collagen-binding peptide (CBP) derived from bone sialoprotein (BSP)

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

Choi, Yoon Jung; Lee, Jue Yeon; Lee, Seung Jin

Highlights: Black-Right-Pointing-Pointer CBP sequence is identified from BSP and has collagen binding activity. Black-Right-Pointing-Pointer CBP directly activates the MAPK signaling, especially ERK1/2. Black-Right-Pointing-Pointer CBP increase osteoblastic differentiation by the activation of Runx2. Black-Right-Pointing-Pointer CBP decrease adipogenic differentiation by the inhibition of PPAR{gamma}. -- Abstract: Bone sialoprotein (BSP) is a mineralized, tissue-specific, non-collagenous protein that is normally expressed only in mineralized tissues such as bone, dentin, cementum, and calcified cartilage, and at sites of new mineral formation. The binding of BSP to collagen is thought to be important for initiating bone mineralization and bone cell adhesion to the mineralized matrix. Severalmore » recent studies have isolated stem cells from muscle tissue, but their functional properties are still unclear. In this study, we examined the effects of a synthetic collagen-binding peptide (CBP) on the differentiation efficiency of muscle-derived stem cells (MDSCs). The CBP sequence (NGVFKYRPRYYLYKHAYFYPHLKRFPVQ) corresponds to residues 35-62 of bone sialoprotein (BSP), which are located within the collagen-binding domain in BSP. Interestingly, this synthetic CBP inhibited adipogenic differentiation but increased osteogenic differentiation in MDSCs. The CBP also induced expression of osteoblastic marker proteins, including alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx2), and osteocalcin; prevented adipogenic differentiation in MDSCs; and down-regulated adipose-specific mRNAs, such as adipocyte protein 2 (aP2) and peroxisome proliferator-activated receptor {gamma}. The CBP increased Extracellular signal-regulated kinases (ERK) 1/2 protein phosphorylation, which is important in lineage determination. These observations suggest that this CBP determines the osteogenic or adipogenic lineage in MDSCs by activating ERK1/2. Taken

Alpha C-telopeptide of type I collagen is associated with subchondral bone turnover and predicts progression of joint space narrowing and osteophytes in osteoarthritis.

PubMed

Huebner, Janet L; Bay-Jensen, Anne C; Huffman, Kim M; He, Yi; Leeming, Diana J; McDaniel, Gary E; Karsdal, Morten A; Kraus, Virginia B

2014-09-01

To evaluate joint tissue remodeling using the urinary collagen biomarkers urinary α-C-telopeptide of type I collagen (α-CTX) and urinary C-telopeptide of type II collagen (CTX-II) and to determine the association of these biomarkers with osteoarthritis (OA) severity, progression, and localized knee bone turnover. Participants (n = 149) with symptomatic and radiographic knee OA underwent fixed-flexion knee radiography at baseline and 3 years, and late-phase bone scintigraphy of both knees at baseline, which were scored semiquantitatively for osteophyte and joint space narrowing (JSN) severity and uptake intensity, with scores summed across knees. Urinary concentrations of α-CTX and CTX-II were determined by enzyme-linked immunosorbent assay. Immunohistochemical analysis of human OA knees was performed to localize the joint tissue origin of the biomarker epitopes. Urinary α-CTX concentrations correlated strongly with the intensity of bone scintigraphic uptake and with JSN progression (risk ratio 13.2) and osteophyte progression (risk ratio 3). Urinary CTX-II concentrations were strongly associated with intensity of bone scintigraphic uptake, with JSN and osteophyte severity, and with OA progression based on osteophyte score. Urinary α-CTX localized primarily to high bone turnover areas in subchondral bone. CTX-II localized to the bone-cartilage interface, the tidemark, and damaged articular cartilage. Baseline urinary α-CTX, which was localized to high turnover areas of subchondral bone, was associated with dynamic bone turnover of knees, as signified by scintigraphy, and progression of both osteophytes and JSN. Urinary CTX-II correlated with JSN and osteophyte severity and progression of osteophytes. To our knowledge, this represents the first report of serologic markers reflecting subchondral bone turnover. These collagen markers may be useful for noninvasive detection and quantification of active subchondral bone turnover and joint remodeling in knee OA

AGE-RELATED EFFECT ON THE CONCENTRATION OF COLLAGEN CROSSLINKS IN HUMAN OSTEONAL AND INTERSTITIAL BONE TISSUE

PubMed Central

Nyman, Jeffry S.; Roy, Anuradha; Acuna, Rae L.; Gayle, Heather J.; Reyes, Michael J.; Tyler, Jerrod H.; Dean, David D.; Wang, Xiaodu

2007-01-01

Collagen crosslinks are important to the quality of bone and may be contributors to the age-related increase in bone fracture. This study was performed to investigate whether age and gender effects on collagen crosslinks are similar in osteonal and interstitial bone tissues. Forty human cadaveric femurs were collected and divided into two age groups: Middle aged (42–63 years of age) and Elderly (69–90 years of age) with ten males and ten females in each group (n = 10). Micro-cores of bone tissue from both secondary osteons (newly formed) and interstitial regions (biologically old) in the medial quadrant of the diaphysis were extracted using a custom-modified, computer numerical controlled machine. The bone specimens were then analyzed using high performance liquid chromatography to determine the effects of age and gender on the concentration of mature, enzymatic crosslinks (hydroxylysyl-pyridinoline – HP and lysylpyridinoline – LP) and a non-enzymatic crosslink (pentosidine – PE) at these two bony sites. The results indicate that age has a significant effect on the concentration of LP and PE, while gender has a significant effect on HP and LP. In addition, the concentration of the crosslinks in the secondary osteons is significantly different from that in the interstitial bone regions. These results suggest that the rate of non-enzymatic crosslinking may increase while the formation of maturate enzymatic crosslinks may decrease with age. Such changes could potentially reduce the inherent quality of the bone tissue in the elderly skeleton. PMID:16962838

A New Bone Substitute Developed from 3D-Prints of Polylactide (PLA) Loaded with Collagen I: An In Vitro Study.

PubMed

Ritz, Ulrike; Gerke, Rebekka; Götz, Hermann; Stein, Stefan; Rommens, Pol Maria

2017-11-29

Although a lot of research has been performed, large segmental bone defects caused by trauma, infection, bone tumors or revision surgeries still represent big challenges for trauma surgeons. New and innovative bone substitutes are needed. Three-dimensional (3D) printing is a novel procedure to create 3D porous scaffolds that can be used for bone tissue engineering. In the present study, solid discs as well as porous cage-like 3D prints made of polylactide (PLA) are coated or filled with collagen, respectively, and tested for biocompatibility and endotoxin contamination. Microscopic analyses as well as proliferation assays were performed using various cell types on PLA discs. Stromal-derived factor (SDF-1) release from cages filled with collagen was analyzed and the effect on endothelial cells tested. This study confirms the biocompatibility of PLA and demonstrates an endotoxin contamination clearly below the FDA (Food and Drug Administration) limit. Cells of various cell types (osteoblasts, osteoblast-like cells, fibroblasts and endothelial cells) grow, spread and proliferate on PLA-printed discs. PLA cages loaded with SDF-1 collagen display a steady SDF-1 release, support cell growth of endothelial cells and induce neo-vessel formation. These results demonstrate the potential for PLA scaffolds printed with an inexpensive desktop printer in medical applications, for example, in bone tissue engineering.

A New Bone Substitute Developed from 3D-Prints of Polylactide (PLA) Loaded with Collagen I: An In Vitro Study

PubMed Central

Gerke, Rebekka; Götz, Hermann; Rommens, Pol Maria

2017-01-01

Although a lot of research has been performed, large segmental bone defects caused by trauma, infection, bone tumors or revision surgeries still represent big challenges for trauma surgeons. New and innovative bone substitutes are needed. Three-dimensional (3D) printing is a novel procedure to create 3D porous scaffolds that can be used for bone tissue engineering. In the present study, solid discs as well as porous cage-like 3D prints made of polylactide (PLA) are coated or filled with collagen, respectively, and tested for biocompatibility and endotoxin contamination. Microscopic analyses as well as proliferation assays were performed using various cell types on PLA discs. Stromal-derived factor (SDF-1) release from cages filled with collagen was analyzed and the effect on endothelial cells tested. This study confirms the biocompatibility of PLA and demonstrates an endotoxin contamination clearly below the FDA (Food and Drug Administration) limit. Cells of various cell types (osteoblasts, osteoblast-like cells, fibroblasts and endothelial cells) grow, spread and proliferate on PLA-printed discs. PLA cages loaded with SDF-1 collagen display a steady SDF-1 release, support cell growth of endothelial cells and induce neo-vessel formation. These results demonstrate the potential for PLA scaffolds printed with an inexpensive desktop printer in medical applications, for example, in bone tissue engineering. PMID:29186036

Preparative separation of underivatized amino acids for compound-specific stable isotope analysis and radiocarbon dating of hydrolyzed bone collagen.

PubMed

Tripp, Jennifer A; McCullagh, James S O; Hedges, Robert E M

2006-01-01

Analysis of stable and radioactive isotopes from bone collagen provides useful information to archaeologists about the origin and age of bone artifacts. Isolation and analysis of single amino acids from the proteins can provide additional and more accurate information by removing contamination and separating a bulk isotope signal into its constituent parts. In this paper, we report a new method for the separation and isolation of underivatized amino acids from bone collagen, and their analysis by isotope ratio MS and accelerator MS. RP chromatography is used to separate the amino acids with nonpolar side chains, followed by an ion pair separation to isolate the remaining amino acids. The method produces single amino acids with little or no contamination from the separation process and allows for the measurement of accurate stable isotope ratios and pure samples for radiocarbon dating.

Socket Preservation using Enzyme-treated Equine Bone Granules and an Equine Collagen Matrix: A Case Report with Histological and Histomorphometrical Assessment.

PubMed

Leonida, Alessandro; Todeschini, Giovanni; Lomartire, Giovanni; Cinci, Lorenzo; Pieri, Laura

2016-11-01

To histologically assess the effectiveness of a socket-preservation technique using enzyme-treated equine bone granules as a bone-graft material in combination with an equine collagen matrix as a scaffold for soft-tissue regeneration. Enzyme-treated equine bone granules and equine collagen matrix recently have been developed to help overcome alveolar bone deficiencies that develop in the wake of edentulism. The patient had one mandibular molar extracted and the socket grafted with equine bone granules. The graft was covered with the equine collagen matrix, placed in a double layer. No flap was prepared, and the gingival margins were stabilized with a single stitch, leaving the matrix partially exposed and the site to heal by secondary intention. The adjacent molar was extracted 1 month later, and that socket was left to heal by secondary intention without any further treatment. Three months after each surgery, an implant was placed and a biopsy was collected. The two biopsies underwent histological processing and qualitative evaluation. Histomorphometric analysis was also performed to calculate the percentage of newly formed bone (NFB) in the two cores. Healing at both sites was uneventful, and no inflammation or other adverse reactions were observed in the samples. Soft-tissue healing by secondary intention appeared to occur faster at the grafted site. The corresponding core showed a marked separation between soft and hard tissue that was not observed in the core from the nongrafted site, where soft-tissue hypertrophy could be observed. Newly formed bone at the grafted and nongrafted sites was not significantly different (27.2 ± 7.1 and 29.4 ± 6.2% respectively, p = 0.45). The surgical technique employed in this case appeared to facilitate postextraction soft-tissue healing by second intention and simplify soft-tissue management. Using a collagen-based matrix to cover a postextraction grafted site may facilitate second intention soft-tissue healing and proper

Bone Tissue Collagen Maturity and Mineral Content Increase With Sustained Hyperglycemia in the KK-Ay Murine Model of Type 2 Diabetes.

PubMed

Hunt, Heather B; Pearl, Jared C; Diaz, David R; King, Karen B; Donnelly, Eve

2018-05-01

Type 2 diabetes mellitus (T2DM) increases fracture risk for a given bone mineral density (BMD), which suggests that T2DM changes bone tissue properties independently of bone mass. In this study, we assessed the effects of hyperglycemia on bone tissue compositional properties, enzymatic collagen crosslinks, and advanced glycation end-products (AGEs) in the KK-Ay murine model of T2DM using Fourier transform infrared (FTIR) imaging and high-performance liquid chromatography (HPLC). Compared to KK-aa littermate controls (n = 8), proximal femoral bone tissue of KK-Ay mice (n = 14) exhibited increased collagen maturity, increased mineral content, and less heterogeneous mineral properties. AGE accumulation assessed by the concentration of pentosidine, as well as the concentrations of the nonenzymatic crosslinks hydroxylysylpyridinoline (HP) and lysyl pyridinoline (LP), did not differ in the proximal femurs of KK-Ay mice compared to controls. The observed differences in tissue-level compositional properties in the KK-Ay mice are consistent with bone that is older and echo observations of reduced remodeling in T2DM. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

Promising efficacy of Escherichia coli recombinant human bone morphogenetic protein-2 in collagen sponge for ectopic and orthotopic bone formation and comparison with mammalian cell recombinant human bone morphogenetic protein-2.

PubMed

Kim, In Sook; Lee, Eui Nam; Cho, Tae Hyung; Song, Yun Mi; Hwang, Soon Jung; Oh, Ji Hye; Park, Eun Kyung; Koo, Tai Young; Seo, Young-Kwon

2011-02-01

Nonglycosylated recombinant human bone morphogenetic protein (rhBMP)-2 prepared in Escherichia coli (E. coli rhBMP-2) has recently been considered as an alternative to mammalian cell rhBMP-2. However, its clinical use is still limited owing to lack of evidence for osteogenic activity comparable with that of mammalian cell rhBMP-2 via microcomputed tomography-based analysis. Therefore, this study aimed to evaluate the ability of E. coli rhBMP-2 in absorbable collagen sponge to form ectopic and orthotopic bone and to compare it to that of mammalian rhBMP-2. In vitro investigation was performed to study osteoblast differentiation of human mesenchymal stromal cells. Both types of rhBMP-2 enhanced proliferation, alkaline phosphatase activity, and matrix mineralization of human mesenchymal stromal cells at similar levels. Similar tendencies were observed in microcomputed tomography analysis, which determined bone volume, fractional bone volume, trabecular thickness, trabecular separation, bone mineral density, and other characteristics. Histology from an in vivo osteoinductivity test and from a rat calvarial defect model demonstrated a dose-dependent increase in local bone formation. The E. coli rhBMP-2 group (5 μg) not only induced complete regeneration of an 8-mm critical-sized defect at 4 weeks, but also led to new bone with the same bone mineral density as normal bone at 8 weeks, with the same efficiency as that of mammalian cell rhBMP-2 (5 μg). These uniformly favorable results provide evidence that the osteogenic activity of E. coli rhBMP-2 is not inferior to that of mammalian cell rhBMP-2 despite its low solubility and lack of gylcosylation. These results suggest that the application of E. coli rhBMP-2 in absorbable collagen sponge may be a promising equivalent to mammalian cell rhBMP-2 in bone tissue engineering.

Altered distributions of bone tissue mineral and collagen properties in women with fragility fractures.

PubMed

Wang, Zhen Xiang; Lloyd, Ashley A; Burket, Jayme C; Gourion-Arsiquaud, Samuel; Donnelly, Eve

2016-03-01

Heterogeneity of bone tissue properties is emerging as a potential indicator of altered bone quality in pathologic tissue. The objective of this study was to compare the distributions of tissue properties in women with and without histories of fragility fractures using Fourier transform infrared (FTIR) imaging. We extended a prior study that examined the relationship of the mean FTIR properties to fracture risk by analyzing in detail the widths and the tails of the distributions of FTIR properties in biopsies from fracture and non-fracture cohorts. The mineral and matrix properties of cortical and trabecular iliac crest tissue were compared in biopsies from women with a history of fragility fracture (+Fx; n=21, age: mean 54±SD 15y) and with no history of fragility fracture (-Fx; n=12, age: 57±5y). A subset of the patients included in the -Fx group were taking estrogen-plus-progestin hormone replacement therapy (HRT) (-Fx+HRT n=8, age: 58±5y) and were analyzed separately from patients with no history of HRT (-Fx-HRT n=4, age: 56±7y). When the FTIR parameter mean values were examined by treatment group, the trabecular tissue of -Fx-HRT patients had a lower mineral:matrix ratio (M:M) and collagen maturity (XLR) than that of -Fx+HRT patients (-22% M:M, -18% XLR) and +Fx patients (-17% M:M, -18% XLR). Across multiple FTIR parameters, tissue from the -Fx-HRT group had smaller low-tail (5th percentile) values than that from the -Fx+HRT or +Fx groups. In trabecular collagen maturity and crystallinity (XST), the -Fx-HRT group had smaller low-tail values than those in the -Fx+HRT group (-16% XLR, -5% XST) and the +Fx group (-17% XLR, -7% XST). The relatively low values of trabecular mineral:matrix ratio and collagen maturity and smaller low-tail values of collagen maturity and crystallinity observed in the -Fx-HRT group are characteristic of younger tissue. Taken together, our data suggest that the presence of newly formed tissue that includes small/imperfect crystals

Experimental model of bone response to collagenized xenografts of porcine origin (OsteoBiol® mp3): a radiological and histomorphometric study.

PubMed

Calvo Guirado, Jose Luis; Ramírez Fernández, Maria Piedad; Negri, Bruno; Delgado Ruiz, Rafael Arcesio; Maté Sánchez de-Val, José Eduardo; Gómez-Moreno, Gerardo

2013-02-01

Adequate alveolar ridges are fundamental to successful rehabilitation with implants. There are diverse techniques for reconstructing atrophied ridges, of which bone substitute grafts is one possibility. The aim of this study was to carry out radiological and histomorphometric evaluations of bone response to collagenized porcine bone xenografts over a 4-month period following their insertion in rabbits' tibiae. Twenty New Zealand rabbits were used. Twenty collagenized porcine bone xenografts (Osteobiol® mp3, Tecnoss Dental s.r.l., Torino, Italy), in granulated form of 600 to 1,000 µm, were inserted in the proximal metaphyseal area of the animals' tibiae and 20 control areas were created. Following implantation, the animals were sacrificed in four groups of five, after 1, 2, 3, and 4 months, respectively. Radiological and histomorphometric studies were made. After 4 months, radiological images revealed bone defects with a decrease in graft volume and the complete repair of the osseous defect. No healed or residual bone alterations attributable to the presence of the implants were observed. Histomorphometric analysis at 4 months found mean values for newly formed bone, residual graft material, and non-mineralized connective tissue of 25.4 ± 1.8%, 36.37 ± 3.0%, and 38.22 ± 2.5%, respectively. There were no statistical differences in the length of cortical formation with collagenized porcine xenograft (98.9 ± 1.1%) compared with the control samples (99.1 ± 0.7%) at the end of the study period. The biomaterial used proved to be biocompatible, bioabsorbable, and osteoconductive and as such, a possible bone substitute that did not interfere with the bone's normal reparative processes. © 2011 Wiley Periodicals, Inc.

Collagen membranes: a review.

PubMed

Bunyaratavej, P; Wang, H L

2001-02-01

Collagen materials have been utilized in medicine and dentistry because of their proven biocompatability and capability of promoting wound healing. For guided tissue regeneration (GTR) procedures, collagen membranes have been shown to be comparable to non-absorbable membranes with regard to probing depth reduction, clinical attachment gain, and percent of bone fill. Although these membranes are absorbable, collagen membranes have been demonstrated to prevent epithelial down-growth along the root surfaces during the early phase of wound healing. The use of grafting material in combination with collagen membranes seems to improve clinical outcomes for furcation, but not intrabony, defects when compared to the use of membranes alone. Recently, collagen materials have also been applied in guided bone regeneration (GBR) and root coverage procedures with comparable success rates to non-absorbable expanded polytetrafluoroethylene (ePTFE) membranes and conventional subepithelial connective tissue grafts, respectively. Long-term clinical trials are still needed to further evaluate the benefits of collagen membranes in periodontal and peri-implant defects. This article will review the rationale for each indication and its related literature, both in vitro and in vivo studies. The properties that make collagen membranes attractive for use in regenerative therapy will be addressed. In addition, varieties of cross-linking techniques utilized to retard the degradation rate of collagen membranes will be discussed.

Socket augmentation using a commercial collagen-based product--an animal study in pigs.

PubMed

Kunert-Keil, Christiane; Gredes, Tomasz; Heinemann, Friedhelm; Dominiak, Marzena; Botzenhart, Ute; Gedrange, Tomasz

2015-01-01

The aim of the present study was to identify properties of pure collagen for augmentation techniques and compare to a proved xenogenic material and natural bone regeneration. For that the osteogenesis of extraction alveoli after augmentation with a collagen cone covered with an absorbable collagen membrane in a single product (PARASORB Sombrero®, Resorba) was evaluated in a pig model. Extraction alveoli were treated with the collagen cone and the collagen membrane in a single product (test group; n=7) or demineralized bovine bone mineral and a collagen membrane (two separate products; positive control; n=7). Untreated alveoli were used (n=6) as negative controls.(1) Bone specimens were extracted 1 and 3 months after teeth extraction. Serial longitudinal sections were stained with Masson Goldner trichrome. Furthermore, bone specimens were examined using X-ray analyses. Significant differences of bone atrophy were detected 12 weeks after material insertion using X-ray analyses. The bone atrophy was reduced by approximately 32% after insertion of the positive control (P=0.046). Bone atrophy reached 37.6% of those from untreated alveoli (P=0.002) using the test group. After 4 weeks, bone formation was noticeable in most sites, whereas after 12 weeks of healing, specimens of all groups exhibited nearly complete osseous organization of the former defected area. The mandibulary bone texture showed typical spongious bone structures. Histomorphometric analyses revealed after 4 and 12 weeks significant higher levels of bone marrow in test and negative control than in positive control. Quantification of bone tissue and osteoid does not show any significant difference. The present study confirms reduced bone resorption following socket augmentation with an absorbable collagen membrane with collagen cone while the resulting bone structure is similar to natural bone regeneration. Pure collagen can be used for bone augmentation, and shows over other xenogenic materials, a clear

Biomimetic mineralization of recombinant collagen type I derived protein to obtain hybrid matrices for bone regeneration.

PubMed

Ramírez-Rodríguez, Gloria Belén; Delgado-López, José Manuel; Iafisco, Michele; Montesi, Monica; Sandri, Monica; Sprio, Simone; Tampieri, Anna

2016-11-01

Understanding the mineralization mechanism of synthetic protein has recently aroused great interest especially in the development of advanced materials for bone regeneration. Herein, we propose the synthesis of composite materials through the mineralization of a recombinant collagen type I derived protein (RCP) enriched with RGD sequences in the presence of magnesium ions (Mg) to closer mimic bone composition. The role of both RCP and Mg ions in controlling the precipitation of the mineral phase is in depth evaluated. TEM and X-ray powder diffraction reveal the crystallization of nanocrystalline apatite (Ap) in all the evaluated conditions. However, Raman spectra point out also the precipitation of amorphous calcium phosphate (ACP). This amorphous phase is more evident when RCP and Mg are at work, indicating the synergistic role of both in stabilizing the amorphous precursor. In addition, hybrid matrices are prepared to tentatively address their effectiveness as scaffolds for bone tissue engineering. SEM and AFM imaging show an homogeneous mineral distribution on the RCP matrix mineralized in presence of Mg, which provides a surface roughness similar to that found in bone. Preliminary in vitro tests with pre-osteoblast cell line show good cell-material interaction on the matrices prepared in the presence of Mg. To the best of our knowledge this work represents the first attempt to mineralize recombinant collagen type I derived protein proving the simultaneous effect of the organic phase (RCP) and Mg on ACP stabilization. This study opens the possibility to engineer, through biomineralization process, advanced hybrid matrices for bone regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

Guided bone regeneration produced by new mineralized and reticulated collagen membranes in critical-sized rat calvarial defects

PubMed Central

Leitão, Renata FC; Figueiró, Sônia D; Góes, Júlio C; Lima, Vilma; Silveira, Charles O; Brito, Gerly AC

2015-01-01

The aim of this study was to evaluate the bone regenerative effect of glutaraldehyde (GA) cross-linking on mineralized polyanionic collagen membranes in critical-sized defects on rat calvarias. Bone calvarial defects were induced in Wistar rats, which were then divided into five groups: a sham group; a control group, which received a commercial membrane; and GA, 25GA, and 75GA groups, which received one of three different polyanionic collagen membranes mineralized by 0, 25, or 75 hydroxyapatite cycles and then cross-linked by GA. Bone formation was evaluated based on digital radiography and computerized tomography. Histological analyses were performed 4 and 12 weeks after the surgical procedure to observe bone formation, membrane resorption, and fibrous tissue surrounding the membranes. Measurement of myeloperoxidase activity, tumor necrosis factor alpha, and interleukin 1beta production was performed 24 h after surgery. The percentage of new bone formation in the GA, 25GA, and 75GA groups was higher compared with the control and sham groups. In the GA and 25 GA groups, the membranes were still in place and were contained in a thick fibrous capsule after 12 weeks. No significant difference was found among the groups regarding myeloperoxidase activity and interleukin 1beta levels, although the GA, 25GA, and 75GA groups presented decreased levels of tumor necrosis factor alpha compared with the control group. These new GA cross-linked membranes accelerated bone healing of the calvarium defects and did not induce inflammation. In addition, unlike the control membrane, the experimental membranes were not absorbed during the analyzed period, so they may offer advantages in large bone defects where prolonged membrane barrier functions are desirable. PMID:25245073

The use of collagen-based scaffolds to simulate prostate cancer bone metastases with potential for evaluating delivery of nanoparticulate gene therapeutics.

PubMed

Fitzgerald, Kathleen A; Guo, Jianfeng; Tierney, Erica G; Curtin, Caroline M; Malhotra, Meenakshi; Darcy, Raphael; O'Brien, Fergal J; O'Driscoll, Caitriona M

2015-10-01

Prostate cancer bone metastases are a leading cause of cancer-related death in men with current treatments offering only marginally improved rates of survival. Advances in the understanding of the genetic basis of prostate cancer provide the opportunity to develop gene-based medicines capable of treating metastatic disease. The aim of this work was to establish a 3D cell culture model of prostate cancer bone metastasis using collagen-based scaffolds, to characterise this model, and to assess the potential of the model to evaluate delivery of gene therapeutics designed to target bone metastases. Two prostate cancer cell lines (PC3 and LNCaP) were cultured in 2D standard culture and compared to 3D cell growth on three different collagen-based scaffolds (collagen and composites of collagen containing either glycosaminoglycan or nanohydroxyapatite). The 3D model was characterised for cell proliferation, viability and for matrix metalloproteinase (MMP) enzyme and Prostate Specific Antigen (PSA) secretion. Chemosensitivity to docetaxel treatment was assessed in 2D in comparison to 3D. Nanoparticles (NPs) containing siRNA formulated using a modified cyclodextrin were delivered to the cells on the scaffolds and gene silencing was quantified. Both prostate cancer cell lines actively infiltrated and proliferated on the scaffolds. Cell culture in 3D resulted in reduced levels of MMP1 and MMP9 secretion in PC3 cells. In contrast, LNCaP cells grown in 3D secreted elevated levels of PSA, particularly on the scaffold composed of collagen and glycosaminoglycans. Both cell lines grown in 3D displayed increased resistance to docetaxel treatment. The cyclodextrin.siRNA nanoparticles achieved cellular uptake and knocked down the endogenous GAPDH gene in the 3D model. In conclusion, development of a novel 3D cell culture model of prostate cancer bone metastasis has been initiated resulting, for the first time, in the successful delivery of gene therapeutics in a 3D in vitro model

Secondary closure of alveolar cleft with resorbable collagen membrane and a combination of intraoral autogenous bone graft and deproteinized anorganic bovine bone

PubMed Central

Aly, Lobna Abdel Aziz; Hammouda, Nelly

2016-01-01

Objects: Secondary alveolar bone grafting is a method that enables an excellent oral rehabilitation of the patients having alveolar cleft. The aim of this work is to report the closure of the alveolar cleft with the use of harvested autogenous bone graft combined with deproteinized anorganic bovine bone (Bio-Oss) under local anesthesia. Settings and Sample Population: Nine patients with age range, 8–11 years were consulted for their unilateral alveolar cleft. Materials and Methods: A combination of symphyseal bone and deproteinized bovine bone mineral (DBBM) was placed into the alveolar cleft defect. Clinical and radiographical assessments were performed at 1, 3, and 6 months postoperatively. Results: The healing period was uneventful in all cases, and no complications, such as membrane exposure, infection, or harvest site morbidity, were observed. All treated defect sites exhibited excellent bone formation, with an average of 5.45 mm (range, 2–9 mm; standard deviation 1.93 mm) of augmentation achieved overall. Conclusion: The treatment of vertically deficient alveolar ridges with guided bone regeneration using a mixture of autogenous bone and DBBM and resorbable collagen membrane can be considered successful, using this technique in an out-patient office setting. PMID:28299252

Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone

PubMed Central

Galli, Giorgio Giacomo; Honnens de Lichtenberg, Kristian; Carrara, Matteo; Hans, Wolfgang; Wuelling, Manuela; Mentz, Bettina; Multhaupt, Hinke Arnolda; Fog, Cathrine Kolster; Jensen, Klaus Thorleif; Rappsilber, Juri; Vortkamp, Andrea; Coulton, Les; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabě de Angelis, Martin; Calogero, Raffaele Adolfo; Couchman, John Robert; Lund, Anders Henrik

2012-01-01

PRDM family members are transcriptional regulators involved in tissue specific differentiation. PRDM5 has been reported to predominantly repress transcription, but a characterization of its molecular functions in a relevant biological context is lacking. We demonstrate here that Prdm5 is highly expressed in developing bones; and, by genome-wide mapping of Prdm5 occupancy in pre-osteoblastic cells, we uncover a novel and unique role for Prdm5 in targeting all mouse collagen genes as well as several SLRP proteoglycan genes. In particular, we show that Prdm5 controls both Collagen I transcription and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining the transcriptional program necessary to the proper assembly of osteoblastic extracellular matrix. PMID:22589746

Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation.

PubMed

Sekiguchi, Hiroyuki; Uchida, Kentaro; Matsushita, Osamu; Inoue, Gen; Nishi, Nozomu; Masuda, Ryo; Hamamoto, Nana; Koide, Takaki; Shoji, Shintaro; Takaso, Masashi

2018-01-01

Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly) 10 , induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly) 12 -NH 2 , a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly) 10 /bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

Enhanced bone regeneration using an insulin-loaded nano-hydroxyapatite/collagen/PLGA composite scaffold.

PubMed

Wang, Xing; Zhang, Guilan; Qi, Feng; Cheng, Yongfeng; Lu, Xuguang; Wang, Lu; Zhao, Jing; Zhao, Bin

2018-01-01

Insulin is widely considered as a classical hormone and drug in maintaining energy and glucose homeostasis. Recently, insulin has been increasingly recognized as an indispensable factor for osteogenesis and bone turnover, but its applications in bone regeneration have been restricted because of the short periods of activity and uncontrolled release. In this study, we incorporated insulin-loaded poly lactic-co-glycolic-acid (PLGA) nanospheres into nano-hydroxyapatite/collagen (nHAC) scaffolds and investigated the bioactivity of the composite scaffolds in vitro and in vivo. Bioactive insulin was successfully released from the nanospheres within the scaffold, and the release kinetics of insulin could be efficiently controlled by uniform-sized nanospheres. The physical characterizations of the composite scaffolds demonstrated that incorporation of nanospheres in nHAC scaffolds using this method did not significantly change the porosity, pore diameters, and compressive strengths of nHAC. In vitro, the insulin-loaded nHAC/PLGA composite scaffolds possessed favorable biological function for bone marrow mesenchymal stem cells adhesion and proliferation, as well as the differentiation into osteoblasts. In vivo, the optimized bone regenerative capability of this composite scaffold was confirmed in rabbit mandible critical size defects. These results demonstrated successful development of a functional insulin-PLGA-nHAC composite scaffold that enhances the bone regeneration capability of nHAC.

Alteration of mineral crystallinity and collagen cross-linking of bones in osteopetrotic toothless (tl/tl) rats and their improvement after treatment with colony stimulating factor-1

NASA Technical Reports Server (NTRS)

Wojtowicz, A.; Dziedzic-Goclawska, A.; Kaminski, A.; Stachowicz, W.; Wojtowicz, K.; Marks, S. C. Jr; Yamauchi, M.

1997-01-01

A common feature of various types of mammalian osteopetroses is a marked increase in bone mass accompanied by spontaneous bone fractures. The toothless (tl/tl) rat osteopetrotic mutation is characterized by drastically reduced bone resorption due to a profound deficiency of osteoclasts and their precursors. An altered bone morphology has also been observed. The mutants cannot be cured by bone marrow transplantation, but skeletal defects are greatly reduced after treatment with colony stimulating factor 1 (CSF-1). The objectives of this study were to characterize mineral and collagen matrices in cancellous and compact bone isolated from long bones of 6-week-old normal littermates, tl/tl osteopetrotic mutants and mutants (tl/tl) treated with CSF-1. There were no differences in bone mineral content, but a significant decrease in the crystallinity of mineral evaluated by the method based on electron paramagnetic resonance spectrometry was observed in all bones of tl/tl mutants as compared to that of controls. Within the collagen matrix, slight decreases in the labile cross-links, but significant increases in the content of the stable cross-links, pyridinoline, and deoxypyridinoline, were observed in both cancellous and compact bone of osteopetrotic mutants. In tl/tl mutants treated with human recombinant CSF-1, the normalization of the crystallinity of bone mineral as well as collagen cross-links was found. Our results indicate that remodeling of bone matrix in tl/tl mutants is highly suppressed, but that after treatment with CSF-1, this activity recovers significantly. Taken together, these data provide further support for the hypothesis that CSF-1 is an essential factor for normal osteoclast differentiation and bone remodelling.

The Ratio 1660/1690 cm−1 Measured by Infrared Microspectroscopy Is Not Specific of Enzymatic Collagen Cross-Links in Bone Tissue

PubMed Central

Farlay, Delphine; Duclos, Marie-Eve; Gineyts, Evelyne; Bertholon, Cindy; Viguet-Carrin, Stéphanie; Nallala, Jayakrupakar; Sockalingum, Ganesh D.; Bertrand, Dominique; Roger, Thierry; Hartmann, Daniel J.; Chapurlat, Roland; Boivin, Georges

2011-01-01

In postmenopausal osteoporosis, an impairment in enzymatic cross-links (ECL) occurs, leading in part to a decline in bone biomechanical properties. Biochemical methods by high performance liquid chromatography (HPLC) are currently used to measure ECL. Another method has been proposed, by Fourier Transform InfraRed Imaging (FTIRI), to measure a mature PYD/immature DHLNL cross-links ratio, using the 1660/1690 cm−1 area ratio in the amide I band. However, in bone, the amide I band composition is complex (collagens, non-collagenous proteins, water vibrations) and the 1660/1690 cm−1 by FTIRI has never been directly correlated with the PYD/DHLNL by HPLC. A study design using lathyritic rats, characterized by a decrease in the formation of ECL due to the inhibition of lysyl oxidase, was used in order to determine the evolution of 1660/1690 cm−1 by FTIR Microspectroscopy in bone tissue and compare to the ECL quantified by HPLC. The actual amount of ECL was quantified by HPLC on cortical bone from control and lathyritic rats. The lathyritic group exhibited a decrease of 78% of pyridinoline content compared to the control group. The 1660/1690 cm−1 area ratio was increased within center bone compared to inner bone, and this was also correlated with an increase in both mineral maturity and mineralization index. However, no difference in the 1660/1690 cm−1 ratio was found between control and lathyritic rats. Those results were confirmed by principal component analysis performed on multispectral infrared images. In bovine bone, in which PYD was physically destructed by UV-photolysis, the PYD/DHLNL (measured by HPLC) was strongly decreased, whereas the 1660/1690 cm−1 was unmodified. In conclusion, the 1660/1690 cm−1 is not related to the PYD/DHLNL ratio, but increased with age of bone mineral, suggesting that a modification of this ratio could be mainly due to a modification of the collagen secondary structure related to the mineralization process. PMID:22194900

The ratio 1660/1690 cm(-1) measured by infrared microspectroscopy is not specific of enzymatic collagen cross-links in bone tissue.

PubMed

Farlay, Delphine; Duclos, Marie-Eve; Gineyts, Evelyne; Bertholon, Cindy; Viguet-Carrin, Stéphanie; Nallala, Jayakrupakar; Sockalingum, Ganesh D; Bertrand, Dominique; Roger, Thierry; Hartmann, Daniel J; Chapurlat, Roland; Boivin, Georges

2011-01-01

In postmenopausal osteoporosis, an impairment in enzymatic cross-links (ECL) occurs, leading in part to a decline in bone biomechanical properties. Biochemical methods by high performance liquid chromatography (HPLC) are currently used to measure ECL. Another method has been proposed, by Fourier Transform InfraRed Imaging (FTIRI), to measure a mature PYD/immature DHLNL cross-links ratio, using the 1660/1690 cm(-1) area ratio in the amide I band. However, in bone, the amide I band composition is complex (collagens, non-collagenous proteins, water vibrations) and the 1660/1690 cm(-1) by FTIRI has never been directly correlated with the PYD/DHLNL by HPLC. A study design using lathyritic rats, characterized by a decrease in the formation of ECL due to the inhibition of lysyl oxidase, was used in order to determine the evolution of 1660/1690 cm(-1) by FTIR Microspectroscopy in bone tissue and compare to the ECL quantified by HPLC. The actual amount of ECL was quantified by HPLC on cortical bone from control and lathyritic rats. The lathyritic group exhibited a decrease of 78% of pyridinoline content compared to the control group. The 1660/1690 cm(-1) area ratio was increased within center bone compared to inner bone, and this was also correlated with an increase in both mineral maturity and mineralization index. However, no difference in the 1660/1690 cm(-1) ratio was found between control and lathyritic rats. Those results were confirmed by principal component analysis performed on multispectral infrared images. In bovine bone, in which PYD was physically destructed by UV-photolysis, the PYD/DHLNL (measured by HPLC) was strongly decreased, whereas the 1660/1690 cm(-1) was unmodified. In conclusion, the 1660/1690 cm(-1) is not related to the PYD/DHLNL ratio, but increased with age of bone mineral, suggesting that a modification of this ratio could be mainly due to a modification of the collagen secondary structure related to the mineralization process.

Differences in Non-Enzymatic Glycation and Collagen Crosslinks between Human Cortical and Cancellous Bone

PubMed Central

Karim, Lamya; Tang, Simon Y.; Sroga, Grażyna E.; Vashishth, Deepak

2015-01-01

Purpose Accumulation of collagen crosslinks (advanced glycation end products [AGEs]) produced by non-enzymatic glycation deteriorates bone's mechanical properties and fracture resistance. Although a single AGE, pentosidine, is commonly used as a representative marker, it is unclear whether it quantitatively reflects total fluorescent AGEs in bone. The goal of this study was to establish the relationship between pentosidine and total AGEs in cancellous and cortical bone. Methods Pentosidine and total AGEs were quantified in 170 human bone samples. Total fluorescent AGEs were measured in 28 additional cancellous and cortical bone specimens of the same apparent volume that were incubated in control or in vitro glycation solutions. Correlations between pentosidine and total AGEs and differences between cortical and cancellous groups were determined. Results Pentosidine was correlated with total AGEs in cancellous bone (r=0.53, p<0.0001) and weakly correlated in cortical bone (r=0.23, p<0.05). There was more pentosidine (p<0.01) and total AGEs (p<0.001) in cancellous than in cortical bone. The in vitro glycation sub-study showed that cancellous bone accumulated more AGEs than cortical bone (p<0.05). Conclusion The relationship between pentosidine and total AGEs and their magnitude of accumulation differed in cancellous and cortical bone of the same apparent volume, and were dependent on the surface-to-volume ratios of each sample. It is important to consider the bone types as two separate entities, and it is crucial to quantify total AGEs in addition to pentosidine to allow for more comprehensive analysis of the effects of non-enzymatic glycation in bone. PMID:23471564

Evaluation of autogenous PRGF+β-TCP with or without a collagen membrane on bone formation and implant osseointegration in large size bone defects. A preclinical in vivo study.

PubMed

Batas, Leonidas; Stavropoulos, Andreas; Papadimitriou, Serafim; Nyengaard, Jens R; Konstantinidis, Antonios

2016-08-01

The aim of this study was to evaluate whether the adjunctive use of a collagen membrane enhances bone formation and implant osseointegration in non-contained defects grafted with chair-side prepared autologous platelet-rich growth factor (PRGF) adsorbed on a β-TCP particulate carrier. Large box-type defects (10 × 6 mm; W × D) were prepared in the edentulated and completely healed mandibles of six Beagles dogs. An implant with moderately rough surface was placed in the center of each defect leaving the coronal 6 mm of the implant not covered with bone. The remaining defect space was then filled out with chair-side prepared autologous PRGF adsorbed on β-TCP particles and either covered with a collagen membrane (PRGF/β-TCP+CM) (6 defects) or left without a membrane (PRGF/β-TCP) (5 defects). Histology 4 months post-op showed new lamellar and woven bone formation encompassing almost entirely the defect and limited residual β-TCP particles. Extent of osseointegration of the previously exposed portion of the implants varied, but in general was limited. Within the defect, new mineralized bone (%) averaged 43.2 ± 9.86 vs. 39.9 ± 13.7 in the PRGF/β-TCP+CM and PRGF/β-TCP group (P = 0.22) and relative mineralized bone-to-implant contact (%) averaged 26.2 ± 16.45 vs. 35.91 ± 24.45, respectively (P = 0.5). First, bone-to-implant contact from the implant top was 4.1 ± 1.5 and 3.2 ± 2.3 (P = 0.9), in the PRGF/β-TCP+CM and PRGF/β-TCP group, respectively. Implantation of chair-side prepared autologous PRGF adsorbed on a β-TCP carrier in non-contained peri-implant defects resulted in large amounts of bone regeneration, but osseointegration was limited. Provisions for GBR with a collagen membrane did not significantly enhance bone regeneration or implant osseointegration. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Osmotically driven tensile stress in collagen-based mineralized tissues.

PubMed

Bertinetti, Luca; Masic, Admir; Schuetz, Roman; Barbetta, Aurelio; Seidt, Britta; Wagermaier, Wolfgang; Fratzl, Peter

2015-12-01

Collagen is the most abundant protein in mammals and its primary role is to serve as mechanical support in many extracellular matrices such as those of bones, tendons, skin or blood vessels. Water is an integral part of the collagen structure, but its role is still poorly understood, though it is well-known that the mechanical properties of collagen depend on hydration. Recently, it was shown that the conformation of the collagen triple helix changes upon water removal, leading to a contraction of the molecule with considerable forces. Here we investigate the influence of mineralization on this effect by studying bone and turkey leg tendon (TLT) as model systems. Indeed, TLT partially mineralizes so that well-aligned collagen with various mineral contents can be found in the same tendon. We show that water removal leads to collagen contraction in all cases generating tensile stresses up to 80MPa. Moreover, this contraction of collagen puts mineral particles under compression leading to strains of around 1%, which implies localized compressive loads in mineral of up to 800MPa. This suggests that collagen dehydration upon mineralization is at the origin of the compressive pre-strains commonly observed in bone mineral. Copyright © 2015 Elsevier Ltd. All rights reserved.

Socket preservation using bovine bone mineral and collagen membrane: a randomized controlled clinical trial with histologic analysis.

PubMed

Cardaropoli, Daniele; Tamagnone, Lorenzo; Roffredo, Alessandro; Gaveglio, Lorena; Cardaropoli, Giuseppe

2012-08-01

After tooth extraction, varying amounts of bone resorption occur because of qualitative and quantitative changes at the edentulous site of the alveolar process. The aims of this randomized controlled clinical trial were (1) to compare the postextraction changes in residual ridge dimensions during spontaneous healing with those during socket preservation, (2) to analyze the histologic and histomorphometric aspects of the grafted sockets, and (3) to compare probing procket depth (PPD) and clinical attachment level (CAL) changes at teeth adjacent to extraction sites. Forty-eight teeth were extracted from 41 patients referred for extraction of 1 or more maxillary or mandibular premolars or molars. The edentulous sites were randomly assigned to the control (EXT, extraction alone) or experimental groups (SP, extraction and socket preservation). In the SP group, the sockets were filled with bovine bone mineral and covered with porcine collagen membrane. At baseline and after 4 months, PPD, gingival recession (REC), and CAL were measured at teeth adjacent to the edentulous sites. The changes in ridge dimensions from baseline to 4 months were assessed on dental casts. At 4 months, bone was harvested from the grafted areas in the SP group and the edentulous areas in the EXT group. PPD, REC, and CAL were comparable between groups. However, from baseline to 4 months, the SP group showed significantly less reduction in ridge width (1.04 ± 1.08 mm vs 4.48 ± 0.65 mm, P < .001) and height (0.46 ± 0.46 mm vs 1.54 ± 0.33 mm, P < .001). Histologically, the grafted sockets exhibited various stages of bone maturation and formation without inflammatory responses. No significant difference in the mineralized and nonmineralized fractions was noted between the groups. Socket preservation using bovine bone mineral and porcine collagen membrane considerably limits the amount of horizontal and vertical bone resorption when compared with extraction alone.

Tunability of collagen matrix mechanical properties via multiple modes of mineralization

PubMed Central

Smith, Lester J.; Deymier, Alix C.; Boyle, John J.; Li, Zhen; Linderman, Stephen W.; Pasteris, Jill D.; Xia, Younan; Genin, Guy M.; Thomopoulos, Stavros

2016-01-01

Functionally graded, mineralized collagen tissues exist at soft-to-hard material attachments throughout the body. However, the details of how collagen and hydroxyapatite mineral (HA) interact are not fully understood, hampering efforts to develop tissue-engineered constructs that can assist with repair of injuries at the attachments of soft tissues to bone. In this study, spatial control of mineralization was achieved in collagen matrices using simulated body fluids (SBFs). Based upon previous observations of poor bonding between reconstituted collagen and HA deposited using SBF, we hypothesized that mineralizing collagen in the presence of fetuin (which inhibits surface mineralization) would lead to more mineral deposition within the scaffold and therefore a greater increase in stiffness and toughness compared with collagen mineralized without fetuin. We tested this hypothesis through integrated synthesis, mechanical testing and modelling of graded, mineralized reconstituted collagen constructs. Results supported the hypothesis, and further suggested that mineralization on the interior of reconstituted collagen constructs, as promoted by fetuin, led to superior bonding between HA and collagen. The results provide us guidance for the development of mineralized collagen scaffolds, with implications for bone and tendon-to-bone tissue engineering. PMID:26855755

Influence of 45S5 Bioactive Glass in A Standard Calcium Phosphate Collagen Bone Graft Substitute on the Posterolateral Fusion of Rabbit Spine.

PubMed

Pugely, Andrew J; Petersen, Emily B; DeVries-Watson, Nicole; Fredericks, Douglas C

2017-01-01

Spinal fusion surgery is an effective but costly treatment for select spinal pathology. Historically iliac crest bone graft (ICBG) has remained the gold standard for achieving successful arthrodesis. Given well-established morbidity autograft harvest, multiple bone graft replacements, void fillers, and extenders have been developed. The objective of this study was to evaluate the in vivo efficacy and safety of two mineralized collagen bone void filler materials similar in composition. Both bone void fillers were composed of hydroxyapatite (HA), tricalcium phosphate (TCP) and bovine collagen. The first test article (Bi-Ostetic bioactive glass foam or "45S5") also contained 45S5 bioactive glass particles while the second test article (Formagraft or "FG") did not. 45S5 and FG were combined with bone marrow aspirate and iliac crest autograft and compared to ICBG in an established posterolateral spine fusion rabbit model. Sixty-nine mature New Zealand White rabbits were divided into 3 test cohorts: ICBG, 45S5, and FG. A Posterolateral fusion model previous validated was utilized to assess fusion efficacy. The test groups were evaluated for spine fusion rate, new bone formation, graft resorption and inflammatory response using radiographic, μCT, biomechanical and histological endpoints at 4, 8 and 12 weeks following implantation. There were 4 clinical complications unrelated to the graft materials and were evenly split between groups (ICBG graft harvest complications; hind limb mobility, chronic pain) and were euthanized. These omissions did not affect the overall outcome of the study. Radiographic scoring of the fusion sites indicated a normal healing response in all test groups, with no adverse reactions and similar progressions of new bone formation observed over time. All groups demonstrated significantly less range of motion in both flexion/extension and lateral bending compared to normal not-fused controls, which supports fusion results observed in the other

Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro

NASA Astrophysics Data System (ADS)

Tamaddon, M.; Burrows, M.; Ferreira, S. A.; Dazzi, F.; Apperley, J. F.; Bradshaw, A.; Brand, D. D.; Czernuszka, J.; Gentleman, E.

2017-03-01

Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS - which was released from scaffolds quickly - significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

Collagen: Biochemistry, biomechanics, biotechnology

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

Nimni, M.E.

1988-01-01

This book is an up-to-date reference for new ideas, information, and concepts in collagen research. The first volume emphasizes the relationship between the molecular structure and function of collagen, including descriptions of collagen types which exist in tissues as well as how these molecules organize into fibrils and the nature of the chemical crosslinks which stabilize them. In Volume II the biomechanical behavior of various specialized tissues, abnormal accumulation of collagen in the form of scars of fibrous infiltration are examined/and wound healing, tissue regulation and repair are covered in detail. Volume III explores the increasing application of collagen technologymore » to the field of bioprosthesis, including the production of heart valve bioprosthesis, blood vessels, ligament substitutes, and bone substitutes.« less

Understanding the Structure of Bones

MedlinePlus

... bend. The same organization is true of bone. Collagen rods in bone are similar to the steel ... These minerals give the bones strength while the collagen rods provide resiliency. Diseases that interfere with the ...

Biophysicochemical evaluation of chitosan-hydroxyapatite-marine sponge collagen composite for bone tissue engineering.

PubMed

Pallela, Ramjee; Venkatesan, Jayachandran; Janapala, Venkateswara Rao; Kim, Se-Kwon

2012-02-01

Tricomponent scaffold systems prepared by natural materials especially of marine origin are gaining much attention nowadays for the application in bone tissue engineering. A novel scaffold (Chi-HAp-MSCol) containing chitosan (Chi), hydroxyapatite (HAp) derived from Thunnus obesus bone and marine sponge (Ircinia fusca) collagen (MSCol) was prepared using freeze-drying and lyophilization method. This biomimetic scaffold, along with the Chi and Chi-HAp scaffolds were characterized biophysicochemically for their comparative significance in bone grafting applications. The structural composition of the chitosan, Chi-Hap, and Chi-HAp-MSCol scaffolds were characterized by Fourier Transform Infrared spectroscopy. The porosity, water uptake, and retention abilities of the composite scaffolds decreased, whereas Thermogravimetric and Differential Thermal Analyses results revealed the increase in thermal stability in the scaffold because of the highly stable HAp and MSCol. Homogeneous dispersion of HAp and MSCol in chitosan matrix with interconnected porosity of 60-180 μm (Chi-HAp) and 50-170 μm (Chi-HAp-MSCol) was observed by Scanning Electron Microscopy, X-ray diffraction, and optical microscopy. Cell proliferation in composite scaffolds was relatively higher than pure chitosan when observed by MTT assay and Hoechst staining in vitro using MG-63 cell line. These observations suggest that the novel Chi-HAp-MSCol composite scaffolds are promising biomaterials for matrix-based bone repair and bone augmentation. Copyright © 2011 Wiley Periodicals, Inc.

Evaluation of multi-scale mineralized collagen-polycaprolactone composites for bone tissue engineering.

PubMed

Weisgerber, D W; Erning, K; Flanagan, C L; Hollister, S J; Harley, B A C

2016-08-01

A particular challenge in biomaterial development for treating orthopedic injuries stems from the need to balance bioactive design criteria with the mechanical and geometric constraints governed by the physiological wound environment. Such trade-offs are of particular importance in large craniofacial bone defects which arise from both acute trauma and chronic conditions. Ongoing efforts in our laboratory have demonstrated a mineralized collagen biomaterial that can promote human mesenchymal stem cell osteogenesis in the absence of osteogenic media but that possesses suboptimal mechanical properties in regards to use in loaded wound sites. Here we demonstrate a multi-scale composite consisting of a highly bioactive mineralized collagen-glycosaminoglycan scaffold with micron-scale porosity and a polycaprolactone support frame (PCL) with millimeter-scale porosity. Fabrication of the composite was performed by impregnating the PCL support frame with the mineral scaffold precursor suspension prior to lyophilization. Here we evaluate the mechanical properties, permeability, and bioactivity of the resulting composite. Results indicated that the PCL support frame dominates the bulk mechanical response of the composite resulting in a 6000-fold increase in modulus compared to the mineral scaffold alone. Similarly, the incorporation of the mineral scaffold matrix into the composite resulted in a higher specific surface area compared to the PCL frame alone. The increased specific surface area in the collagen-PCL composite promoted increased initial attachment of porcine adipose derived stem cells versus the PCL construct. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of nanohydroxyapaptite (nano-HA) coated epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes.

PubMed

Chu, Chenyu; Deng, Jia; Man, Yi; Qu, Yili

2017-09-01

Collagen is the main component of extracellular matrix (ECM) with desirable biological activities and low antigenicity. Collagen materials have been widely utilized in guided bone regeneration (GBR) surgery due to its abilities to maintain space for hard tissue growth. However, pure collagen lacks optimal mechanical properties. In our previous study, epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes, with better biological activities and enhanced mechanical properties, may promote osteoblast proliferation, but their effect on osteoblast differentiation is not very significant. Nanohydroxyapatite (nano-HA) is the main component of mineral bone, which possesses exceptional bioactivity properties including good biocompatibility, high osteoconductivity and osteoinductivity, non-immunogenicity and non-inflammatory behavior. Herein, by analyzing the physical and chemical properties as well as the effects on promoting bone regeneration, we have attempted to present a novel EGCG-modified collagen membrane with nano-HA coating, and have found evidence that the novel collagen membrane may promote bone regeneration with a better surface morphology, without destroying collagen backbone. To evaluate the surface morphologies, chemical and mechanical properties of pure collagen membranes, epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes, nano-HA coated collagen membranes, nano-HA coated EGCG-collagen membranes, (ii) to evaluate the bone regeneration promoted by theses membranes. In the present study, collagen membranes were divided into 4 groups: (1) untreated collagen membranes (2) EGCG cross-linked collagen membranes (3) nano-HA modified collagen membranes (4) nano-HA modified EGCG-collagen membranes. Scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR) were used to evaluate surface morphologies and chemical properties, respectively. Mechanical properties were determined by differential scanning calorimeter (DSC

Nanoscale Morphology of Type I Collagen is Altered in the Brtl Mouse Model of Osteogenesis Imperfecta

PubMed Central

Wallace, Joseph M.; Orr, Bradford G.; Marini, Joan C.; Banaszak Holl, Mark M.

2010-01-01

Bone has a complex hierarchical structure that has evolved to serve structural and metabolic roles in the body. Due to the complexity of bone structure and the number of diseases which affect the ultrastructural constituents of bone, it is important to develop quantitative methods to assess bone nanoscale properties. Autosomal dominant Osteogenesis Imperfecta results predominantly from glycine substitutions (80%) and splice site mutations (20%) in the genes encoding the α1 or α2 chains of Type I collagen. Genotype-phenotype correlations using over 830 collagen mutations have revealed that lethal mutations are located in regions crucial for collagen-ligand binding in the matrix. However, few of these correlations have been extended to collagen structure in bone. Here, an atomic force microscopy-based approach was used to image and quantitatively analyze the D-periodic spacing of Type I collagen fibrils in femora from heterozygous (Brtl/+) mice (α1(I)G349C), compared to wild type (WT) littermates. This disease system has a well-defined change in the col1α1 allele, leading to a well characterized alteration in collagen protein structure, which are directly related to altered Type I collagen nanoscale morphology, as measured by the D-periodic spacing. In Brtl/+ bone, the D-periodic spacing shows significantly greater variability on average and along the length of the bone compared to WT, although the average spacing was unchanged. Brtl/+ bone also had a significant difference in the population distribution of collagen D-period spacings. These changes may be due to the mutant collagen structure, or to the heterogeneity of collagen monomers in the Brtl/+ matrix. These observations at the nanoscale level provide insight into the structural basis for changes present in bone composition, geometry and mechanical integrity in Brtl/+ bones. Further studies are necessary to link these morphological observations to nanoscale mechanical integrity. PMID:20696252

Raman spectral markers of collagen denaturation and hydration in human cortical bone tissue are affected by radiation sterilization and high cycle fatigue damage.

PubMed

Flanagan, Christopher D; Unal, Mustafa; Akkus, Ozan; Rimnac, Clare M

2017-11-01

Thermal denaturation and monotonic mechanical damage alter the organic and water-related compartments of cortical bone. These changes can be detected using Raman spectroscopy. However, less is known regarding Raman sensitivity to detect the effects of cyclic fatigue damage and allograft sterilization doses of gamma radiation. To determine if Raman spectroscopic biomarkers of collagen denaturation and hydration are sensitive to the effects of (a) high cycle fatigue damage and (b) 25kGy irradiation. Unirradiated and gamma-radiation sterilized human cortical bone specimens previously tested in vitro under high-cycle (> 100,000 cycles) fatigue conditions at 15MPa, 25MPa, 35MPa, 45MPa, and 55MPa cyclic stress levels were studied. Cortical bone Raman spectral profiles from wavenumber ranges of 800-1750cm -1 and 2700-3800cm -1 were obtained and compared from: a) non-fatigue vs fatigue fracture sites and b) radiated vs. unirradiated states. Raman biomarker ratios 1670/1640 and 3220/2949, which reflect collagen denaturation and organic matrix (mainly collagen)-bound water, respectively, were assessed. One- and two-way ANOVA analyses were utilized to identify differences between groups along with interaction effects between cyclic fatigue and radiation-induced damage. Cyclic fatigue damage resulted in increases in collagen denaturation (1670/1640: 1.517 ± 0.043 vs 1.579 ± 0.021, p < 0.001) and organic matrix-bound water (3220/2949: 0.109 ± 0.012 vs 0.131 ± 0.008, p < 0.001). Organic matrix-bound water increased secondary to 25kGy irradiation (3220/2949: 0.105 ± 0.010 vs 0.1161 ± 0.009, p = 0.003). Organic matrix-bound water was correlated positively with collagen denaturation (r = 0.514, p < 0.001). Raman spectroscopy can detect the effects of cyclic fatigue damage and 25kGy irradiation via increases in organic matrix (mainly collagen)-bound water. A Raman measure of collagen denaturation was sensitive to cyclic fatigue damage but not 25kGy irradiation. Collagen

Collagen-gelatin-genipin-hydroxyapatite composite scaffolds colonized by human primary osteoblasts are suitable for bone tissue engineering applications: in vitro evidences.

PubMed

Vozzi, G; Corallo, C; Carta, S; Fortina, M; Gattazzo, F; Galletti, M; Giordano, N

2014-05-01

The application of porous hydroxyapatite (HAp)-collagen as a bone tissue engineering scaffold represents a new trend of mimicking the specific bone extracellular matrix (ECM). The use of HAp in reconstructive surgery has shown that it is slowly invaded by host tissue. Therefore, implant compatibility may be augmented by seeding cells before implantation. Human primary osteoblasts were seeded onto innovative collagen-gelatin-genipin (GP)-HAp scaffolds containing respectively 10%, 20%, and 30% HAp. Cellular adhesion, proliferation, alkaline phosphatase (ALP) activity, osteopontin (OPN), and osteocalcin (OC) expressions were evaluated after 3, 7, 15, and 21 days. The three types of scaffolds showed increased cellular proliferation over time in culture (maximum at 21 days) but the highest was recorded in 10% HAp scaffolds. ALP activity was the highest in 10% HAp scaffolds in all the times of evaluation. OC and OPN resulted in higher concentration in 10% HAp scaffolds compared to 20% and 30% HAp (maximum at 21 days). Finally, scanning electron microscopy analysis showed progressive scaffolds adhesion and colonization from the surface to the inside from day 3 to day 21. In vitro attachment, proliferation, and colonization of human primary osteoblasts on collagen-GP-HAp scaffolds with different percentages of HAp (10%, 20%, and 30%) all increased over time in culture, but comparing different percentages of HAp, they seem to increase with decreasing of HAp component. Therefore, the mechanical properties (such as the stiffness due to the HAp%) coupled with a good biomimetic component (collagen) are the parameters to set up in composite scaffolds design for bone tissue engineering. Copyright © 2013 Wiley Periodicals, Inc.

Raloxifene microsphere-embedded collagen/chitosan/β-tricalcium phosphate scaffold for effective bone tissue engineering.

PubMed

Zhang, Ming-Lei; Cheng, Ji; Xiao, Ye-Chen; Yin, Ruo-Feng; Feng, Xu

2017-02-25

Engineering novel scaffolds that can mimic the functional extracellular matrix (ECM) would be a great achievement in bone tissue engineering. This paper reports the fabrication of novel collagen/chitosan/β-tricalcium phosphate (CCTP) based tissue engineering scaffold. In order to improve the regeneration ability of scaffold, we have embedded raloxifene (RLX)-loaded PLGA microsphere in the CCTP scaffold. The average pore of scaffold was in the range of 150-200μm with ideal mechanical strength and swelling/degradation characteristics. The release rate of RLX from the microsphere (MS) embedded scaffold was gradual and controlled. Also a significantly enhanced cell proliferation was observed in RLX-MS exposed cell group suggesting that microsphere/scaffold could be an ideal biomaterial for bone tissue engineering. Specifically, RLX-MS showed a significantly higher Alizarin red staining indicating the higher mineralization capacity of this group. Furthermore, a high alkaline phosphatase (ALP) activity for RLX-MS exposed group after 15days incubation indicates the bone regeneration capacity of MC3T3-E1 cells. Overall, present study showed that RLX-loaded microsphere embedded scaffold has the promising potential for bone tissue engineering applications. Copyright © 2016. Published by Elsevier B.V.

Identification of Pyridinoline Trivalent Collagen Cross-Links by Raman Microspectroscopy.

PubMed

Gamsjaeger, Sonja; Robins, Simon P; Tatakis, Dimitris N; Klaushofer, Klaus; Paschalis, Eleftherios P

2017-06-01

Intermolecular cross-linking of bone collagen is intimately related to the way collagen molecules are arranged in a fibril, imparts certain mechanical properties to the fibril, and may be involved in the initiation of mineralization. Raman microspectroscopy allows the analysis of minimally processed bone blocks and provides simultaneous information on both the mineral and organic matrix (mainly type I collagen) components, with a spatial resolution of ~1 μm. The aim of the present study was to validate Raman spectroscopic parameters describing one of the major mineralizing type I trivalent cross-links, namely pyridinoline (PYD). To achieve this, a series of collagen cross-linked peptides with known PYD content (as determined by HPLC analysis), human bone, porcine skin, predentin and dentin animal model tissues were analyzed by Raman microspectroscopy. The results of the present study confirm that it is feasible to monitor PYD trivalent collagen cross-links by Raman spectroscopic analysis in mineralized tissues, exclusively through a Raman band ~1660 wavenumbers. This allows determination of the relative PYD content in undecalcified bone tissues with a spatial resolution of ~1 μm, thus enabling correlations with histologic and histomorphometric parameters.

The ability of a collagen/calcium phosphate scaffold to act as its own vector for gene delivery and to promote bone formation via transfection with VEGF(165).

PubMed

Keeney, Michael; van den Beucken, Jeroen J J P; van der Kraan, Peter M; Jansen, John A; Pandit, Abhay

2010-04-01

Collagen/calcium phosphate scaffolds have been used for bone reconstruction due to their inherent similarities to the bone extracellular matrix. Calcium phosphate alone has also been used as a non-viral vector for gene delivery. The aim of this study was to determine the capability of a collagen/calcium phosphate scaffold to deliver naked plasmid DNA and mediate transfection in vivo. The second goal of the study was to deliver a plasmid encoding vascular endothelial growth factor(165) (pVEGF(165)) to promote angiogenesis, and hence bone formation, in a mouse intra-femoral model. The delivery of naked plasmid DNA resulted in a 7.6-fold increase in mRNA levels of beta-Galactosidase compared to the delivery of plasmid DNA complexed with a partially degraded PAMAM dendrimer (dPAMAM) in a subcutaneous murine model. When implanted in a muirne intra-femoral model, the delivery of pVEGF(165) resulted in a 2-fold increase in bone volume at the defect site relative to control scaffolds without pVEGF(165). It was concluded that a collagen/calcium phosphate scaffold can mediate transfection without the use of additional transfection vectors and can promote bone formation in a mouse model via the delivery of pVEGF(165). 2009 Elsevier Ltd. All rights reserved.

Changes in Structural-Mechanical Properties and Degradability of Collagen during Aging-associated Modifications*

PubMed Central

Panwar, Preety; Lamour, Guillaume; Mackenzie, Neil C. W.; Yang, Heejae; Ko, Frank; Li, Hongbin; Brömme, Dieter

2015-01-01

During aging, changes occur in the collagen network that contribute to various pathological phenotypes in the skeletal, vascular, and pulmonary systems. The aim of this study was to investigate the consequences of age-related modifications on the mechanical stability and in vitro proteolytic degradation of type I collagen. Analyzing mouse tail and bovine bone collagen, we found that collagen at both fibril and fiber levels varies in rigidity and Young's modulus due to different physiological changes, which correlate with changes in cathepsin K (CatK)-mediated degradation. A decreased susceptibility to CatK-mediated hydrolysis of fibrillar collagen was observed following mineralization and advanced glycation end product-associated modification. However, aging of bone increased CatK-mediated osteoclastic resorption by ∼27%, and negligible resorption was observed when osteoclasts were cultured on mineral-deficient bone. We observed significant differences in the excavations generated by osteoclasts and C-terminal telopeptide release during bone resorption under distinct conditions. Our data indicate that modification of collagen compromises its biomechanical integrity and affects CatK-mediated degradation both in bone and tissue, thus contributing to our understanding of extracellular matrix aging. PMID:26224630

Collagen production of osteoblasts revealed by ultra-high voltage electron microscopy.

PubMed

Hosaki-Takamiya, Rumiko; Hashimoto, Mana; Imai, Yuichi; Nishida, Tomoki; Yamada, Naoko; Mori, Hirotaro; Tanaka, Tomoyo; Kawanabe, Noriaki; Yamashiro, Takashi; Kamioka, Hiroshi

2016-09-01

In the bone, collagen fibrils form a lamellar structure called the "twisted plywood-like model." Because of this unique structure, bone can withstand various mechanical stresses. However, the formation of this structure has not been elucidated because of the difficulty of observing the collagen fibril production of the osteoblasts via currently available methods. This is because the formation occurs in the very limited space between the osteoblast layer and bone matrix. In this study, we used ultra-high-voltage electron microscopy (UHVEM) to observe collagen fibril production three-dimensionally. UHVEM has 3-MV acceleration voltage and enables us to use thicker sections. We observed collagen fibrils that were beneath the cell membrane of osteoblasts elongated to the outside of the cell. We also observed that osteoblasts produced collagen fibrils with polarity. By using AVIZO software, we observed collagen fibrils produced by osteoblasts along the contour of the osteoblasts toward the bone matrix area. Immediately after being released from the cell, the fibrils run randomly and sparsely. But as they recede from the osteoblast, the fibrils began to run parallel to the definite direction and became thick, and we observed a periodical stripe at that area. Furthermore, we also observed membrane structures wrapped around filamentous structures inside the osteoblasts. The filamentous structures had densities similar to the collagen fibrils and a columnar form and diameter. Our results suggested that collagen fibrils run parallel and thickly, which may be related to the lateral movement of the osteoblasts. UHVEM is a powerful tool for observing collagen fibril production.

Perinatal collagen turnover markers in intrauterine growth restriction.

PubMed

Gourgiotis, Demetrios; Briana, Despina D; Georgiadis, Anestis; Boutsikou, Maria; Baka, Stavroula; Marmarinos, Antonios; Hassiakos, Demetrios; Malamitsi-Puchner, Ariadne

2012-09-01

To investigate bone and connective tissue collagen turnover in intrauterine growth restricted (IUGR) pregnancies, by determining circulating markers of type I collagen synthesis (carboxy-terminal propeptide of type I procollagen [PICP], representing bone formation) and degradation (cross-linked telopeptide of type I collagen [ICTP], representing bone resorption) as well as type III collagen synthesis (N-terminal propeptide of type-III procollagen [PIIINP], reflecting growth and tissue maturity). Plasma PICP, ICTP and PIIINP concentrations were measured in 40 mothers and their 20 asymmetric IUGR and 20 appropriate for gestational age (AGA) full-term fetuses and neonates on postnatal day 1-(N1) and 4-(N4). Fetal PICP, fetal and N4 ICTP, as well as fetal, N1 and N4 PIIINP concentrations were higher in the IUGR group (p ≤ 0.038, in all cases). In both groups, maternal PICP, ICTP and PIIINP concentrations were lower than fetal, N1 and N4 ones (p<0.001, in each case). Type I collagen turnover is enhanced in IUGR than AGA fetuses/neonates. Similarly, fetal/neonatal PIIINP concentrations are elevated in IUGR, probably due to stress, responsible for induction of tissue maturation, and/or to impaired excretory renal function, leading to reduced protein clearance. Fetal/neonatal PICP, ICTP and PIIINP concentrations are higher than maternal concentrations, possibly reflecting increased skeletal growth and collagen turnover in the former.

Regional alterations of type I collagen in rat tibia induced by skeletal unloading

NASA Technical Reports Server (NTRS)

Shiiba, Masashi; Arnaud, Sara B.; Tanzawa, Hideki; Kitamura, Eiji; Yamauchi, Mitsuo

2002-01-01

Skeletal unloading induces loss of mineral density in weight-bearing bones that leads to inferior bone mechanical strength. This appears to be caused by a failure of bone formation; however, its mechanisms still are not well understood. The objective of this study was to characterize collagen, the predominant matrix protein in bone, in various regions of tibia of rats that were subjected to skeletal unloading by 4 weeks tail suspension. Sixteen male Sprague-Dawley rats (4 months old) were divided into tail suspension and ambulatory controls (eight rats each). After the tail suspension, tibias from each animal were collected and divided into five regions and collagen was analyzed. The collagen cross-linking and the extent of lysine (Lys) hydroxylation in unloaded bones were significantly altered in proximal epiphysis, diaphysis, and, in particular, proximal metaphysis but not in distal regions. The pool of immature/nonmineralized collagen measured by its extractability with a chaotropic solvent was significantly increased in proximal metaphysis. These results suggest that skeletal unloading induced an accumulation of post-translationally altered nonmineralized collagen and that these changes are bone region specific. These alterations might be caused by impaired osteoblastic function/differentiation resulting in a mineralization defect.

Bioglass incorporation improves mechanical properties and enhances cell-mediated mineralization on electrochemically aligned collagen threads.

PubMed

Nijsure, Madhura P; Pastakia, Meet; Spano, Joseph; Fenn, Michael B; Kishore, Vipuil

2017-09-01

Bone tissue engineering mandates the development of a functional scaffold that mimics the physicochemical properties of native bone. Bioglass 45S5 (BG) is a highly bioactive material known to augment bone formation and restoration. Hybrid scaffolds fabricated using collagen type I and BG resemble the organic and inorganic composition of the bone extracellular matrix and hence have been extensively investigated for bone tissue engineering applications. However, collagen-BG scaffolds developed thus far do not recapitulate the aligned structure of collagen found in native bone. In this study, an electrochemical fabrication method was employed to synthesize BG-incorporated electrochemically aligned collagen (BG-ELAC) threads that are compositionally similar to native bone. Further, aligned collagen fibrils within BG-ELAC threads mimic the anisotropic arrangement of collagen fibrils in native bone. The effect of BG incorporation on the mechanical properties and cell-mediated mineralization on ELAC threads was investigated. The results indicated that BG can be successfully incorporated within ELAC threads, without disturbing collagen fibril alignment. Further, BG incorporation significantly increased the ultimate tensile stress (UTS) and modulus of ELAC threads (p < 0.05). SBF conditioning showed extensive mineralization on BG-ELAC threads that increased over time demonstrating the bone bioactivity of BG-ELAC threads. Additionally, BG incorporation into ELAC threads resulted in increased cell proliferation (p < 0.05) and deposition of a highly dense and continuous mineralized matrix. In conclusion, incorporation of BG into ELAC threads is a viable strategy for the development of an osteoconductive material for bone tissue engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2429-2440, 2017. © 2017 Wiley Periodicals, Inc.

Calcified cartilage or bone? Collagens in the tessellated endoskeletons of cartilaginous fish (sharks and rays).

PubMed

Seidel, Ronald; Blumer, Michael; Pechriggl, Elisabeth-Judith; Lyons, Kady; Hall, Brian K; Fratzl, Peter; Weaver, James C; Dean, Mason N

2017-10-01

The primary skeletal tissue in elasmobranchs -sharks, rays and relatives- is cartilage, forming both embryonic and adult endoskeletons. Only the skeletal surface calcifies, exhibiting mineralized tiles (tesserae) sandwiched between a cartilage core and overlying fibrous perichondrium. These two tissues are based on different collagens (Coll II and I, respectively), fueling a long-standing debate as to whether tesserae are more like calcified cartilage or bone (Coll 1-based) in their matrix composition. We demonstrate that stingray (Urobatis halleri) tesserae are bipartite, having an upper Coll I-based 'cap' that merges into a lower Coll II-based 'body' zone, although tesserae are surrounded by cartilage. We identify a 'supratesseral' unmineralized cartilage layer, between tesserae and perichondrium, distinguished from the cartilage core in containing Coll I and X (a common marker for mammalian mineralization), in addition to Coll II. Chondrocytes within tesserae appear intact and sit in lacunae filled with Coll II-based matrix, suggesting tesserae originate in cartilage, despite comprising a diversity of collagens. Intertesseral joints are also complex in their collagenous composition, being similar to supratesseral cartilage closer to the perichondrium, but containing unidentified fibrils nearer the cartilage core. Our results indicate a unique potential for tessellated cartilage in skeletal biology research, since it lacks features believed diagnostic for vertebrate cartilage mineralization (e.g. hypertrophic and apoptotic chondrocytes), while offering morphologies amenable for investigating the regulation of complex mineralized ultrastructure and tissues patterned on multiple collagens. Copyright © 2017 Elsevier Inc. All rights reserved.

Ridge preservation of extraction sockets with chronic pathology using Bio-Oss® Collagen with or without collagen membrane: an experimental study in dogs.

PubMed

Kim, Jung-Ju; Schwarz, Frank; Song, Hyun Young; Choi, YoonMi; Kang, Kyung-Rim; Koo, Ki-Tae

2017-06-01

This study aimed to evaluate the dynamics of newly bone formation and dimensional change in diseased extraction sockets using Bio-Oss ® Collagen with or without a collagen membrane. In six beagle dogs, right and left 3rd and 4th mandibular premolars were hemisected and the distal roots were removed. Combined endodontic-periodontic lesions were induced in all sites using black silk, collagen sponge, endodontic files, and application of Porphyromonas gingivalis. After 4 months, among 4 premolars, three teeth were randomly selected per dog and allocated to the following experimental groups: Control group (no treatment but debridement), Test 1 group (only Bio-Oss ® Collagen graft), and Test 2 group (Bio-Oss ® Collagen graft with a collagen membrane). After 7 months from the baseline, the beagle dogs were sacrificed for histomorphometric and Micro-CT analysis. The vertical distance between buccal and lingual crests in the Control group (2.22 ± 0.26 mm) and Test 2 group (1.80 ± 0.16 mm) was significantly different. The socket of the Test 2 group (27.04 ± 5.25%) was occupied by a greater quantity of bone graft compared to the Test 1 group (18.49 ± 2.11%). Ridge preservation in diseased extraction sockets could compensate for buccal bone resorption by contact osteogenesis surrounding the bone graft particles at the bucco-coronal area during socket healing, and the application of a collagen membrane at the entrance of the socket is useful for preserving graft material at the coronal part of the socket. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effects of cell-attachment and extracellular matrix on bone formation in vivo in collagen-hydroxyapatite scaffolds.

PubMed

Villa, Max M; Wang, Liping; Rowe, David W; Wei, Mei

2014-01-01

Cell-based tissue engineering can be used to replace missing or damaged bone, but the optimal methods for delivering therapeutic cells to a bony defect have not yet been established. Using transgenic reporter cells as a donor source, two different collagen-hydroxyapatite (HA) scaffolds, and a critical-size calvarial defect model, we investigated the effect of a cell-attachment period prior to implantation, with or without an extracellular matrix-based seeding suspension, on cell engraftment and osteogenesis. When quantitatively compared, the in-house scaffold implanted immediately had a higher mean radiopacity than in-house scaffolds incubated overnight. Both scaffold types implanted immediately had significantly higher area fractions of donor cells, while the in-house collagen-HA scaffolds implanted immediately had higher area fractions of the mineralization label compared with groups incubated overnight. When the cell loading was compared in vitro for each delivery method using the in-house scaffold, immediate loading led to higher numbers of delivered cells. Immediate loading may be preferable in order to ensure robust bone formation in vivo. The use of a secondary ECM carrier improved the distribution of donor cells only when a pre-attachment period was applied. These results have improved our understanding of cell delivery to bony defects in the context of in vivo outcomes.

Biodegradation, biocompatibility, and osteoconduction evaluation of collagen-nanohydroxyapatite cryogels for bone tissue regeneration.

PubMed

Salgado, Christiane Laranjo; Grenho, Liliana; Fernandes, Maria Helena; Colaço, Bruno Jorge; Monteiro, Fernando Jorge

2016-01-01

Designing biomimetic biomaterials inspired by the natural complex structure of bone and other hard tissues is still a challenge nowadays. The control of the biomineralization process onto biomaterials should be evaluated before clinical application. Aiming at bone regeneration applications, this work evaluated the in vitro biodegradation and interaction between human bone marrow stromal cells (HBMSC) cultured on different collagen/nanohydroxyapatite cryogels. Cell proliferation, differentiation, morphology, and metabolic activity were assessed through different protocols. All the biocomposite materials allowed physiologic apatite deposition after incubation in simulated body fluid and the cryogel with the highest nanoHA content showed to have the highest mechanical strength (DMA). The study clearly showed that the highest concentration of nanoHA granules on the cryogels were able to support cell type's survival, proliferation, and individual functionality in a monoculture system, for 21 days. In fact, the biocomposites were also able to differentiate HBMSCs into osteoblastic phenotype. The composites behavior was also assessed in vivo through subcutaneous and bone implantation in rats to evaluate its tissue-forming ability and degradation rate. The cryogels Coll/nanoHA (30 : 70) promoted tissue regeneration and adverse reactions were not observed on subcutaneous and bone implants. The results achieved suggest that scaffolds of Coll/nanoHA (30 : 70) should be considered promising implants for bone defects that present a grotto like appearance with a relatively small access but a wider hollow inside. This material could adjust to small dimensions and when entering into the defect, it could expand inside and remain in close contact with the defect walls, thus ensuring adequate osteoconductivity. © 2015 Wiley Periodicals, Inc.

Quantification of nitrogenous bases, DNA and Collagen type I for the estimation of the postmortem interval in bone remains.

PubMed

Pérez-Martínez, Cristina; Pérez-Cárceles, María D; Legaz, Isabel; Prieto-Bonete, Gemma; Luna, Aurelio

2017-12-01

Estimating the postmortem interval (PMI) is an important goal in forensic medicine and continues to be one of the most difficult tasks of the forensic investigator. Few accurate methods exist to determine the time since death of skeletonized human remains due to the great number of intrinsic and external factors that may alter the normal course of postmortem change. The purpose of this research was to assess the usefulness of various biochemical parameters, such as nitrogenous bases (adenine, guanine, purines, cytosine, thymine, pyrimidines, hypoxanthine and xanthine), DNA and Collagen Type I peptides to estimate PMI. These parameters were analysed in cortical bone for the establishment of data in a total of 80 long bones of 80 corpses (50 males, 30 females) with a mean age of 68.31 years (S.D.=18.021, range=20-97). The bones were removed from the cement niches of a cemetery in Murcia (south-eastern Spain), where they had lain for between 5 and 47 years (mean time 23.83 years, S.D.=10.85). Our results show a significant decrease in adenine (p=0.0004), guanine (p=0.0001), purines (p=0.0001), cytosine (p=0.0001), thymine (p=0.0226), pyrimidines (p=0.0002) and the number of peptides of Collagen type I (p=0.0053) in those with a PMI≥20 years. In a curvilinear regression analysis the results show that 30.6% of the variable PMI could be explained by guanine concentration, in bones with a PMI<20 years, while in cases of a PMI≥20 years, the variable that best explained membership of this group was adenine (38.0%). In the discriminant analysis applied to the all the variables as a function of PMI when two groups were established, 86.7% of the cases were correctly classified. These results show that the quantification of Collagen type I proteins and nitrogenous bases could be used as a complementary tool, together with other analyses, in the estimation of PMI. Copyright © 2017 Elsevier B.V. All rights reserved.

Enigmatic insight into collagen

PubMed Central

Deshmukh, Shrutal Narendra; Dive, Alka M; Moharil, Rohit; Munde, Prashant

2016-01-01

Collagen is a unique, triple helical molecule which forms the major part of extracellular matrix. It is the most abundant protein in the human body, representing 30% of its dry weight. It is the fibrous structural protein that makes up the white fibers (collagen fibers) of skin, tendons, bones, cartilage and all other connective tissues. Collagens are not only essential for the mechanical resistance and resilience of multicellular organisms, but are also signaling molecules defining cellular shape and behavior. The human body has at least 16 types of collagen, but the most prominent types are I, II and III. Collagens are produced by several cell types and are distinguishable by their molecular compositions, morphologic characteristics, distribution, functions and pathogenesis. This is the major fibrous glycoprotein present in the extracellular matrix and in connective tissue and helps in maintaining the structural integrity of these tissues. It has a triple helical structure. Various studies have proved that mutations that modify folding of the triple helix result in identifiable genetic disorders. Collagen diseases share certain similarities with autoimmune diseases, because autoantibodies specific to each collagen disease are produced. Therefore, this review highlights the role of collagen in normal health and also the disorders associated with structural and functional defects in collagen. PMID:27601823

Modelling the mechanics of partially mineralized collagen fibrils, fibres and tissue

PubMed Central

Liu, Yanxin; Thomopoulos, Stavros; Chen, Changqing; Birman, Victor; Buehler, Markus J.; Genin, Guy M.

2014-01-01

Progressive stiffening of collagen tissue by bioapatite mineral is important physiologically, but the details of this stiffening are uncertain. Unresolved questions about the details of the accommodation of bioapatite within and upon collagen's hierarchical structure have posed a central hurdle, but recent microscopy data resolve several major questions. These data suggest how collagen accommodates bioapatite at the lowest relevant hierarchical level (collagen fibrils), and suggest several possibilities for the progressive accommodation of bioapatite at higher hierarchical length scales (fibres and tissue). We developed approximations for the stiffening of collagen across spatial hierarchies based upon these data, and connected models across hierarchies levels to estimate mineralization-dependent tissue-level mechanics. In the five possible sequences of mineralization studied, percolation of the bioapatite phase proved to be an important determinant of the degree of stiffening by bioapatite. The models were applied to study one important instance of partially mineralized tissue, which occurs at the attachment of tendon to bone. All sequences of mineralization considered reproduced experimental observations of a region of tissue between tendon and bone that is more compliant than either tendon or bone, but the size and nature of this region depended strongly upon the sequence of mineralization. These models and observations have implications for engineered tissue scaffolds at the attachment of tendon to bone, bone development and graded biomimetic attachment of dissimilar hierarchical materials in general. PMID:24352669

The Effect of Interferon-γ and Zoledronate Treatment on Alpha-Tricalcium Phosphate/Collagen Sponge-Mediated Bone-Tissue Engineering

PubMed Central

Li, Peiqi; Hashimoto, Yoshiya; Honda, Yoshitomo; Arima, Yoshiyuki; Matsumoto, Naoyuki

2015-01-01

Inflammatory responses are frequently associated with the expression of inflammatory cytokines and severe osteoclastogenesis, which significantly affect the efficacy of biomaterials. Recent findings have suggested that interferon (IFN)-γ and zoledronate (Zol) are effective inhibitors of osteoclastogenesis. However, little is known regarding the utility of IFN-γ and Zol in bone tissue engineering. In this study, we generated rat models by generating critically sized defects in calvarias implanted with an alpha-tricalcium phosphate/collagen sponge (α-TCP/CS). At four weeks post-implantation, the rats were divided into IFN-γ, Zol, and control (no treatment) groups. Compared with the control group, the IFN-γ and Zol groups showed remarkable attenuation of severe osteoclastogenesis, leading to a significant enhancement in bone mass. Histomorphometric data and mRNA expression patterns in IFN-γ and Zol-injected rats reflected high bone-turnover with increased bone formation, a reduction in osteoclast numbers, and tumor necrosis factor-α expression. Our results demonstrated that the administration of IFN-γ and Zol enhanced bone regeneration of α-TCP/CS implants by enhancing bone formation, while hampering excess bone resorption. PMID:26516841

Bioinspired Collagen Scaffolds in Cranial Bone Regeneration: From Bedside to Bench

PubMed Central

Volpicelli, Elizabeth J.

2018-01-01

Calvarial defects are common reconstructive dilemmas secondary to a variety of etiologies including traumatic brain injury, cerebrovascular disease, oncologic resection, and congenital anomalies. Reconstruction of the calvarium is generally undertaken for the purposes of cerebral protection, contour restoration for psychosocial well-being, and normalization of neurological dysfunction frequently found in patients with massive cranial defects. Current methods for reconstruction using autologous grafts, allogeneic grafts, or allo-plastic materials have significant drawbacks that are unique to each material. The combination of wide medical relevance and the need for a better clinical solution render defects of the cranial skeleton an ideal target for development of regenerative strategies focused on calvarial bone. With the improved understanding of the instructive properties of tissue-specific extracellular matrices and the advent of precise nanoscale modulation in materials science, strategies in regenerative medicine have shifted in paradigm. Previously considered to be simple carriers of stem cells and growth factors, increasing evidence exists for differential materials directing lineage specific differentiation of progenitor cells and tissue regeneration. In this work, we review the clinical challenges for calvarial reconstruction, the anatomy and physiology of bone, and extracellular matrix-inspired, collagen-based materials that have been tested for in vivo cranial defect healing. PMID:28585295

Cooperation of phosphates and carboxylates controls calcium oxalate crystallization in ultrafiltered urine.

PubMed

Grohe, Bernd; Chan, Brian P H; Sørensen, Esben S; Lajoie, Gilles; Goldberg, Harvey A; Hunter, Graeme K

2011-10-01

Osteopontin (OPN) is one of a group of proteins found in urine that are believed to limit the formation of kidney stones. In the present study, we investigate the roles of phosphate and carboxylate groups in the OPN-mediated modulation of calcium oxalate (CaOx), the principal mineral phase found in kidney stones. To this end, crystallization was induced by addition of CaOx solution to ultrafiltered human urine containing either human kidney OPN (kOPN; 7 consecutive carboxylates, 8 phosphates) or synthesized peptides corresponding to residues 65-80 (pSHDHMDDDDDDDDDGD; pOPAR) or 220-235 (pSHEpSTEQSDAIDpSAEK; P3) of rat bone OPN. Sequence 65-80 was also synthesized without the phosphate group (OPAR). Effects on calcium oxalate monohydrate (COM) and dihydrate (COD) formation were studied by scanning electron microscopy. We found that controls form large, partly intergrown COM platelets; COD was never observed. Adding any of the polyelectrolytes was sufficient to prevent intergrowth of COM platelets entirely, inhibiting formation of these platelets strongly, and inducing formation of the COD phase. Strongest effects on COM formation were found for pOPAR and OPAR followed by kOPN and then P3, showing that acidity and hydrophilicity are crucial in polyelectrolyte-affected COM crystallization. At higher concentrations, OPAR also inhibited COD formation, while P3, kOPN and, in particular, pOPAR promoted COD, a difference explainable by the variations of carboxylate and phosphate groups present in the molecules. Thus, we conclude that carboxylate groups play a primary role in inhibiting COM formation, but phosphate and carboxylate groups are both important in initiating and promoting COD formation.

Human fetal bone cells in delivery systems for bone engineering.

PubMed

Tenorio, Diene M H; Scaletta, Corinne; Jaccoud, Sandra; Hirt-Burri, Nathalie; Pioletti, Dominique P; Jaques, Bertrand; Applegate, Lee Ann

2011-11-01

The aim of this study was to culture human fetal bone cells (dedicated cell banks of fetal bone derived from 14 week gestation femurs) within both hyaluronic acid gel and collagen foam, to compare the biocompatibility of both matrices as potential delivery systems for bone engineering and particularly for oral application. Fetal bone cell banks were prepared from one organ donation and cells were cultured for up to 4 weeks within hyaluronic acid (Mesolis®) and collagen foams (TissueFleece®). Cell survival and differentiation were assessed by cell proliferation assays and histology of frozen sections stained with Giemsa, von Kossa and ALP at 1, 2 and 4 weeks of culture. Within both materials, fetal bone cells could proliferate in three-dimensional structure at ∼70% capacity compared to monolayer culture. In addition, these cells were positive for ALP and von Kossa staining, indicating cellular differentiation and matrix production. Collagen foam provides a better structure for fetal bone cell delivery if cavity filling is necessary and hydrogels would permit an injectable technique for difficult to treat areas. In all, there was high biocompatibility, cellular differentiation and matrix deposition seen in both matrices by fetal bone cells, allowing for easy cell delivery for bone stimulation in vivo. Copyright © 2011 John Wiley & Sons, Ltd.

3D diffusion model within the collagen apatite porosity: An insight to the nanostructure of human trabecular bone

PubMed Central

Bini, Fabiano; Pica, Andrada; Marinozzi, Andrea; Marinozzi, Franco

2017-01-01

Bone tissue at nanoscale is a composite mainly made of apatite crystals, collagen molecules and water. This work is aimed to study the diffusion within bone nanostructure through Monte-Carlo simulations. To this purpose, an idealized geometric model of the apatite-collagen structure was developed. Gaussian probability distribution functions were employed to design the orientation of the apatite crystals with respect to the axes (length L, width W and thickness T) of a plate-like trabecula. We performed numerical simulations considering the influence of the mineral arrangement on the effective diffusion coefficient of water. To represent the hindrance of the impermeable apatite crystals on the water diffusion process, the effective diffusion coefficient was scaled with the tortuosity, the constrictivity and the porosity factors of the structure. The diffusion phenomenon was investigated in the three main directions of the single trabecula and the introduction of apatite preferential orientation allowed the creation of an anisotropic medium. Thus, different diffusivities values were observed along the axes of the single trabecula. We found good agreement with previous experimental results computed by means of a genetic algorithm. PMID:29220377

Antioxidative Peptides Derived from Enzyme Hydrolysis of Bone Collagen after Microwave Assisted Acid Pre-Treatment and Nitrogen Protection

PubMed Central

Lin, Yun-Jian; Le, Guo-Wei; Wang, Jie-Yun; Li, Ya-Xin; Shi, Yong-Hui; Sun, Jin

2010-01-01

This study focused on the preparation method of antioxidant peptides by enzymatic hydrolysis of bone collagen after microwave assisted acid pre-treatment and nitrogen protection. Phosphoric acid showed the highest ability of hydrolysis among the four other acids tested (hydrochloric acid, sulfuric acid and/or citric acid). The highest degree of hydrolysis (DH) was 9.5% using 4 mol/L phosphoric acid with a ratio of 1:6 under a microwave intensity of 510 W for 240 s. Neutral proteinase gave higher DH among the four protease tested (Acid protease, neutral protease, Alcalase and papain), with an optimum condition of: (1) ratio of enzyme and substrate, 4760 U/g; (2) concentration of substrate, 4%; (3) reaction temperature, 55 °C and (4) pH 7.0. At 4 h, DH increased significantly (P < 0.01) under nitrogen protection compared with normal microwave assisted acid pre-treatment hydrolysis conditions. The antioxidant ability of the hydrolysate increased and reached its maximum value at 3 h; however DH decreased dramatically after 3 h. Microwave assisted acid pre-treatment and nitrogen protection could be a quick preparatory method for hydrolyzing bone collagen. PMID:21151439

Decreased collagen types I and IV, laminin, CK-19 and α-SMA expression after bone marrow cell transplantation in rats with liver fibrosis.

PubMed

Carvalho, S N; Lira, D C; Oliveira, G P; Thole, A A; Stumbo, A C; Caetano, C E; Marques, R G; Carvalho, L

2010-11-01

Bone marrow cells have frequently been tested in animal models of liver fibrosis to assess their role in hepatic regeneration. The mononuclear fraction of bone marrow cells is of particular interest, as many studies show that these cells may be beneficial to treat hepatic fibrosis. In this study, we used the bile duct ligation model to induce hepatic fibrosis in an irreversible manner, and rats were treated with bone marrow mononuclear (BMMN) cells after fibrosis was established. Analysis of collagen types I and IV, laminin and α-SMA showed a decreased expression of these proteins in fibrotic livers after 7 days of BMMN cell injection. Moreover, cytokeratin-19 analysis showed a reduction in bile ducts in the BMMN cell-treated group. These results were accompanied by ameliorated levels of hepatic enzymes GPT (Glutamic-pyruvic transaminase), GOT (glutamic-oxaloacetic transaminase) and alkaline phosphatase (AP). Therefore, we showed that BMMN cells decrease hepatic fibrosis by significantly reducing myofibroblast numbers and through reduction of the collagen and laminin-rich extracellular matrix of fibrotic septa and hepatic sinusoids.

Biomimetic fabrication of a three-level hierarchical calcium phosphate/collagen/hydroxyapatite scaffold for bone tissue engineering.

PubMed

Zhou, Changchun; Ye, Xingjiang; Fan, Yujiang; Ma, Liang; Tan, Yanfei; Qing, Fangzu; Zhang, Xingdong

2014-09-01

A three-level hierarchical calcium phosphate/collagen/hydroxyapatite (CaP/Col/HAp) scaffold for bone tissue engineering was developed using biomimetic synthesis. Porous CaP ceramics were first prepared as substrate materials to mimic the porous bone structure. A second-level Col network was then composited into porous CaP ceramics by vacuum infusion. Finally, a third-level HAp layer was achieved by biomimetic mineralization. The three-level hierarchical biomimetic scaffold was characterized using scanning electron microscopy, energy-dispersive x-ray spectra, x-ray diffraction and Fourier transform infrared spectroscopy, and the mechanical properties of the scaffold were evaluated using dynamic mechanical analysis. The results show that this scaffold exhibits a similar structure and composition to natural bone tissues. Furthermore, this three-level hierarchical biomimetic scaffold showed enhanced mechanical strength compared with pure porous CaP scaffolds. The biocompatibility and osteoinductivity of the biomimetic scaffolds were evaluated using in vitro and in vivo tests. Cell culture results indicated the good biocompatibility of this biomimetic scaffold. Faster and increased bone formation was observed in these scaffolds following a six-month implantation in the dorsal muscles of rabbits, indicating that this biomimetic scaffold exhibits better osteoinductivity than common CaP scaffolds.

Collagen a natural scaffold for biology and engineering

USDA-ARS?s Scientific Manuscript database

Collagen, the most abundant protein in mammals, constitutes a quarter of the animal's total weight. The unique structure of fibrous collagens, a long triple helix that further associates into fibers, provides an insoluble scaffold that gives strength and form to the skin, tendons, bones, cornea and...

Hydroxyapatite collagen scaffold with autologous bone marrow aspirate for mandibular condylar reconstruction.

PubMed

Howlader, Debraj; Vignesh, U; Bhutia, Dichen P; Pandey, Rahul; Kumar, Sumit; Chandra, Tulika; Mehrotra, Divya

2017-09-01

This study was designed with the aim to assess the efficiency of hydroxyapatite/collagen (HA/Col) bio-scaffold with bone marrow aspirate (BMA) to reconstruct mandibular condyle in patients with temporomandibular joint (TMJ) ankylosis. Seven pediatric patients with TMJ ankylosis, who visited our outpatient clinic and whose parents opted for this procedure, were included in this study. After a history and clinical examination for TMJ movements, computed tomography (CT) scans were obtained. Interposition arthroplasty, with or without coronoidectomy, was performed to gain at least 35 mm of mouth opening. A 2-ml quantity of BMA was aspirated from the posterior iliac crest. A HA/Col block was carved to shape the condyle, and was fixed to the ramus using a plate and screws. A collagen sponge soaked in BMA was interposed in between the graft and ramal end, and the temporal fascia was rotated between the glenoid fossa and graft. Physiotherapy was started on postoperative day 10. All patients were followed up for 1 year. Success was graded on the basis of the mouth opening and TMJ score based on efficiency of chewing, speech, activity, recreation, mood, and anxiety on a five-point ordinal scale. The mean age was 9.71 years (range 5-14 years), and the male-to-female ratio was 5:2. The mean preoperative mouth opening was 4.14 mm, which improved to 34.57 mm at 1-year follow-up. The mean protrusive movement improved from 0 to 2.86 mm. The mean success score was 4.43 out of 5. The mean TMJ score improved from 2.38 to 3.94. A HA/Col bio-scaffold with bone marrow aspirate is a safe and cost-effective alternative for reconstruction of the mandibular condyle, particularly in growing individuals with high osteogenic potential. Copyright © 2017 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Checking collagen preservation in archaeological bone by non-destructive studies (Micro-CT and IBA)

NASA Astrophysics Data System (ADS)

Beck, L.; Cuif, J.-P.; Pichon, L.; Vaubaillon, S.; Dambricourt Malassé, A.; Abel, R. L.

2012-02-01

The material to be studied is a piece of human skull discovered (1999) in Pleistocene sediments from the Orsang river (Gujarat state, India). From anatomical view point, this skull is highly composite: modern Homo sapiens characters are associated to undoubtedly more ancient features. Absolute dating by 14C is critical to understand this discovery. Prior to dating measurements, non-destructive studies have been carried out. Micro-CT reconstruction (X-ray microtomography) and Ion Beam Analysis (IBA) have been undertaken to check the structural preservation of the fossil and the collagen preservation. PIXE elemental map was used to select well-preserved bone area. RBS/EBS and NRA were used for light element quantification, in particular C, N and O contents. We also demonstrate that the PIXE-RBS/EBS combination is a effective tool for the whole characterization of archaeological and recent bones by analysing in one experiment both mineral and organic fractions. We have shown that the archaeological bone, a fragment of the potentially oldest modern Indian, is enough preserved for radiocarbon dating. We propose that Elastic Backscattering Spectrometry (EBS) using 3 MeV protons could be a good non destructive alternative to conventional CHN method using Carbon-Hydrogen-Nitrogen analyzer for measuring C and N before 14C dating.

BMP-2/PLGA delayed-release microspheres composite graft, selection of bone particulate diameters, and prevention of aseptic inflammation for bone tissue engineering.

PubMed

Ji, Ye; Xu, Gong Ping; Zhang, Zhi Peng; Xia, Jing Jun; Yan, Jing Long; Pan, Shang Ha

2010-03-01

Autogenous bone grafts are widely used in the repair of bone defects. Growth factors such as bone morphogenetic protein 2 (BMP-2) can induce bone regeneration and enhance bone growth. The combination of an autogenous bone graft and BMP-2 may provide a better osteogenic effect than either treatment alone, but BMP-2 is easily inactivated in body fluid. The objective of this study was to develop a technique that can better preserve the in vivo activity of BMP-2 incorporated in bone grafts. In this study, we first prepared BMP-2/poly(lactic-co-glycolic acid) (PLGA) delayed-release microspheres, and then combined collagen, the delayed-release microspheres, and rat autologous bone particulates to form four groups of composite grafts with different combinations: collagen in group A; collagen combined with bone particulates in group B; collagen combined with BMP-2/PLGA delayed-release microspheres in group C; and collagen combined with both bone particulates and BMP-2/PLGA delayed-release microspheres in group D. The four groups of composite grafts were implanted into the gluteus maximus pockets in rats. The ectopic osteogenesis and ALP level in group D (experimental group) were compared with those in groups A, B, and C (control groups) to study whether it had higher osteogenic capability. Results showed that the composite graft design increased the utility of BMP-2 and reduced the required dose of BMP-2 and volume of autologous bone. The selection of bone particulate diameter had an impact on the osteogenetic potential of bone grafts. Collagen prevented the occurrence of aseptic inflammation and improved the osteoinductivity of BMP-2. These results showed that this composite graft design is effective and feasible for use in bone repair.

Local administration of calcitriol positively influences bone remodeling and maturation during restoration of mandibular bone defects in rats.

PubMed

Liu, Hongrui; Cui, Jian; Feng, Wei; Lv, Shengyu; Du, Juan; Sun, Jing; Han, Xiuchun; Wang, Zhenming; Lu, Xiong; Yimin; Oda, Kimimitsu; Amizuka, Norio; Li, Minqi

2015-04-01

The aim of this study was to investigate the influence of calcitriol on osteoinduction following local administration into mandibular bone defects. Calcitriol-loaded absorbable collagen membrane scaffolds were prepared using the polydopamine coating method and characterized by scanning electron microscopy. Composite scaffolds were implanted into rat mandibular bone defects in the following groups: no graft material (control), bare collagen membrane (CM group), collagen membrane bearing polydopamine coating (DOP/CM group), and collagen membrane bearing polydopamine coating absorbed with calcitriol (CAL/DOP/CM group). At 1, 2, 4 and 8weeks post-surgery, the osteogenic potential of calcitriol was examined by histological and immunohistochemical methods. Following in vivo implantation, calcitriol-loaded composite scaffolds underwent rapid degradation with pronounced replacement by new bone and induced reunion of the bone marrow cavity. Calcitriol showed strong potential in inhibiting osteoclastogenesis and promotion of osteogenic differentiation at weeks 1, and 2. Furthermore, statistical analysis revealed that the newly formed bone volume in the CAL/DOP/CM group was significantly higher than other groups at weeks 1, and 2. At weeks 4, and 8, the CAL/DOP/CM group showed more mineralized bone and uniform collagen structure. These data suggest that local administration of calcitriol is promising in promoting osteogenesis and mineralization for restoration of mandibular bone defects. Copyright © 2014 Elsevier B.V. All rights reserved.

The effect of complexing phosphoproteins to decalcified collagen on in vitro calcification.

PubMed

Endo, A; Glimcher, M J

1989-01-01

Decalcified samples of chicken bone containing phosphoproteins of varying concentrations were used to assess the effect of phosphoproteins and of protein-bound Ser(P) and Thr(P) in the in vitro nucleation of a Ca-P solid phase from metastable solutions of Ca and P. Phosphoproteins of bone as well as the phosphoproteins from egg yolk (phosvitin) were used. Increasing concentrations of phosphoprotein [as measured by the amount of protein bound Ser(P) and Thr(P)] in the decalcified bone particles significantly reduced the time required for nucleation to occur after exposure to metastable solutions of Ca and P (decreased operational lag times). Treatment with wheat germ acid phosphatase markedly reduced the concentration of Ser(P) and Thr(P) in the decalcified bone samples and in the decalcified bone collagen samples complexed with phosphoproteins (almost to zero). The loss of the organic phosphate groups significantly increased the operational lag time, but did not abolish nucleation of apatite crystals by the bone collagen fibrils essentially devoid of Ser(P) and Thr(P). Bone phosphoproteins were not specific; substitution of phosvitin for bone phosphoproteins as complexes with bone collagen also proved to be effective facilitators of nucleation, which was interesting since both types of phosphoproteins have certain common chemical and structural characteristics. Noncollagenous components other than phosphoproteins were present in the decalcified bone samples. However, the marked dependence of the lag time on the Ser(P) and Thr(P) concentrations and the very marked diminution in the efficacy of the nucleation phenomenon as a result of treatment with wheat germ acid phosphatase, clearly suggests that the organic phosphate residues of the phosphoproteins play a direct and significant role in the process of in vitro nucleation of a solid phase of Ca and P (apatite) by bone collagen, and by implication, possibly in in vivo mineralization as well.

Next generation bone tissue engineering: non-viral miR-133a inhibition using collagen-nanohydroxyapatite scaffolds rapidly enhances osteogenesis

NASA Astrophysics Data System (ADS)

Mencía Castaño, Irene; Curtin, Caroline M.; Duffy, Garry P.; O'Brien, Fergal J.

2016-06-01

Bone grafts are the second most transplanted materials worldwide at a global cost to healthcare systems valued over $30 billion every year. The influence of microRNAs in the regenerative capacity of stem cells offers vast therapeutic potential towards bone grafting; however their efficient delivery to the target site remains a major challenge. This study describes how the functionalisation of porous collagen-nanohydroxyapatite (nHA) scaffolds with miR-133a inhibiting complexes, delivered using non-viral nHA particles, enhanced human mesenchymal stem cell-mediated osteogenesis through the novel focus on a key activator of osteogenesis, Runx2. This study showed enhanced Runx2 and osteocalcin expression, as well as increased alkaline phosphatase activity and calcium deposition, thus demonstrating a further enhanced therapeutic potential of a biomaterial previously optimised for bone repair applications. The promising features of this platform offer potential for a myriad of applications beyond bone repair and tissue engineering, thus presenting a new paradigm for microRNA-based therapeutics.

Resolving the bulk δ 15N values of ancient human and animal bone collagen via compound-specific nitrogen isotope analysis of constituent amino acids

NASA Astrophysics Data System (ADS)

Styring, Amy K.; Sealy, Judith C.; Evershed, Richard P.

2010-01-01

Stable nitrogen isotope analysis is a fundamental tool in assessing dietary preferences and trophic positions within contemporary and ancient ecosystems. In order to assess more fully the dietary contributions to human tissue isotope values, a greater understanding of the complex biochemical and physiological factors which underpin bulk collagen δ 15N values is necessary. Determinations of δ 15N values of the individual amino acids which constitute bone collagen are necessary to unravel these relationships, since different amino acids display different δ 15N values according to their biosynthetic origins. A range of collagen isolates from archaeological faunal and human bone ( n = 12 and 11, respectively), representing a spectrum of terrestrial and marine protein origins and diets, were selected from coastal and near-coastal sites at the south-western tip of Africa. The collagens were hydrolysed and δ 15N values of their constituent amino acids determined as N-acetylmethyl esters (NACME) via gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The analytical approach employed accounts for 56% of bone collagen nitrogen. Reconstruction of bulk bone collagen δ 15N values reveals a 2‰ offset from bulk collagen δ 15N values which is attributable to the δ 15N value of the amino acids which cannot currently be determined by GC-C-IRMS, notably arginine which comprises 53% of the nitrogen unaccounted for (23% of the total nitrogen). The δ 15N values of individual amino acids provide insights into both the contributions of various amino acids to the bulk δ 15N value of collagen and the factors influencing trophic position and the nitrogen source at the base of the food web. The similarity in the δ 15N values of alanine, glutamate, proline and hydroxyproline reflects the common origin of their amino groups from glutamate. The depletion in the δ 15N value of threonine with increasing trophic level indicates a fundamental difference between

Amino acid composition in determination of collagen origin and assessment of physical factors effects.

PubMed

Gauza-Włodarczyk, Marlena; Kubisz, Leszek; Włodarczyk, Dariusz

2017-11-01

The amino acid composition of collagen is a characteristic feature of this protein. Collagen, irrespective of its origin, contains 19 amino acids, including hydroxyproline which does not occur in other proteins. Its atypical amino acid composition is characterized by high content of proline and glycine, as well as the absence of cysteine. This paper shows the comparison of qualitative composition of amino acids of fish skin (FS) collagen, bovine Achilles tendon (BAT) collagen, and bone collagen. Results demonstrate that FS collagen as well as BAT collagen contains no cysteine and significantly different amount of hydroxyproline. In BAT collagen hydroxyproline content is 30% higher than hydroxyproline content of FS collagen. In bone collagen the amount of hydroxyproline is two times more than in FS collagen. Furthermore, it is shown that sensitivity to radiation of individual amino acids varies and depends on the absorbed dose of ionizing radiation. The changes observed in the amino acid composition become very intense for the doses of 500kGy and 1000kGy. Copyright © 2017 Elsevier B.V. All rights reserved.

Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.

PubMed

Cabral, Wayne A; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R; Chang, Weizhong; Perosky, Joseph E; Makareeva, Elena N; Mertz, Edward L; Leikin, Sergey; Tomer, Kenneth B; Kozloff, Kenneth M; Eyre, David R; Yamauchi, Mitsuo; Marini, Joan C

2014-06-01

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink

Evaluation of rhBMP-2/collagen/TCP-HA bone graft with and without bone marrow cells in the canine femoral multi defect model.

PubMed

Luangphakdy, V; Shinohara, K; Pan, H; Boehm, C; Samaranska, A; Muschler, G F

2015-01-12

Recombinant human bone morphogenetic protein-2, when applied to an absorbable type 1 bovine collagen sponge (rhBMP-2/ACS) is an effective therapy in many bone grafting settings. Bone marrow aspirate (BMA) has also been used as a source of transplantable osteogenic connective tissue progenitors. This study was designed to characterize the performance of a scaffold comprising rhBMP-2/ACS in which the sponge wraps around tri-calcium phosphate hydroxyapatite granules (rhBMP-2/ACS/TCP-HA) and to test the hypothesis that addition of BMA will improve the performance of this construct in the Canine Femoral Multi Defect Model. In each subject, two sites were grafted with rhBMP-2/ACS/TCP-HA scaffold loaded with BMA clot and two other sites with rhBMP-2/ACS/TCP-HA scaffold loaded with wound blood (WB). After correction for unresorbed TCP-HA granules, sites grafted with rhBMP-2/ACS/TCP-HA+BMA and rhBMP-2/ACS/TCP-HA+WB were similar, with mean percent bone volumes of 10.9 %±1.2 and 11.2 %±1.2, respectively. No differences were seen in quantitative histomorphometry. While bone formation using both constructs was robust, this study did not support the hypothesis that the addition of unprocessed bone marrow aspirate clot improved bone regeneration in a site engrafted with rhBMP-2/ACS/TCP-HA+BMA. In contrast to prior studies using this model, new bone formation was greater at the center of the defect where TCP-HA was distributed. This finding suggests a potential synergy between rhBMP-2 and the centrally placed ceramic and cellular components of the graft construct. Further optimization may also require more uniform distribution of TCP-HA, alternative cell delivery strategies, and a more rigorous large animal segmental defect model.

Enhanced bone formation in the vicinity of porous β-TCP scaffolds exhibiting slow release of collagen-derived tripeptides.

PubMed

Kamikura, Keita; Minatoya, Tsutomu; Terada-Nakaishi, Michiko; Yamamoto, Shoko; Sakai, Yasuo; Furusawa, Toshitake; Matsushima, Yuta; Unuma, Hidero

2017-09-01

It has been experimentally proven that orally ingested collagen-derived tripeptides (Ctp) are quickly absorbed in the body and effectively promote the regeneration of connective tissues including bone and skin. Ctp are capable to activate osteoblasts and fibroblasts, which eventually promotes tissue regeneration. Based on these findings, a hypothesis was formulated in this study that direct delivery of Ctp to bone defect would also facilitate tissue regeneration as well as oral administration. To test the hypothesis, we prepared a bone augmentation material with the ability to slowly release Ctp, and investigated its in vivo bone regeneration efficacy. The implant material was porous β-tricalcium phosphate (β-TCP) scaffold which was coated with a co-precipitated layer of bone-like hydroxyapatite and Ctp. The β-TCP was impregnated with approximately 0.8%(w/w) Ctp. Then, the Ctp-modified β-TCP was implanted into bone defects of Wistar rats to evaluate in vivo efficacy of Ctp directly delivered from the material to the bone defects. The control was pristine porous β-TCP. In vitro tests showed that Ctp were steadily released from the co-precipitated layer for approximately two weeks. The Ctp-modified scaffolds significantly promoted new bone formation in vivo in their vicinity as compared with pristine β-TCP scaffolds; 6 weeks after the implantation, Ctp-modified scaffolds promoted twice as much bone formation as the control implants. Consequently, we achieved the slow and steady release of Ctp, and found that direct delivery of Ctp from implant materials was effective for bone regeneration as well as oral administration. A β-TCP scaffold capable of slowly releasing bone-enhancing substances significantly promoted bone formation.

Enhancement of healing in osteochondral defects by collagen sponge implants.

PubMed

Speer, D P; Chvapil, M; Volz, R G; Holmes, M D

1979-10-01

Implants of porous, highly cross-linked collagen sponge (CS) were tested for their capacity to enhance the healing of osteochondral defects in rabbits. Comparison was made to the healing of similar defects with polyvinyl alcohol sponge (PVAS) implants and with no implants (CONT). Evaluation was carried out up to 44 weeks following implantation and included observation of host cellular response, biodegradability of implant, gross appearance of restored joint surface, collagenous architecture of repair tissue, and properties of the junctions of implants and host articular cartilage, subchondral bone, and medullary bone. Collagen sponge proved most effective in promoting healing of osteochondral defects with fibrous and fibrocartilaginous tissue over restored subchondral bone. Collagen sponge showed many desirable properties as a potential material for biologic resurfacing of damaged joints. These properties included porosity, biodegradability, biocompatability, ability to mechanically protect cells and matrix while directing cell ingrowth, and an available chemical technology for modifying its biomechanical and biological properties. Comparative analysis of results of healing of CS, PVAS, and CONT osteochondral defects suggest rational design criteria for implant materials to improve their effectiveness in restoration of articular surfaces.

Biochemical changes in bone in a model of weightlessness

NASA Technical Reports Server (NTRS)

Mechanic, Gerald L.

1986-01-01

The amounts of nonmineralized and mineralized collagen in bone from control, immobilized, and immobilized reambulated monkeys were examined. In order to understand structure function relationships of bone collagen and the reponse of a variety of conditions on control of the three dimensional structure of the collagen fibril, the stereochemistry of the cross-linking reactions as well as the stereospecific packing of the collagen molecules were studied.

Single-dose local administration of parathyroid hormone (1-34, PTH) with β-tricalcium phosphate/collagen (β-TCP/COL) enhances bone defect healing in ovariectomized rats.

PubMed

Tao, Zhou-Shan; Zhou, Wan-Shu; Wu, Xin-Jing; Wang, Lin; Yang, Min; Xie, Jia-Bing; Xu, Zhu-Jun; Ding, Guo-Zheng

2018-02-01

Parathyroid hormone (1-34, PTH) combined β-tricalcium phosphate (β-TCP) achieves stable bone regeneration without cell transplantation in previous studies. Recently, with the development of tissue engineering slow release technology, PTH used locally to promote bone defect healing become possible. This study by virtue of collagen with a combination of drugs and has a slow release properties, and investigated bone regeneration by β-TCP/collagen (β-TCP/COL) with the single local administration of PTH. After the creation of a rodent critical-sized femoral metaphyseal bone defect, β-TCP/COL was prepared by mixing sieved granules of β-TCP and atelocollagen for medical use, then β-TCP/COL with dripped PTH solution (1.0 µg) was implanted into the defect of OVX rats until death at 4 and 8 weeks. The defected area in distal femurs of rats was harvested for evaluation by histology, micro-CT, and biomechanics. The results of our study show that single-dose local administration of PTH combined local usage of β-TCP/COL can increase the healing of defects in OVX rats. Furthermore, treatments with single-dose local administration of PTH and β-TCP/COL showed a stronger effect on accelerating the local bone formation than β-TCP/COL used alone. The results from our study demonstrate that combination of single-dose local administration of PTH and β-TCP/COL had an additive effect on local bone formation in osteoporosis rats.

The collagen microfibril model, a tool for biomaterials scientists

USDA-ARS?s Scientific Manuscript database

Animal hides, a major byproduct of the meat industry, are a rich source of collagen, a structural protein of the extracellular matrix that gives strength and form to the skin, tendons and bones of mammals. The structure of fibrous collagen, a long triple helix that self-associates in a staggered arr...

Osteoconductive properties of two different bioactive glass forms (powder and fiber) combined with collagen

NASA Astrophysics Data System (ADS)

Magri, Angela Maria Paiva; Fernandes, Kelly Rossetti; Ueno, Fabio Roberto; Kido, Hueliton Wilian; da Silva, Antonio Carlos; Braga, Francisco José Correa; Granito, Renata Neves; Gabbai-Armelin, Paulo Roberto; Rennó, Ana Claudia Muniz

2017-11-01

Bioactive Glasses (BG) is a group of synthetic silica-based materials with the unique ability to bond to living bone and can be used in bone repair. Although the osteogenic potential of BG, this material may have not present sufficient osteoconductive and osteoinductive properties to allow bone regeneration, especially in compromised situations. In order to overcome this limitation, it was proposed the combination the BG in two forms (powder and fiber) combined with collagen type I (COL-1). The aim of this study was to evaluate the BG/COL-based materials in terms of morphological characteristics, physicochemical features and mineralization. Additionally, the second objective was to investigate and compare the osteoconductive properties of two different bioactive glass forms (powder and fiber) enriched or not with collagen using a tibial bone defect model in rats. For this, four different formulations (BG powder - BGp, BG powder enriched with collagen - BGp/Col, BG fibers - BGf and BGp fibers enriched with collagen - BGf/Col) were developed. The physicochemical and morphological modifications were analyzed by SEM, FTIR, calcium assay and pH measurement. For in vivo evaluations, histopathology, morphometrical and immunohistochemistry were performed in a tibial defect in rats. The FTIR analysis indicated that BGp and BGf maintained the characteristic peaks for this class of material. Furthermore, the calcium assay showed an increased Ca uptake in the BG fibers. The pH measurements revealed that BGp (with or without collagen) presented higher pH values compared to BGf. In addition, the histological analysis demonstrated no inflammation for all groups at the site of the injury, besides a faster material degradation and higher bone ingrowth for groups with collagen. The immunohistochemistry analysis demonstrated Runx-2 and Rank-L expression for all the groups. Those findings support that BGp with collagen can be a promising alternative for treating fracture of difficult

Altered bone material properties in HLA-B27 rats include reduced mineral to matrix ratio and altered collagen cross-links.

PubMed

Gamsjaeger, Sonja; Srivastava, Apurva K; Wergedal, Jon E; Zwerina, Jochen; Klaushofer, Klaus; Paschalis, Eleftherios P; Tatakis, Dimitris N

2014-11-01

Spondyloarthropathy and inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn's disease, are often associated with severe osteopenia/osteoporosis in both children and adults. HLA-B27 transgenic rats present a phenotype that includes severe colitis and severely accelerated alveolar bone loss. The purpose of this study was to evaluate long bone density status, systemic bone metabolic markers, and intrinsic bone material properties in HLA-B27 transgenic (TG) rats, and compare them with those of age- and sex-matched wild-type (WT) animals. The results indicate that in the HLA-B27 rat, an animal susceptible to both alveolar bone loss (ABL) and long bone osteopenia, there is a statistically significant negative correlation between ABL and long bone bone mineral density (BMD), as well as mineral/matrix ratio at active bone-forming trabecular surfaces. The TG animals had a lower mineral/matrix ratio and higher relative proteoglycan and advanced glycation end product (ϵ-N-Carboxymethyl-L-lysine) content and pyridinoline/divalent collagen cross-link ratio compared with WT. These results may provide better understanding of the interrelationship between osteoporosis and oral bone loss, the underlying causes of the inferior bone strength in the HLA-B27 transgenic animals, and could prove to be a useful model in the elucidation of the pathophysiology of spondyloarthropathy and IBD-associated osteopenia/osteoporosis and in the evaluation of pharmacological intervention(s) against such conditions. © 2014 American Society for Bone and Mineral Research.

Bone mineral as an electrical energy reservoir.

PubMed

Nakamura, Miho; Hiratai, Rumi; Yamashita, Kimihiro

2012-05-01

Mechanical stress in bone induces an electrical potential generated by piezoelectricity arising from displacement of collagen fibrils. Where and for how long the potential is stored in bone; however, are still poorly understood. We investigated the electrical properties of collagen fibrils and apatite minerals and found that bone, when polarized electrically by applying an external voltage, depolarizes by two mechanisms. Plots of thermally stimulated depolarization current show two significant peaks: one at 100°C, attributed to collagen fibrils because decalcified bone exhibits depolarization peak at 100°C, and the other at 500°C, attributed to apatite minerals because calcined bone exhibits depolarization peak at 500°C and has activation energy similar to that for synthesized apatite. The crystallographic c-axis orientation of calcined bone depends on the direction in which the bone is cut, either transverse or longitudinal, and strongly affects the polarization efficacy. Copyright © 2012 Wiley Periodicals, Inc.

Abnormal Type I Collagen Post-translational Modification and Crosslinking in a Cyclophilin B KO Mouse Model of Recessive Osteogenesis Imperfecta

PubMed Central

Cabral, Wayne A.; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R.; Chang, Weizhong; Perosky, Joseph E.; Makareeva, Elena N.; Mertz, Edward L.; Leikin, Sergey; Tomer, Kenneth B.; Kozloff, Kenneth M.; Eyre, David R.; Yamauchi, Mitsuo; Marini, Joan C.

2014-01-01

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib−/− mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2–11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib−/− fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered

How tough is Brittle Bone? Investigating Osteogenesis Imperfecta in Mouse Bone††

PubMed Central

Carriero, A.; Zimmermann, E. A.; Paluszny, A.; Tang, S. Y.; Bale, H.; Busse, B.; Alliston, T.; Kazakia, G.

2015-01-01

The multiscale hierarchical structure of bone is naturally optimized to resist fractures. In osteogenesis imperfecta, or brittle bone disease, genetic mutations affect the quality and/or quantity of collagen, dramatically increasing bone fracture risk. Here we reveal how the collagen defect results in bone fragility in a mouse model of osteogenesis imperfecta (oim), which has homotrimeric α1(I) collagen. At the molecular level we attribute the loss in toughness to a decrease in the stabilizing enzymatic crosslinks and an increase in non-enzymatic crosslinks, which may break prematurely inhibiting plasticity. At the tissue level, high vascular canal density reduces the stable crack growth, and extensive woven bone limits the crack-deflection toughening during crack growth. This demonstrates how modifications at the bone molecular level have ramifications at larger length scales affecting the overall mechanical integrity of the bone; thus, treatment strategies have to address multiscale properties in order to regain bone toughness. In this regard, findings from the heterozygous oim bone, where defective as well as normal collagen are present, suggest that increasing the quantity of healthy collagen in these bones helps to recover toughness at the multiple length scales. PMID:24420672

An overview of vertebrate mineralization with emphasis on collagen-mineral interaction

NASA Technical Reports Server (NTRS)

Landis, W. J.

1999-01-01

The nucleation, growth, and development of mineral crystals through their interaction principally with collagen in normal bone and calcifying tendon have been elaborated by applying a number of different techniques for analysis of the inorganic and organic constituents of these tissues. The methods have included conventional and high voltage electron microscopy, electron diffraction, microscopic tomography and 3D image reconstruction, and atomic force microscopy. This summary presents results of these studies that have now characterized the size, shape, and aspects of the chemical nature of the crystals as well as their orientation, alignment, location, and distribution with respect to collagen. These data have provided the means for understanding more completely the formation and strength of the collagen-mineral composite present in most vertebrate calcifying tissues and, from that information, a basis for the adaptation of such tissues under mechanical constraints. In the context of the latter point, other data are given showing effects on collagen in bone cell cultures subjected to the unloading parameters of spaceflight. Implications of these results may be particularly relevant to explaining loss of bone by humans and other vertebrate animals during missions in space, during situations of extended fracture healing, long-term bedrest, physical immobilization, and related conditions. In a broader sense, the data speak to the response of bone and mineralized vertebrate tissues to changes in gravitational loading and applied mechanical forces in general.

Hydrogel-beta-TCP scaffolds and stem cells for tissue engineering bone.

PubMed

Weinand, Christian; Pomerantseva, Irina; Neville, Craig M; Gupta, Rajiv; Weinberg, Eli; Madisch, Ijad; Shapiro, Frederic; Abukawa, Harutsugi; Troulis, Maria J; Vacanti, Joseph P

2006-04-01

Trabecular bone is a material of choice for reconstruction after trauma and tumor resection and for correction of congenital defects. Autologous bone grafts are available in limited shapes and sizes; significant donor site morbidity is another major disadvantage to this approach. To overcome these limitations, we used a tissue engineering approach to create bone replacements in vitro, combining bone-marrow-derived differentiated mesenchymal stem cells (MSCs) suspended in hydrogels and 3-dimensionally printed (3DP) porous scaffolds made of beta-tricalcium-phosphate (beta-TCP). The scaffolds provided support for the formation of bone tissue in collagen I, fibrin, alginate, and pluronic F127 hydrogels during culturing in oscillating and rotating dynamic conditions. Histological evaluation including toluidine blue, alkaline phosphatase, and von Kossa staining was done at 1, 2, 4, and 6 weeks. Radiographic evaluation and high-resolution volumetric CT (VCT) scanning, expression of bone-specific genes and biomechanical compression testing were performed at 6 weeks. Both culture conditions resulted in similar bone tissue formation. Histologically collagen I and fibrin hydrogels specimens had superior bone tissue, although radiopacities were detected only in collagen I samples. VCT scan revealed density values in all but the Pluronic F127 samples, with Houndsfield unit values comparable to native bone in collagen I and fibrin glue samples. Expression of bone-specific genes was significantly higher in the collagen I samples. Pluronic F127 hydrogel did not support formation of bone tissue. All samples cultured in dynamic oscillating conditions had slightly higher mechanical strength than under rotating conditions. Bone tissue can be successfully formed in vitro using constructs comprised of collagen I hydrogel, MSCs, and porous beta-TCP scaffolds.

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

PubMed

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

2017-06-01

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

Development of collagen-hydroxyapatite scaffolds incorporating PLGA and alginate microparticles for the controlled delivery of rhBMP-2 for bone tissue engineering.

PubMed

Quinlan, Elaine; López-Noriega, Adolfo; Thompson, Emmet; Kelly, Helena M; Cryan, Sally Ann; O'Brien, Fergal J

2015-01-28

The spatiotemporally controlled delivery of the pro-osteogenic factor rhBMP-2 would overcome most of the severe secondary effects linked to the products delivering this protein for bone regeneration. With this in mind, the aim of the present work was to develop a controlled rhBMP-2 release system using collagen-hydroxyapatite (CHA) scaffolds, which had been previously optimized for bone regeneration, as delivery platforms to produce a device with enhanced capacity for bone repair. Spray-drying and emulsion techniques were used to encapsulate bioactive rhBMP-2 in alginate and PLGA microparticles, with a high encapsulation efficiency. After incorporation of these microparticles into the scaffolds, rhBMP-2 was delivered in a sustained fashion for up to 28days. When tested in vitro with osteoblasts, these eluting materials showed an enhanced pro-osteogenic effect. From these results, an optimal rhBMP-2 eluting scaffold composition was selected and implanted in critical-sized calvarial defects in a rat model, where it demonstrated an excellent healing capacity in vivo. These platforms have an immense potential in the field of tissue regeneration; by tuning the specific therapeutic molecule to the tissue of interest and by utilizing different collagen-based scaffolds, similar systems can be developed for enhancing the healing of a diverse range of tissues and organs. Copyright © 2014 Elsevier B.V. All rights reserved.

Influence of bone morphogenetic protein and proportion of hydroxyapatite on new bone formation in biphasic calcium phosphate graft: two pilot studies in animal bony defect model.

PubMed

Yun, Pil-Young; Kim, Young-Kyun; Jeong, Kyung-In; Park, Ju-Cheol; Choi, Yeon-Jo

2014-12-01

The purpose of these two pilot studies using animal bony defect models was to evaluate the influence of bone morphogenetic protein (BMP) and proportion of hydroxyapatite (HA)/beta-tricalcium phosphate (β-TCP) in biphasic calcium phosphate (BCP) graft on new bone formation. In this study, four kinds of synthetic osteoconductive bone materials known for bone growth scaffold, OSTEON™II(HA:β-TCP 30:70), OSTEON™III (HA:β-TCP 20:80), OSTEON™II Collagen, and OSTEON™III Collagen, were prepared as BCP graft materials. In pilot study 1, three BCP materials (OSTEON™II, OSTEON™III, and OSTEON™II Collagen) were grafted in rabbit calvarial defects after impregnating in rhBMP-2. OSTEON™II without the rhBMP-2 impregnation was included in the study as the control. The amount of new bone was examined and measured histologically at 2, 4, and 8 weeks. In pilot study 2, four BCP materials (OSTEON™II, OSTEON™III, OSTEON™II Collagen, and OSTEON™III Collagen) were grafted in beagle dog mandibular defects after soaking in the rhBMP-2. The amount of total bone and new bone were measured three-dimensionally using microCT and healing process was examined histologically at 2, 4, and 8 weeks. In pilot study 1, rhBMP-2 impregnated groups showed more new bone formation than the rhBMP-2 free group. In pilot study 2, increased new bone formation was observed in time-dependent manner after graft of BCP and BCP-collagen (OSTEON™II, OSTEON™III, OSTEON™II Collagen, and OSTEON™III Collagen) impregnated with rhBMP-2. Also, BCP with a higher proportion of HA (30% HA) showed more favorable result in new bone formation and space maintenance, especially at the 8 weeks. From the results of the pilot studies, rhBMP-2 played positive roles in new bone formation and BCP could become a scaffold candidate for rhBMP-2 impregnation to induce new bone formation. Moreover, BCP with a higher proportion of HA (30% HA) could be considered more appropriate for rhBMP-2 carrier. Copyright

Efficacy of Mucograft vs Conventional Resorbable Collagen Membranes in Guided Bone Regeneration Around Standardized Calvarial Defects in Rats: A Histologic and Biomechanical Assessment.

PubMed

Ramalingam, Sundar; Basudan, Amani; Babay, Nadir; Al-Rasheed, Abdulaziz; Nooh, Nasser; Nagshbandi, Jafar; Aldahmash, Abdullah; Atteya, Muhammad; Al-Hezaimi, Khalid

2016-01-01

Guided bone regeneration (GBR) using a porcine-derived collagen matrix (Mucograft [MG], Geistlich) has not yet been reported. The aim of this histologic and biomechanical study was to compare the efficacy of MG versus resorbable collagen membranes (RCMs) in facilitating GBR around standardized rat calvarial defects. Forty female Wistar albino rats with a mean age and weight of 6 to 9 weeks and 250 to 300 g, respectively, were used. With the rats under general anesthesia, the skin over the calvaria was exposed using a full-thickness flap. A 4.6-mm-diameter standardized calvarial defect was created in the left parietal bone. For treatment, the rats were randomly divided into four groups (n = 10 per group): (1) MG group: the defect was covered with MG; (2) RCM group: the defect was covered with an RCM; (3) MG + bone group: the defect was filled with bone graft particles and covered by MG; and (4) RCM + bone group: the defect was filled with bone graft particles and covered by an RCM. Primary closure was achieved using interrupted resorbable sutures. The animals were sacrificed at 8 weeks after the surgical procedures. Qualitative histologic analysis and biomechanical assessment to identify hardness and elastic modulus of newly formed bone (NFB) were performed. Collected data were statistically analyzed using one-way analysis of variance. Histologic findings revealed NFB with fibrous connective tissue in all groups. The quantity of NFB was highest in the RCM + bone group. Statistically significant differences in the hardness (F = 567.69, dfN = 3, dfD = 36, P < .001) and elastic modulus (F = 294.19, dfN = 3, dfD = 36, P < .001) of NFB were found between the groups. Although the RCM + bone group had the highest mean ± standard deviation (SD) hardness of NFB (531.4 ± 24.9 MPa), the RCM group had the highest mean ± SD elastic modulus of NFB (18.63 ± 1.89 GPa). The present study demonstrated that RCMs are better than MG at enhancing new bone formation in standardized

Energetic basis for the molecular-scale organization of bone

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

Tao, Jinhui; Battle, Keith C.; Pan, Haihua

2014-12-24

The remarkable properties of bone derive from a highly organized arrangement of co-aligned nm-scale apatite platelets within a fibrillar collagen matrix. The origin of this arrangement is poorly understood and the crystal structures of hydroxyapatite (HAP) and the non-mineralized collagen fibrils alone do not provide an explanation. Moreover, little is known about collagen-apatite interaction energies, which should strongly influence both the molecular-scale organization and the resulting mechanical properties of the composite. We investigated collagen-mineral interactions by combining dynamic force spectroscopy (DFS) measurements of binding energies with molecular dynamics (MD) simulations of binding and AFM observations of collagen adsorption on singlemore » crystals of calcium phosphate for four mineral phases of potential importance in bone formation. In all cases, we observe a strong preferential orientation of collagen binding, but comparison between the observed orientations and TEM analyses native tissues shows only calcium-deficient apatite (CDAP) provides an interface with collagen that is consistent with both. MD simulations predict preferred collagen orientations that agree with observations and results from both MD and DFS reveal large values for the binding energy due to multiple binding sites. These findings reconcile apparent contradictions inherent in a hydroxyapatite or carbonated apatite (CAP) model of bone mineral and provide an energetic rationale for the molecular scale organization of bone.« less

Energetic basis for the molecular-scale organization of bone

DOE PAGES

Tao, Jinhui; Battle, Keith C.; Pan, Haihua; ...

2014-12-24

Here, the remarkable properties of bone derive from a highly organized arrangement of co-aligned nm-scale apatite platelets within a fibrillar collagen matrix. The origin of this arrangement is poorly understood and the crystal structures of hydroxyapatite (HAP) and the non-mineralized collagen fibrils alone do not provide an explanation. Moreover, little is known about collagen-apatite interaction energies, which should strongly influence both the molecular-scale organization and the resulting mechanical properties of the composite. We investigated collagen-mineral interactions by combining dynamic force spectroscopy (DFS) measurements of binding energies with molecular dynamics (MD) simulations of binding and AFM observations of collagen adsorption onmore » single crystals of calcium phosphate for four mineral phases of potential importance in bone formation. In all cases, we observe a strong preferential orientation of collagen binding, but comparison between the observed orientations and TEM analyses native tissues shows only calcium-deficient apatite (CDAP) provides an interface with collagen that is consistent with both. MD simulations predict preferred collagen orientations that agree with observations and results from both MD and DFS reveal large values for the binding energy due to multiple binding sites. These findings reconcile apparent contradictions inherent in a hydroxyapatite or carbonated apatite (CAP) model of bone mineral and provide an energetic rationale for the molecular scale organization of bone.« less

Energetic basis for the molecular-scale organization of bone.

PubMed

Tao, Jinhui; Battle, Keith C; Pan, Haihua; Salter, E Alan; Chien, Yung-Ching; Wierzbicki, Andrzej; De Yoreo, James J

2015-01-13

The remarkable properties of bone derive from a highly organized arrangement of coaligned nanometer-scale apatite platelets within a fibrillar collagen matrix. The origin of this arrangement is poorly understood and the crystal structures of hydroxyapatite (HAP) and the nonmineralized collagen fibrils alone do not provide an explanation. Moreover, little is known about collagen-apatite interaction energies, which should strongly influence both the molecular-scale organization and the resulting mechanical properties of the composite. We investigated collagen-mineral interactions by combining dynamic force spectroscopy (DFS) measurements of binding energies with molecular dynamics (MD) simulations of binding and atomic force microscopy (AFM) observations of collagen adsorption on single crystals of calcium phosphate for four mineral phases of potential importance in bone formation. In all cases, we observe a strong preferential orientation of collagen binding, but comparison between the observed orientations and transmission electron microscopy (TEM) analyses of native tissues shows that only calcium-deficient apatite (CDAP) provides an interface with collagen that is consistent with both. MD simulations predict preferred collagen orientations that agree with observations, and results from both MD and DFS reveal large values for the binding energy due to multiple binding sites. These findings reconcile apparent contradictions inherent in a hydroxyapatite or carbonated apatite (CAP) model of bone mineral and provide an energetic rationale for the molecular-scale organization of bone.

Energetic basis for the molecular-scale organization of bone

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

Tao, Jinhui; Battle, Keith C.; Pan, Haihua

The remarkable properties of bone derive from a highly organized arrangement of co-aligned nm-scale apatite platelets within a fibrillar collagen matrix. The origin of this arrangement is poorly understood and the crystal structures of hydroxyapatite (HAP) and the non-mineralized collagen fibrils alone do not provide an explanation. Moreover, little is known about collagen-apatite interaction energies, which should strongly influence both the molecular-scale organization and the resulting mechanical properties of the composite. We investigated collagen-mineral interactions by combining dynamic force spectroscopy (DFS) measurements of binding energies with molecular dynamics (MD) simulations of binding and AFM observations of collagen adsorption on singlemore » crystals of calcium phosphate for four mineral phases of potential importance in bone formation. In all cases, we observe a strong preferential orientation of collagen binding, but comparison between the observed orientations and TEM analyses native tissues shows only calcium-deficient apatite (CDAP) provides an interface with collagen that is consistent with both. MD simulations predict preferred collagen orientations that agree with observations and results from both MD and DFS reveal large values for the binding energy due to multiple binding sites. These findings reconcile apparent contradictions inherent in a hydroxyapatite or carbonated apatite (CAP) model of bone mineral and provide an energetic rationale for the molecular scale organization of bone.« less

Multifunctional role of osteopontin in directing intrafibrillar mineralization of collagen and activation of osteoclasts

PubMed Central

Rodriguez, Douglas E.; Thula-Mata, Taili; Toro, Edgardo J.; Yeh, Ya-Wen; Holt, Carl; Holliday, L. Shannon; Gower, Laurie B.

2013-01-01

Mineralized collagen composites are of interest because they have the potential to provide a bone-like scaffold that stimulates the natural processes of resorption and remodeling. Working toward this goal, our group has previously shown that the nanostructure of bone can be reproduced using a polymer-induced liquid-precursor (PILP) process, which enables intrafibrillar mineralization of collagen with hydroxyapatite (HA) to be achieved. This prior work used polyaspartic acid (pASP), a simple mimic for acidic non-collagenous proteins (NCPs), to generate nanodroplets/nanoparticles of an amorphous mineral precursor which can infiltrate the interstices of type-I collagen fibrils. In this study we show that osteopontin (OPN) can similarly serve as a process-directing agent for the intrafibrillar mineralization of collagen, even though OPN is generally considered a mineralization inhibitor. We also found that inclusion of OPN in the mineralization process promotes the interaction of mouse marrow-derived osteoclasts with PILP-remineralized bone that was previously demineralized, as measured by actin ring formation. While osteoclast activation occurred when pASP was used as the process-directing agent, using OPN resulted in a dramatic effect on osteoclast activation, presumably because of the inherent arginine-glycine-aspartate acid (RGD) ligands of OPN. By capitalizing on the multifunctionality of OPN, these studies may lead the way to producing biomimetic bone substitutes with the capability of tailorable bioresorption rates. PMID:24140612

The supramolecular structure of bone: X-ray scattering analysis and lateral structure modeling

PubMed Central

Zhou, Hong-Wen; Burger, Christian; Wang, Hao; Hsiao, Benjamin S.; Chu, Benjamin; Graham, Lila

2016-01-01

The evolution of vertebrates required a key development in supramolecular evolution: internally mineralized collagen fibrils. In bone, collagen molecules and mineral crystals form a nanocomposite material comparable to cast iron in tensile strength, but several times lighter and more flexible. Current understanding of the internal nanoscale structure of collagen fibrils, derived from studies of rat tail tendon (RTT), does not explain how nucleation and growth of mineral crystals can occur inside a collagen fibril. Experimental obstacles encountered in studying bone have prevented a solution to this problem for several decades. This report presents a lateral packing model for collagen molecules in bone fibrils, based on the unprecedented observation of multiple resolved equatorial reflections for bone tissue using synchrotron small-angle X-ray scattering (SAXS; ∼1 nm resolution). The deduced structure for pre-mineralized bone fibrils includes features that are not present in RTT: spatially discrete microfibrils. The data are consistent with bone microfibrils similar to pentagonal Smith microfibrils, but are not consistent with the (nondiscrete) quasi-hexagonal microfibrils reported for RTT. These results indicate that collagen fibrils in bone and tendon differ in their internal structure in a manner that allows bone fibrils, but not tendon fibrils, to internally mineralize. In addition, the unique pattern of collagen cross-link types and quantities in mineralized tissues can be can be accounted for, in structural/functional terms, based on a discrete microfibril model. PMID:27599731

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, PHYSICAL REMOVAL OF MICROBIAL CONTAMINATION AGENTS IN DRINKING WATER, ECOWATER SYSTEMS, INC., SEARS KENMORE ULTRAFILTER 500 DRINKING WATER TREATMENT SYSTEM (POU)

EPA Science Inventory

The Sears Kenmore Ultrafilter 500 RO system was tested for removal of bacteria and viruses at NSF International's Drinking Water Treatment Systems Laboratory. EcoWater Systems submitted ten units for testing, which were split into two groups of five. One group received 25 days ...

The bcl-2 knockout mouse exhibits marked changes in osteoblast phenotype and collagen deposition in bone as well as a mild growth plate phenotype

PubMed Central

BOOT-HANDFORD, R. P.; MICHAELIDIS, T. M.; HILLARBY, M. C.; ZAMBELLI, A.; DENTON, J.; HOYLAND, J. A.; FREEMONT, A. J.; GRANT, M. E.; WALLIS, G. A.

1998-01-01

Histological examination of long bones from 1-day-old bcl-2 knockout and age-matched control mice revealed no obvious differences in length of bone, growth plate architecture or stage of endochondral ossification. In 35-day-old bcl-2 knockout mice that are growth retarded or ‘dwarfed’, the proliferative zone of the growth plate appeared slightly thinner and the secondary centres of ossification less well developed than their age-matched wild-type controls. The most marked histological effects of bcl-2 ablation were on osteoblasts and bone. 35-day-old knockout mouse bones exhibited far greater numbers of osteoblasts than controls and the osteoblasts had a cuboidal phenotype in comparison with the normal flattened cell appearance. In addition, the collagen deposited by the osteoblasts in the bcl-2 knockout mouse bone was disorganized in comparison with control tissue and had a pseudo-woven appearance. The results suggest an important role for Bcl-2 in controlling osteoblast phenotype and bone deposition in vivo. PMID:10193316

Defective Endochondral Ossification-Derived Matrix and Bone Cells Alter the Lymphopoietic Niche in Collagen X Mouse Models

PubMed Central

Sweeney, Elizabeth; Roberts, Douglas; Lin, Angela; Guldberg, Robert

2013-01-01

Despite the appreciated interdependence of skeletal and hematopoietic development, the cell and matrix components of the hematopoietic niche remain to be fully defined. Utilizing mice with disrupted function of collagen X (ColX), a major hypertrophic cartilage matrix protein associated with endochondral ossification, our data identified a cytokine defect in trabecular bone cells at the chondro-osseous hematopoietic niche as a cause for aberrant B lymphopoiesis in these mice. Specifically, analysis of ColX transgenic and null mouse chondro-osseous regions via micro-computed tomography revealed an altered trabecular bone environment. Additionally, cocultures with hematopoietic and chondro-osseous cell types highlighted impaired hematopoietic support by ColX transgenic and null mouse derived trabecular bone cells. Further, cytokine arrays with conditioned media from the trabecular osteoblast cocultures suggested an aberrant hematopoietic cytokine milieu within the chondro-osseous niche of the ColX deficient mice. Accordingly, B lymphopoiesis was rescued in the ColX mouse derived trabecular osteoblast cocultures with interlukin-7, stem cell factor, and stromal derived factor-1 supplementation. Moreover, B cell development was restored in vivo after injections of interlukin-7. These data support our hypothesis that endrochondrally-derived trabecular bone cells and matrix constituents provide cytokine-rich niches for hematopoiesis. Furthermore, this study contributes to the emerging concept that niche defects may underlie certain immuno-osseous and hematopoietic disorders. PMID:23656481

Defective endochondral ossification-derived matrix and bone cells alter the lymphopoietic niche in collagen X mouse models.

PubMed

Sweeney, Elizabeth; Roberts, Douglas; Lin, Angela; Guldberg, Robert; Jacenko, Olena

2013-10-01

Despite the appreciated interdependence of skeletal and hematopoietic development, the cell and matrix components of the hematopoietic niche remain to be fully defined. Utilizing mice with disrupted function of collagen X (ColX), a major hypertrophic cartilage matrix protein associated with endochondral ossification, our data identified a cytokine defect in trabecular bone cells at the chondro-osseous hematopoietic niche as a cause for aberrant B lymphopoiesis in these mice. Specifically, analysis of ColX transgenic and null mouse chondro-osseous regions via micro-computed tomography revealed an altered trabecular bone environment. Additionally, cocultures with hematopoietic and chondro-osseous cell types highlighted impaired hematopoietic support by ColX transgenic and null mouse derived trabecular bone cells. Further, cytokine arrays with conditioned media from the trabecular osteoblast cocultures suggested an aberrant hematopoietic cytokine milieu within the chondro-osseous niche of the ColX deficient mice. Accordingly, B lymphopoiesis was rescued in the ColX mouse derived trabecular osteoblast cocultures with interlukin-7, stem cell factor, and stromal derived factor-1 supplementation. Moreover, B cell development was restored in vivo after injections of interlukin-7. These data support our hypothesis that endrochondrally-derived trabecular bone cells and matrix constituents provide cytokine-rich niches for hematopoiesis. Furthermore, this study contributes to the emerging concept that niche defects may underlie certain immuno-osseous and hematopoietic disorders.

Biomimetics of Bone Implants: The Regenerative Road.

PubMed

Brett, Elizabeth; Flacco, John; Blackshear, Charles; Longaker, Michael T; Wan, Derrick C

2017-01-01

The current strategies for healing bone defects are numerous and varied. At the core of each bone healing therapy is a biomimetic mechanism, which works to enhance bone growth. These range from porous scaffolds, bone mineral usage, collagen, and glycosaminoglycan substitutes to transplanted cell populations. Bone defects face a range of difficulty in their healing, given the composite of dense outer compact bone and blood-rich inner trabecular bone. As such, the tissue possesses a number of inherent characteristics, which may be clinically harnessed as promoters of bone healing. These include mechanical characteristics, mineral composition, native collagen content, and cellular fraction of bone. This review charts multiple biomimetic strategies to help heal bony defects in large and small osseous injury sites, with a special focus on cell transplantation.

Collagen in Human Tissues: Structure, Function, and Biomedical Implications from a Tissue Engineering Perspective

NASA Astrophysics Data System (ADS)

Balasubramanian, Preethi; Prabhakaran, Molamma P.; Sireesha, Merum; Ramakrishna, Seeram

The extracellular matrix is a complex biological structure encoded with various proteins, among which the collagen family is the most significant and abundant of all, contributing 30-35% of the whole-body protein. "Collagen" is a generic term for proteins that forms a triple-helical structure with three polypeptide chains, and around 29 types of collagen have been identified up to now. Although most of the members of the collagen family form such supramolecular structures, extensive diversity exists between each type of collagen. The diversity is not only based on the molecular assembly and supramolecular structures of collagen types but is also observed within its tissue distribution, function, and pathology. Collagens possess complex hierarchical structures and are present in various forms such as collagen fibrils (1.5-3.5 nm wide), collagen fibers (50-70 nm wide), and collagen bundles (150-250 nm wide), with distinct properties characteristic of each tissue providing elasticity to skin, softness of the cartilage, stiffness of the bone and tendon, transparency of the cornea, opaqueness of the sclera, etc. There exists an exclusive relation between the structural features of collagen in human tissues (such as the collagen composition, collagen fibril length and diameter, collagen distribution, and collagen fiber orientation) and its tissue-specific mechanical properties. In bone, a transverse collagen fiber orientation prevails in regions of higher compressive stress whereas longitudinally oriented collagen fibers correlate to higher tensile stress. The immense versatility of collagen compels a thorough understanding of the collagen types and this review discusses the major types of collagen found in different human tissues, highlighting their tissue-specific uniqueness based on their structure and mechanical function. The changes in collagen during a specific tissue damage or injury are discussed further, focusing on the many tissue engineering applications for

Effects of Different Concentrations of Collagenous Peptide from Fish Scales on Osteoblast Proliferation and Osteoclast Resorption.

PubMed

Hu, Chung-Hsuan; Yao, Chun-Hsu; Chan, Tzu-Min; Huang, Teng-Le; Sen, Yang; Huang, Chih-Yang; Ho, Chien-Yi

2016-08-31

The incidence of osteoporosis has increased among the elderly population. Establishing a model of bone remodeling for screening new drugs is critical to identify safe and effective treatments for osteoporosis. In this study, we established a platform to investigate the therapeutic effects of collagenous peptides extracted from scales of two kinds of fish, namely, sparidae and chanos. These peptides were prepared using seven concentrations of collagenous peptide: 100, 80, 60, 40, 20, 10 and 1 mg/ml. Experimental results indicated that collagenous peptides promoted the proliferation of osteoblasts and inhibited the proliferation of mature osteoclasts; the effective concentration of collagenous peptide-sparidae was 10 mg/ml and that of collagenous peptide-chanos was 40 mg/ml. These findings demonstrate that, to a certain extent, collagenous peptides extracted from fish scales can be used to prevent osteoporosis to assist bone remodeling.

Bone-Inspired Spatially Specific Piezoelectricity Induces Bone Regeneration

PubMed Central

Yu, Peng; Ning, Chengyun; Zhang, Yu; Tan, Guoxin; Lin, Zefeng; Liu, Shaoxiang; Wang, Xiaolan; Yang, Haoqi; Li, Kang; Yi, Xin; Zhu, Ye; Mao, Chuanbin

2017-01-01

The extracellular matrix of bone can be pictured as a material made of parallel interspersed domains of fibrous piezoelectric collagenous materials and non-piezoelectric non-collagenous materials. To mimic this feature for enhanced bone regeneration, a material made of two parallel interspersed domains, with higher and lower piezoelectricity, respectively, is constructed to form microscale piezoelectric zones (MPZs). The MPZs are produced using a versatile and effective laser-irradiation technique in which K0.5Na0.5NbO3 (KNN) ceramics are selectively irradiated to achieve microzone phase transitions. The phase structure of the laser-irradiated microzones is changed from a mixture of orthorhombic and tetragonal phases (with higher piezoelectricity) to a tetragonal dominant phase (with lower piezoelectricity). The microzoned piezoelectricity distribution results in spatially specific surface charge distribution, enabling the MPZs to bear bone-like microscale electric cues. Hence, the MPZs induce osteogenic differentiation of stem cells in vitro and bone regeneration in vivo even without being seeded with stem cells. The concept of mimicking the spatially specific piezoelectricity in bone will facilitate future research on the rational design of tissue regenerative materials. PMID:28900517

Investigating the potential of electrospun gelatin and collagen scaffolds for tissue engineering applications

NASA Astrophysics Data System (ADS)

Sisson, Kristin M.

Electrospinning provides an avenue to explore tissue engineering with the ability to produce nano- and micro-sized fibers in a non-woven construct with properties ideal for a tissue engineered scaffold including: small diameter fibers, which create a large surface to volume ratio, and an interconnected porous network that enables cell migration, good mechanical integrity and a three-dimensional structure. A tissue engineered scaffold also must be biocompatible, biodegradable, non-toxic and able to be sterilized. All of these requirements can be satisfied by choosing an appropriate polymer and solvent system for electrospinning. The main objective of this research is to create a non-toxic, flat, bone tissue engineered scaffold to place into a non-immune compromised mouse. The current bone tissue repair and replacement methodologies include using metal and ceramic replacements or autologous and autogenous bone grafts. Each of these has its own set of disadvantages. Autologous grafts are bone harvested in one location in a patient and used in another location. This procedure is expensive, often results in pain and infection at the replacement site, and the actual harvesting procedure can cause problems for the patient. Autogenous grafts are bone harvested in one patient and used in another patient. The shortcomings include low donor availability and the possibility of rejection of the implant. The other options include using metal and ceramics to create replacement bone. However, metals provide good mechanical stability but can fail due to infection and also have poor integration into natural tissue. Ceramics, on the other hand, are brittle and have very low tensile strength. The natural extracellular matrix (ECM) of bone consists mainly of collagen type I. Electrospun fiber diameters closely resemble those of the natural ECM of bone. Thus, electrospinning a natural polymer like collagen type I for bone tissue engineering could make sense. Applications for these

Multiscale Homogenization Theory: An Analysis Tool for Revealing Mechanical Design Principles in Bone and Bone Replacement Materials

NASA Astrophysics Data System (ADS)

Hellmich, Christian; Fritsch, Andreas; Dormieux, Luc

Biomimetics deals with the application of nature-made "design solutions" to the realm of engineering. In the quest to understand mechanical implications of structural hierarchies found in biological materials, multiscale mechanics may hold the key to understand "building plans" inherent to entire material classes, here bone and bone replacement materials. Analyzing a multitude of biophysical hierarchical and biomechanical experiments through homogenization theories for upscaling stiffness and strength properties reveals the following design principles: The elementary component "collagen" induces, right at the nanolevel, the mechanical anisotropy of bone materials, which is amplified by fibrillar collagen-based structures at the 100-nm scale, and by pores in the micrometer-to-millimeter regime. Hydroxyapatite minerals are poorly organized, and provide stiffness and strength in a quasi-brittle manner. Water layers between hydroxyapatite crystals govern the inelastic behavior of the nanocomposite, unless the "collagen reinforcement" breaks. Bone replacement materials should mimic these "microstructural mechanics" features as closely as possible if an imitation of the natural form of bone is desired (Gebeshuber et al., Adv Mater Res 74:265-268, 2009).

Low-Temperature Additive Manufacturing of Biomimic Three-Dimensional Hydroxyapatite/Collagen Scaffolds for Bone Regeneration.

PubMed

Lin, Kai-Feng; He, Shu; Song, Yue; Wang, Chun-Mei; Gao, Yi; Li, Jun-Qin; Tang, Peng; Wang, Zheng; Bi, Long; Pei, Guo-Xian

2016-03-23

Low-temperature additive manufacturing (AM) holds promise for fabrication of three-dimensional (3D) scaffolds containing bioactive molecules and/or drugs. Due to the strict technical limitations of current approaches, few materials are suitable for printing at low temperature. Here, a low-temperature robocasting method was employed to print biomimic 3D scaffolds for bone regeneration using a routine collagen-hydroxyapatite (CHA) composite material, which is too viscous to be printed via normal 3D printing methods at low temperature. The CHA scaffolds had excellent 3D structure and maintained most raw material properties after printing. Compared to nonprinted scaffolds, printed scaffolds promoted bone marrow stromal cell proliferation and improved osteogenic outcome in vitro. In a rabbit femoral condyle defect model, the interconnecting pores within the printed scaffolds facilitated cell penetration and mineralization before the scaffolds degraded and enhanced repair, compared to nonprinted CHA scaffolds. Additionally, the optimal printing parameters for 3D CHA scaffolds were investigated; 600-μm-diameter rods were optimal in terms of moderate mechanical strength and better repair outcome in vivo. This low-temperature robocasting method could enable a variety of bioactive molecules to be incorporated into printed CHA materials and provides a method of bioprinting biomaterials without compromising their natural properties.

Clay-Enriched Silk Biomaterials for Bone Formation

PubMed Central

Mieszawska, Aneta J.; Llamas, Jabier Gallego; Vaiana, Christopher A.; Kadakia, Madhavi P.; Naik, Rajesh R.; Kaplan, David L.

2011-01-01

The formation of silk protein/clay composite biomaterials for bone tissue formation is described. Silk fibroin serves as an organic scaffolding material offering mechanical stability suitable for bone specific uses. Clay montmorillonite (Cloisite ® Na+) and sodium silicate are sources of osteoinductive silica-rich inorganic species, analogous to bioactive bioglass-like bone repair biomaterial systems. Different clay particle-silk composite biomaterial films were compared to silk films doped with sodium silicate as controls for support of human bone marrow derived mesenchymal stem cells (hMSCs) in osteogenic culture. The cells adhered and proliferated on the silk/clay composites over two weeks. Quantitative real-time RT-PCR analysis revealed increased transcript levels for alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col I) osteogenic markers in the cells cultured on the silk/clay films in comparison to the controls. Early evidence for bone formation based on collagen deposition at the cell-biomaterial interface was also found, with more collagen observed for the silk films with higher contents of clay particles. The data suggest that the silk/clay composite systems may be useful for further study toward bone regenerative needs. PMID:21549864

Collagen type I as a ligand for receptor-mediated signaling

NASA Astrophysics Data System (ADS)

Boraschi-Diaz, Iris; Wang, Jennifer; Mort, John S.; Komarova, Svetlana V.

2017-05-01

Collagens form the fibrous component of the extracellular matrix in all multi-cellular animals. Collagen type I is the most abundant collagen present in skin, tendons, vasculature, as well as the organic portion of the calcified tissue of bone and teeth. This review focuses on numerous receptors for which collagen acts as a ligand, including integrins, discoidin domain receptors DDR1 and 2, OSCAR, GPVI, G6b-B and Lair-1 of the leukocyte receptor complex and mannose family receptor uPARAP/Endo 180. We explore the process of collagen production and self-assembly, as well as its degradation by collagenases and gelatinases in order to predict potential temporal and spatial sites of action of different collagen receptors. While the interactions of the mature collagen matrix with integrins and DDR are well-appreciated, potential signals from immature matrix as well as collagen degradation products are possible but not yet described. The role of multiple collagen receptors in physiological processes and their contribution to pathophysiology of diseases affecting collagen homeostasis require further studies.

Comparison of thermal properties of fish collagen and bovine collagen in the temperature range 298-670K.

PubMed

Gauza-Włodarczyk, Marlena; Kubisz, Leszek; Mielcarek, Sławomir; Włodarczyk, Dariusz

2017-11-01

The increased interest in fish collagen is a consequence of the risk of exposure to Creutzfeld-Jacob disease (CJD) and the bovine spongiform encephalopathy (BSE), whose occurrence is associated with prions carried by bovine collagen. Collagen is the main biopolymer in living organisms and the main component of the skin and bones. Until the discovery of the BSE, bovine collagen had been widely used. The BSE epidemic increased the interest in new sources of collagen such as fish skin collagen (FSC) and its properties. Although the thermal properties of collagen originating from mammals have been well described, less attention has been paid to the thermal properties of FSC. Denaturation temperature is a particularly important parameter, depending on the collagen origin and hydration level. In the reported experiment, the free water and bound water release processes along with thermal denaturation process were studied by means of the differential scanning calorimetry (DSC). Measurements were carried out using a DSC 7 instrument (Elmer-Perkin), in the temperature range 298-670K. The study material was FSC derived by acidic hydration method. The bovine Achilles tendon (BAT) collagen type I was used as the control material. The thermograms recorded revealed both, exothermic and endothermic peaks. For both materials, the peaks in the temperature range of 330-360K were assigned to the release of free water and bound water. The denaturation temperatures of FSC and BAT collagen were determined as 420K and 493K, respectively. Thermal decomposition process was observed at about 500K for FSC and at about 510K for BAT collagen. These results show that FSC is less resistant to high temperature than BAT collagen. Copyright © 2017 Elsevier B.V. All rights reserved.

Collagen cross-link excretion during space flight and bed rest

NASA Technical Reports Server (NTRS)

Smith, S. M.; Nillen, J. L.; Leblanc, A.; Lipton, A.; Demers, L. M.; Lane, H. W.; Leach, C. S.; LeBlanc, A. (Principal Investigator)

1998-01-01

Extended exposure to weightlessness results in bone loss. However, little information exists as to the precise nature or time course of this bone loss. Bone resorption results in the release of collagen breakdown products, including N-telopeptide and the pyridinium (PYD) cross-links, pyridinoline and deoxypyridinoline. Urinary pyridinoline and deoxypyridinoline are known to increase during bed rest. We assessed excretion of PYD cross-links and N-telopeptide before, during, and after long (28-day, 59-day, and 84-day) Skylab missions, as well as during short (14-day) and long (119-day) bed-rest studies. During space flight, the urinary cross-link excretion level was twice those observed before flight. Urinary excretion levels of the collagen breakdown products were also 40-50% higher, during short and long bed rest, than before. These results clearly show that the changes in bone metabolism associated with space flight involve increased resorption. The rate of response (i.e. within days to weeks) suggests that alterations in bone metabolism are an early effect of weightlessness. These studies are important for a better understanding of bone metabolism in space crews and in those who are bedridden.

Hydroxyapatite reinforced collagen scaffolds with improved architecture and mechanical properties.

PubMed

Kane, Robert J; Weiss-Bilka, Holly E; Meagher, Matthew J; Liu, Yongxing; Gargac, Joshua A; Niebur, Glen L; Wagner, Diane R; Roeder, Ryan K

2015-04-01

Hydroxyapatite (HA) reinforced collagen scaffolds have shown promise for synthetic bone graft substitutes and tissue engineering scaffolds. Freeze-dried HA-collagen scaffolds are readily fabricated and have exhibited osteogenicity in vivo, but are limited by an inherent scaffold architecture that results in a relatively small pore size and weak mechanical properties. In order to overcome these limitations, HA-collagen scaffolds were prepared by compression molding HA reinforcements and paraffin microspheres within a suspension of concentrated collagen fibrils (∼ 180 mg/mL), cross-linking the collagen matrix, and leaching the paraffin porogen. HA-collagen scaffolds exhibited an architecture with high porosity (85-90%), interconnected pores ∼ 300-400 μm in size, and struts ∼ 3-100 μm in thickness containing 0-80 vol% HA whisker or powder reinforcements. HA reinforcement enabled a compressive modulus of up to ∼ 1 MPa, which was an order of magnitude greater than unreinforced collagen scaffolds. The compressive modulus was also at least one order of magnitude greater than comparable freeze-dried HA-collagen scaffolds and two orders of magnitude greater than absorbable collagen sponges used clinically. Moreover, scaffolds reinforced with up to 60 vol% HA exhibited fully recoverable elastic deformation upon loading to 50% compressive strain for at least 100,000 cycles. Thus, the scaffold mechanical properties were well-suited for surgical handling, fixation, and bearing osteogenic loads during bone regeneration. The scaffold architecture, permeability, and composition were shown to be conducive to the infiltration and differentiation of adipose-derive stromal cells in vitro. Acellular scaffolds were demonstrated to induce angiogenesis and osteogenesis after subcutaneous ectopic implantation by recruiting endogenous cell populations, suggesting that the scaffolds were osteoinductive. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights

Evaluation of maxillary alveolar reconstruction using a resorbable collagen sponge with recombinant human bone morphogenetic protein-2 in cleft lip and palate patients.

PubMed

Alonso, Nivaldo; Tanikawa, Daniela Yukie Sakai; Freitas, Renato da Silva; Canan, Lady; Ozawa, Terumi Okada; Rocha, Diógenes Laércio

2010-10-01

A resorbable collagen matrix with recombinant human bone morphogenetic protein (rhBMP-2) was compared with traditional iliac crest bone graft for the closure of alveolar defects during secondary dental eruption. Sixteen patients with unilateral cleft lip and palate, aged 8 to 12 years, were selected and randomly assigned to group 1 (rhBMP-2) or group 2 (iliac crest bone graft). Computed tomography was performed to assess both groups preoperatively and at months 6 and 12 postoperatively. Bone height and defect volume were calculated through Osirix Dicom Viewer (Pixmeo, Apple Inc.). Overall morbidity was recorded. Preoperative and follow-up examinations revealed progressive alveolar bone union in all patients. For group 1, final completion of the defect with a 65.0% mean bone height was detected 12 months postoperatively. For group 2, final completion of the defect with an 83.8% mean bone height was detected 6 months postoperatively. Dental eruption routinely occurred in both groups. Clinical complications included significant swelling in three group 1 patients (37.5%) and significant donor-site pain in seven group 2 patients (87.5%). For this select group of patients with immature skeleton, rhBMP-2 therapy resulted in satisfactory bone healing and reduced morbidity compared with traditional iliac crest bone grafting.

Ribose mediated crosslinking of collagen-hydroxyapatite hybrid scaffolds for bone tissue regeneration using biomimetic strategies.

PubMed

Krishnakumar, Gopal Shankar; Gostynska, Natalia; Campodoni, Elisabetta; Dapporto, Massimiliano; Montesi, Monica; Panseri, Silvia; Tampieri, Anna; Kon, Elizaveta; Marcacci, Maurilio; Sprio, Simone; Sandri, Monica

2017-08-01

This study explores for the first time the application of ribose as a highly biocompatible agent for the crosslinking of hybrid mineralized constructs, obtained by bio-inspired mineralization of self-assembling Type I collagen matrix with magnesium-doped-hydroxyapatite nanophase, towards a biomimetic mineralized 3D scaffolds (MgHA/Coll) with excellent compositional and structural mimicry of bone tissue. To this aim, two different crosslinking mechanisms in terms of pre-ribose glycation (before freeze drying) and post-ribose glycation (after freeze drying) were investigated. The obtained results explicate that with controlled freeze-drying, highly anisotropic porous structures with opportune macro-micro porosity are obtained. The physical-chemical features of the scaffolds characterized by XRD, FTIR, ICP and TGA demonstrated structural mimicry analogous to the native bone. The influence of ribose greatly assisted in decreasing solubility and increased enzymatic resistivity of the scaffolds. In addition, enhanced mechanical behaviour in response to compressive forces was achieved. Preliminary cell culture experiments reported good cytocompatibility with extensive cell adhesion, proliferation and colonization. Overall, scaffolds developed by pre-ribose glycation process are preferred, as the related crosslinking technique is more facile and robust to obtain functional scaffolds. As a proof of concept, we have demonstrated that ribose crosslinking is cost-effective, safe and functionally effective. This study also offers new insights and opportunities in developing promising scaffolds for bone tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

Ascorbic acid induces alkaline phosphatase, type X collagen, and calcium deposition in cultured chick chondrocytes.

PubMed

Leboy, P S; Vaias, L; Uschmann, B; Golub, E; Adams, S L; Pacifici, M

1989-10-15

During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic chondrocytes, which then deposit a mineralized matrix to form calcified cartilage. Chondrocyte hypertrophy and matrix mineralization are associated with expression of type X collagen and the induction of high levels of the bone/liver/kidney isozyme of alkaline phosphatase. To determine what role vitamin C plays in these processes, chondrocytes derived from the cephalic portion of 14-day chick embryo sternae were grown in the absence or presence of exogenous ascorbic acid. Control untreated cells displayed low levels of type X collagen and alkaline phosphatase activity throughout the culture period. However, cells grown in the presence of ascorbic acid produced increasing levels of alkaline phosphatase activity and type X collagen mRNA and protein. Both alkaline phosphatase activity and type X collagen mRNA levels began to increase within 24 h of ascorbate treatment; by 9 days, the levels of both alkaline phosphatase activity and type X collagen mRNA were 15-20-fold higher than in non-ascorbate-treated cells. Ascorbate treatment also increased calcium deposition in the cell layer and decreased the levels of types II and IX collagen mRNAs; these effects lagged significantly behind the elevation of alkaline phosphatase and type X collagen. Addition of beta-glycerophosphate to the medium increased calcium deposition in the presence of ascorbate but had no effect on levels of collagen mRNAs or alkaline phosphatase. The results suggest that vitamin C may play an important role in endochondral bone formation by modulating gene expression in hypertrophic chondrocytes.

Contributions of Raman spectroscopy to the understanding of bone strength.

PubMed

Mandair, Gurjit S; Morris, Michael D

2015-01-01

Raman spectroscopy is increasingly commonly used to understand how changes in bone composition and structure influence tissue-level bone mechanical properties. The spectroscopic technique provides information on bone mineral and matrix collagen components and on the effects of various matrix proteins on bone material properties as well. The Raman spectrum of bone not only contains information on bone mineral crystallinity that is related to bone hardness but also provides information on the orientation of mineral crystallites with respect to the collagen fibril axis. Indirect information on collagen cross-links is also available and will be discussed. After a short introduction to bone Raman spectroscopic parameters and collection methodologies, advances in in vivo Raman spectroscopic measurements for animal and human subject studies will be reviewed. A discussion on the effects of aging, osteogenesis imperfecta, osteoporosis and therapeutic agents on bone composition and mechanical properties will be highlighted, including genetic mouse models in which structure-function and exercise effects are explored. Similarly, extracellular matrix proteins, proteases and transcriptional proteins implicated in the regulation of bone material properties will be reviewed.

Molecular crowding of collagen: a pathway to produce highly-organized collagenous structures.

PubMed

Saeidi, Nima; Karmelek, Kathryn P; Paten, Jeffrey A; Zareian, Ramin; DiMasi, Elaine; Ruberti, Jeffrey W

2012-10-01

Collagen in vertebrate animals is often arranged in alternating lamellae or in bundles of aligned fibrils which are designed to withstand in vivo mechanical loads. The formation of these organized structures is thought to result from a complex, large-area integration of individual cell motion and locally-controlled synthesis of fibrillar arrays via cell-surface fibripositors (direct matrix printing). The difficulty of reproducing such a process in vitro has prevented tissue engineers from constructing clinically useful load-bearing connective tissue directly from collagen. However, we and others have taken the view that long-range organizational information is potentially encoded into the structure of the collagen molecule itself, allowing the control of fibril organization to extend far from cell (or bounding) surfaces. We here demonstrate a simple, fast, cell-free method capable of producing highly-organized, anistropic collagen fibrillar lamellae de novo which persist over relatively long-distances (tens to hundreds of microns). Our approach to nanoscale organizational control takes advantage of the intrinsic physiochemical properties of collagen molecules by inducing collagen association through molecular crowding and geometric confinement. To mimic biological tissues which comprise planar, aligned collagen lamellae (e.g. cornea, lamellar bone or annulus fibrosus), type I collagen was confined to a thin, planar geometry, concentrated through molecular crowding and polymerized. The resulting fibrillar lamellae show a striking resemblance to native load-bearing lamellae in that the fibrils are small, generally aligned in the plane of the confining space and change direction en masse throughout the thickness of the construct. The process of organizational control is consistent with embryonic development where the bounded planar cell sheets produced by fibroblasts suggest a similar confinement/concentration strategy. Such a simple approach to nanoscale

Type I Collagen and Strontium-Containing Mesoporous Glass Particles as Hybrid Material for 3D Printing of Bone-Like Materials.

PubMed

Montalbano, Giorgia; Fiorilli, Sonia; Caneschi, Andrea; Vitale-Brovarone, Chiara

2018-04-28

Bone tissue engineering offers an alternative promising solution to treat a large number of bone injuries with special focus on pathological conditions, such as osteoporosis. In this scenario, the bone tissue regeneration may be promoted using bioactive and biomimetic materials able to direct cell response, while the desired scaffold architecture can be tailored by means of 3D printing technologies. In this context, our study aimed to develop a hybrid bioactive material suitable for 3D printing of scaffolds mimicking the natural composition and structure of healthy bone. Type I collagen and strontium-containing mesoporous bioactive glasses were combined to obtain suspensions able to perform a sol-gel transition under physiological conditions. Field emission scanning electron microscopy (FESEM) analyses confirmed the formation of fibrous nanostructures homogeneously embedding inorganic particles, whereas bioactivity studies demonstrated the large calcium phosphate deposition. The high-water content promoted the strontium ion release from the embedded glass particles, potentially enhancing the osteogenic behaviour of the composite. Furthermore, the suspension printability was assessed by means of rheological studies and preliminary extrusion tests, showing shear thinning and fast material recovery upon deposition. In conclusion, the reported results suggest that promising hybrid systems suitable for 3D printing of bioactive scaffolds for bone tissue engineering have been developed.

Morphological assessment of bone mineralization in tibial metaphyses of ascorbic acid-deficient ODS rats.

PubMed

Hasegawa, Tomoka; Li, Minqi; Hara, Kuniko; Sasaki, Muneteru; Tabata, Chihiro; de Freitas, Paulo Henrique Luiz; Hongo, Hiromi; Suzuki, Reiko; Kobayashi, Masatoshi; Inoue, Kiichiro; Yamamoto, Tsuneyuki; Oohata, Noboru; Oda, Kimimitsu; Akiyama, Yasuhiro; Amizuka, Norio

2011-08-01

Osteogenic disorder shionogi (ODS) rats carry a hereditary defect in ascorbic acid synthesis, mimicking human scurvy when fed with an ascorbic acid-deficient (aa-def) diet. As aa-def ODS rats were shown to feature disordered bone formation, we have examined the bone mineralization in this rat model. A fibrous tissue layer surrounding the trabeculae of tibial metaphyses was found in aa-def ODS rats, and this layer showed intense alkaline phosphatase activity and proliferating cell nuclear antigen-immunopositivity. Many osteoblasts detached from the bone surfaces and were characterized by round-shaped rough endoplasmic reticulum (rER), suggesting accumulation of malformed collagen inside the rER. Accordingly, fine, fragile fibrillar collagenous structures without evident striation were found in aa-def bones, which may result from misassembling of the triple helices of collagenous α-chains. Despite a marked reduction in bone formation, ascorbic acid deprivation seemed to have no effect on mineralization: while reduced in number, normal matrix vesicles and mineralized nodules could be seen in aa-def bones. Fine needle-like mineral crystals extended from these mineralized nodules, and were apparently bound to collagenous fibrillar structures. In summary, collagen mineralization seems unaffected by ascorbic acid deficiency in spite of the fine, fragile collagenous fibrils identified in the bones of our animal model.

Polarization sensitive optical coherence tomography in equine bone

NASA Astrophysics Data System (ADS)

Jacobs, J. W.; Matcher, S. J.

2009-02-01

Optical coherence tomography (OCT) has been used to image equine bone samples. OCT and polarization sensitive OCT (PS-OCT) images of equine bone samples, before and after demineralization, are presented. Using a novel approach, taking a series of images at different angles of illumination, the polar angle and true birefringence of collagen within the tissue is determined, at one site in the sample. The images were taken before and after the bones were passed through a demineralization process. The images show an improvement in depth penetration after demineralization allowing better visualization of the internal structure of the bone and the optical orientation of the collagen. A quantitative measurement of true birefringence has been made of the bone; true birefringence was shown to be 1.9x10-3 before demineralization increasing to 2.7x10-3 after demineralization. However, determined collagen fiber orientation remains the same before and after demineralization. The study of bone is extensive within the field of tissue engineering where an understanding of the internal structures is essential. OCT in bone, and improved depth penetration through demineralization, offers a useful approach to bone analysis.

Cortical bone growth and maturational changes in dwarf rats induced by recombinant human growth hormone

NASA Technical Reports Server (NTRS)

Martinez, D. A.; Orth, M. W.; Carr, K. E.; Vanderby, R. Jr; Vailas, A. C.

1996-01-01

The growth hormone (GH)-deficient dwarf rat was used to investigate recombinant human (rh) GH-induced bone formation and to determine whether rhGH facilitates simultaneous increases in bone formation and bone maturation during rapid growth. Twenty dwarf rats, 37 days of age, were randomly assigned to dwarf plus rhGH (GH; n = 10) and dwarf plus vehicle (n = 10) groups. The GH group received 1.25 mg rhGH/kg body wt two times daily for 14 days. Biochemical, morphological, and X-ray diffraction measurements were performed on the femur middiaphysis. rhGH stimulated new bone growth in the GH group, as demonstrated by significant increases (P < 0.05) in longitudinal bone length (6%), middiaphyseal cross-sectional area (20%), and the amount of newly accreted bone collagen (28%) in the total pool of middiaphyseal bone collagen. Cortical bone density, mean hydroxyapatite crystal size, and the calcium and collagen contents (microgram/mm3) were significantly smaller in the GH group (P < 0.05). Our findings suggest that the processes regulating new collagen accretion, bone collagen maturation, and mean hydroxyapatite crystal size may be independently regulated during rapid growth.

Functional adaptation of long bone extremities involves the localized ``tuning'' of the cortical bone composition; evidence from Raman spectroscopy

NASA Astrophysics Data System (ADS)

Buckley, Kevin; Kerns, Jemma G.; Birch, Helen L.; Gikas, Panagiotis D.; Parker, Anthony W.; Matousek, Pavel; Goodship, Allen E.

2014-11-01

In long bones, the functional adaptation of shape and structure occurs along the whole length of the organ. This study explores the hypothesis that adaptation of bone composition is also site-specific and that the mineral-to-collagen ratio of bone (and, thus, its mechanical properties) varies along the organ's length. Raman spectroscopy was used to map the chemical composition of long bones along their entire length in fine spatial resolution (1 mm), and then biochemical analysis was used to measure the mineral, collagen, water, and sulfated glycosaminoglycan content where site-specific differences were seen. The results show that the mineral-to-collagen ratio of the bone material in human tibiae varies by <5% along the mid-shaft but decreases by >10% toward the flared extremities of the bone. Comparisons with long bones from other large animals (horses, sheep, and deer) gave similar results with bone material composition changing across tens of centimeters. The composition of the bone apatite also varied with the phosphate-to-carbonate ratio decreasing toward the ends of the tibia. The data highlight the complexity of adaptive changes and raise interesting questions about the biochemical control mechanisms involved. In addition to their biological interest, the data provide timely information to researchers developing Raman spectroscopy as a noninvasive tool for measuring bone composition in vivo (particularly with regard to sampling and measurement protocol).

Functional adaptation of long bone extremities involves the localized "tuning" of the cortical bone composition; evidence from Raman spectroscopy.

PubMed

Buckley, Kevin; Kerns, Jemma G; Birch, Helen L; Gikas, Panagiotis D; Parker, Anthony W; Matousek, Pavel; Goodship, Allen E

2014-01-01

In long bones, the functional adaptation of shape and structure occurs along the whole length of the organ. This study explores the hypothesis that adaptation of bone composition is also site-specific and that the mineral-to-collagen ratio of bone (and, thus, its mechanical properties) varies along the organ's length. Raman spectroscopy was used to map the chemical composition of long bones along their entire length in fine spatial resolution (1 mm), and then biochemical analysis was used to measure the mineral, collagen, water, and sulfated glycosaminoglycan content where site-specific differences were seen. The results show that the mineral-to-collagen ratio of the bone material in human tibiae varies by <5% along the mid-shaft but decreases by >10% toward the flared extremities of the bone. Comparisons with long bones from other large animals (horses, sheep, and deer) gave similar results with bone material composition changing across tens of centimeters. The composition of the bone apatite also varied with the phosphate-to-carbonate ratio decreasing toward the ends of the tibia. The data highlight the complexity of adaptive changes and raise interesting questions about the biochemical control mechanisms involved. In addition to their biological interest, the data provide timely information to researchers developing Raman spectroscopy as a noninvasive tool for measuring bone composition in vivo (particularly with regard to sampling and measurement protocol).

Peroxidase Enzymes Regulate Collagen Biosynthesis and Matrix Mineralization by Cultured Human Osteoblasts.

PubMed

DeNichilo, Mark O; Shoubridge, Alexandra J; Panagopoulos, Vasilios; Liapis, Vasilios; Zysk, Aneta; Zinonos, Irene; Hay, Shelley; Atkins, Gerald J; Findlay, David M; Evdokiou, Andreas

2016-03-01

The early recruitment of inflammatory cells to sites of bone fracture and trauma is a critical determinant in successful fracture healing. Released by infiltrating inflammatory cells, myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, whose functional involvement in bone repair has mainly been studied in the context of providing a mechanism for oxidative defense against invading microorganisms. We report here novel findings that show peroxidase enzymes have the capacity to stimulate osteoblastic cells to secrete collagen I protein and generate a mineralized extracellular matrix in vitro. Mechanistic studies conducted using cultured osteoblasts show that peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl hydroxylase-dependent manner, which does not require ascorbic acid. Our studies demonstrate that osteoblasts rapidly bind and internalize both MPO and EPO, and the catalytic activity of these peroxidase enzymes is essential to support collagen I biosynthesis and subsequent release of collagen by osteoblasts. We show that EPO is capable of regulating osteogenic gene expression and matrix mineralization in culture, suggesting that peroxidase enzymes may play an important role not only in normal bone repair, but also in the progression of pathological states where infiltrating inflammatory cells are known to deposit peroxidases.

Improving the permeability of lyophilized collagen-hydroxyapatite scaffolds for cell-based bone regeneration with a gelatin porogen.

PubMed

Villa, Max M; Wang, Liping; Huang, Jianping; Rowe, David W; Wei, Mei

2016-11-01

Bone tissue engineering using biomaterial scaffolds and culture-expanded osteoprogenitor cells has been demonstrated in several studies; however, it is not yet a clinical reality. One challenge is the optimal design of scaffolds for cell delivery and the identification of scaffold parameters that can delineate success and failure in vivo. Motivated by a previous experiment in which a batch of lyophilized collagen-hydroxyapatite (HA) scaffolds displayed modest bone formation in vivo, despite having large pores and high porosity, we began to investigate the effect of scaffold permeability on bone formation. Herein, we fabricated scaffolds with a permeability of 2.17 ± 1.63 × 10 -9 m 4 /(N s) and fourfold higher using a sacrificial gelatin porogen. Scaffolds were seeded with mouse bone marrow stromal cells carrying a fluorescent reporter for osteoblast differentiation and implanted into critical-size calvarial defects in immunodeficient mice. The porogen scaffold group containing a 1:1 ratio of solids to beads was significantly more radiopaque than the scaffold group without the bead porogen 3 weeks after implantation. Quantitative histomorphometry uncovered the same trend between the 1:1 group and scaffolds without porogen found in the radiographic data; however, this was not statistically significant here. Taken together, the X-ray and histology suggest that the 1:1 ratio of porogen to scaffold solids, resulting in a fourfold increase in permeability, may enhance bone formation when compared to scaffolds without porogen. Scaffold permeability can be a useful quality control measure before implantation and this practice should improve the consistency and efficacy of cell-based bone tissue engineering. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1580-1590, 2016. © 2015 Wiley Periodicals, Inc.

Molecular dynamics simulations on networks of heparin and collagen.

PubMed

Kulke, Martin; Geist, Norman; Friedrichs, Wenke; Langel, Walter

2017-06-01

Synthetic scaffolds containing collagen (Type I) are of increasing interest for bone tissue engineering, especially for highly porous biomaterials in combination with glycosaminoglycans. In experiments the integration of heparin during the fibrillogenesis resulted in different types of collagen fibrils, but models for this aggregation on a molecular scale were only tentative. We conducted molecular dynamic simulations investigating the binding of heparin to collagen and the influence of the telopeptides during collagen aggregation. This aims at explaining experimental findings on a molecular level. Novel structures for N- and C-telopeptides were developed with the TIGER2 replica exchange algorithm and dihedral principle component analysis. We present an extended statistical analysis of the mainly electrostatic interaction between heparin and collagen and identify several binding sites. Finally, we propose a molecular mechanism for the influence of glycosaminoglycans on the morphology of collagen fibrils. Proteins 2017; 85:1119-1130. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Tomographic imaging of bone composition using coherently scattered x rays

NASA Astrophysics Data System (ADS)

Batchelar, Deidre L.; Dabrowski, W.; Cunningham, Ian A.

2000-04-01

Bone tissue consists primarily of calcium hydroxyapatite crystals (bone mineral) and collagen fibrils. Bone mineral density (BMD) is commonly used as an indicator of bone health. Techniques available at present for assessing bone health provide a measure of BMD, but do not provide information about the degree of mineralization of the bone tissue. This may be adequate for assessing diseases in which the collagen-mineral ratio remains constant, as assumed in osteoporosis, but is insufficient when the mineralization state is known to change, as in osteomalacia. No tool exists for the in situ examination of collagen and hydroxyapatite density distributions independently. Coherent-scatter computed tomography (CSCT) is a technique we are developing that produces images of the low- angle scatter properties of tissue. These depend on the molecular structure of the scatterer making it possible to produce material-specific maps of each component in a conglomerate. After corrections to compensate for exposure fluctuations, self-attenuation of scatter and the temporal response of the image intensifier, material-specific images of mineral, collagen, fat and water distributions are obtained. The gray-level in these images provides the volumetric density of each component independently.

Combination of Root Surface Modification with BMP-2 and Collagen Hydrogel Scaffold Implantation for Periodontal Healing in Beagle Dogs

PubMed Central

Kato, Akihito; Miyaji, Hirofumi; Ishizuka, Ryosuke; Tokunaga, Keisuke; Inoue, Kana; Kosen, Yuta; Yokoyama, Hiroyuki; Sugaya, Tsutomu; Tanaka, Saori; Sakagami, Ryuji; Kawanami, Masamitsu

2015-01-01

Objective : Biomodification of the root surface plays a major role in periodontal wound healing. Root surface modification with bone morphogenetic protein (BMP) stimulates bone and cementum-like tissue formation; however, severe ankylosis is simultaneously observed. Bio-safe collagen hydrogel scaffolds may therefore be useful for supplying periodontal ligament cells and preventing ankylosis. We examined the effects of BMP modification in conjunction with collagen hydrogel scaffold implantation on periodontal wound healing in dogs. Material and Methods: The collagen hydrogel scaffold was composed of type I collagen sponge and collagen hydrogel. One-wall infrabony defects (5 mm in depth, 3 mm in width) were surgically created in six beagle dogs. In the BMP/Col group, BMP-2 was applied to the root surface (loading dose; 1 µg/µl), and the defects were filled with collagen hydrogel scaffold. In the BMP or Col group, BMP-2 coating or scaffold implantation was performed. Histometric parameters were evaluated at 4 weeks after surgery. Results: Single use of BMP stimulated formation of alveolar bone and ankylosis. In contrast, the BMP/Col group frequently enhanced reconstruction of periodontal attachment including cementum-like tissue, periodontal ligament and alveolar bone. The amount of new periodontal ligament in the BMP/Col group was significantly greater when compared to all other groups. In addition, ankylosis was rarely observed in the BMP/Col group. Conclusion: The combination method using root surface modification with BMP and collagen hydrogel scaffold implantation facilitated the reestablishment of periodontal attachment. BMP-related ankylosis was suppressed by implantation of collagen hydrogel. PMID:25674172

Evaluation of Anterior Vertebral Interbody Fusion Using Osteogenic Mesenchymal Stem Cells Transplanted in Collagen Sponge.

PubMed

Yang, Wencheng; Dong, Youhai; Hong, Yang; Guang, Qian; Chen, Xujun

2016-05-01

The study used a rabbit model to achieve anterior vertebral interbody fusion using osteogenic mesenchymal stem cells (OMSCs) transplanted in collagen sponge. We investigated the effectiveness of graft material for anterior vertebral interbody fusion using a rabbit model by examining the OMSCs transplanted in collagen sponge. Anterior vertebral interbody fusion is commonly performed. Although autogenous bone graft remains the gold-standard fusion material, it requires a separate surgical procedure and is associated with significant short-term and long-term morbidity. Recently, mesenchymal stem cells from bone marrow have been studied in various fields, including posterolateral spinal fusion. Thus, we hypothesized that cultured OMSCs transplanted in porous collagen sponge could be used successfully even in anterior vertebral interbody fusion. Forty mature male White Zealand rabbits (weight, 3.5-4.5 kg) were randomly allocated to receive one of the following graft materials: porous collagen sponge plus cultured OMSCs (group I); porous collagen sponge alone (group II); autogenous bone graft (group III); and nothing (group IV). All animals underwent anterior vertebral interbody fusion at the L4/L5 level. The lumbar spine was harvested en bloc, and the new bone formation and spinal fusion was evaluated using radiographic analysis, microcomputed tomography, manual palpation test, and histologic examination at 8 and 12 weeks after surgery. New bone formation and bony fusion was evident as early as 8 weeks in groups I and III. And there was no statistically significant difference between 8 and 12 weeks. At both time points, by microcomputed tomography and histologic analysis, new bone formation was observed in both groups I and III, fibrous tissue was observed and there was no new bone in both groups II and IV; by manual palpation test, bony fusion was observed in 40% (4/10) of rabbits in group I, 70% (7/10) of rabbits in group III, and 0% (0/10) of rabbits in both groups

Effects of mechanical repetitive load on bone quality around implants in rat maxillae

PubMed Central

Uto, Yusuke; Nakano, Takayoshi; Ishimoto, Takuya; Inaba, Nao; Uchida, Yusuke; Sawase, Takashi

2017-01-01

Greater understanding and acceptance of the new concept “bone quality”, which was proposed by the National Institutes of Health and is based on bone cells and collagen fibers, are required. The novel protein Semaphorin3A (Sema3A) is associated with osteoprotection by regulating bone cells. The aims of this study were to investigate the effects of mechanical loads on Sema3A production and bone quality based on bone cells and collagen fibers around implants in rat maxillae. Grade IV-titanium threaded implants were placed at 4 weeks post-extraction in maxillary first molars. Implants received mechanical loads (10 N, 3 Hz for 1800 cycles, 2 days/week) for 5 weeks from 3 weeks post-implant placement to minimize the effects of wound healing processes by implant placement. Bone structures, bone mineral density (BMD), Sema3A production and bone quality based on bone cells and collagen fibers were analyzed using microcomputed tomography, histomorphometry, immunohistomorphometry, polarized light microscopy and birefringence measurement system inside of the first and second thread (designated as thread A and B, respectively), as mechanical stresses are concentrated and differently distributed on the first two threads from the implant neck. Mechanical load significantly increased BMD, but not bone volume around implants. Inside thread B, but not thread A, mechanical load significantly accelerated Sema3A production with increased number of osteoblasts and osteocytes, and enhanced production of both type I and III collagen. Moreover, mechanical load also significantly induced preferential alignment of collagen fibers in the lower flank of thread B. These data demonstrate that mechanical load has different effects on Sema3A production and bone quality based on bone cells and collagen fibers between the inside threads of A and B. Mechanical load-induced Sema3A production may be differentially regulated by the type of bone structure or distinct stress distribution, resulting in

Effects of mechanical repetitive load on bone quality around implants in rat maxillae.

PubMed

Uto, Yusuke; Kuroshima, Shinichiro; Nakano, Takayoshi; Ishimoto, Takuya; Inaba, Nao; Uchida, Yusuke; Sawase, Takashi

2017-01-01

Greater understanding and acceptance of the new concept "bone quality", which was proposed by the National Institutes of Health and is based on bone cells and collagen fibers, are required. The novel protein Semaphorin3A (Sema3A) is associated with osteoprotection by regulating bone cells. The aims of this study were to investigate the effects of mechanical loads on Sema3A production and bone quality based on bone cells and collagen fibers around implants in rat maxillae. Grade IV-titanium threaded implants were placed at 4 weeks post-extraction in maxillary first molars. Implants received mechanical loads (10 N, 3 Hz for 1800 cycles, 2 days/week) for 5 weeks from 3 weeks post-implant placement to minimize the effects of wound healing processes by implant placement. Bone structures, bone mineral density (BMD), Sema3A production and bone quality based on bone cells and collagen fibers were analyzed using microcomputed tomography, histomorphometry, immunohistomorphometry, polarized light microscopy and birefringence measurement system inside of the first and second thread (designated as thread A and B, respectively), as mechanical stresses are concentrated and differently distributed on the first two threads from the implant neck. Mechanical load significantly increased BMD, but not bone volume around implants. Inside thread B, but not thread A, mechanical load significantly accelerated Sema3A production with increased number of osteoblasts and osteocytes, and enhanced production of both type I and III collagen. Moreover, mechanical load also significantly induced preferential alignment of collagen fibers in the lower flank of thread B. These data demonstrate that mechanical load has different effects on Sema3A production and bone quality based on bone cells and collagen fibers between the inside threads of A and B. Mechanical load-induced Sema3A production may be differentially regulated by the type of bone structure or distinct stress distribution, resulting in

A novel three-dimensional bone chip organ culture.

PubMed

Kuttenberger, Johannes; Polska, Elzbieta; Schaefer, Birgit M

2013-07-01

The objective of this study was to develop a 3D bone chip organ culture model. We aimed to collect in vitro evidence of the ability of vital bone chips to promote new bone formation. We developed a 3D in vitro hypoxic bone chip organ culture model. Histology of the bone chips was performed before and after culture and immunohistochemistry after 3-week culture. The 3D culture supernatants were tested for the presence of pro-angiogenic growth factors, TGFβ1, GADPH, bone alkaline phosphatase, osteocalcin, osteonectin, osteopontin, bone sialoprotein and collagen type I. Histology after culture revealed bone chips in a matrix of fibrin remnants and a fibrous-appearing matter. Collagen type I- and IV-positive structures were also identified. Cells could be seen on the surface of the bone chips, with spindle-shaped cells bridging the bone chip particles. Pro-angiogenic growth factors and TGFβ1were detected in the 3D cell culture supernatants. The transcripts for osteocalcin, bone sialoprotein and collagen type I were revealed only via PCR. Our results indicate that bone chips in our 3D organ culture remain vital and may stimulate the growth of a bone-forming matrix. The use of autogenous bone chips for oral and maxillofacial bone augmentation procedures is widespread in clinical practice. The rationale for this is that if bone chips remain vital in vivo, they could provide an environment promoting new bone formation through growth factors and cells. This 3D culture method is an essential tool for investigating the behaviour of bone chips.

Demineralized Bone Matrix Scaffolds Modified by CBD-SDF-1α Promote Bone Regeneration via Recruiting Endogenous Stem Cells.

PubMed

Shi, Jiajia; Sun, Jie; Zhang, Wen; Liang, Hui; Shi, Qin; Li, Xiaoran; Chen, Yanyan; Zhuang, Yan; Dai, Jianwu

2016-10-07

The reconstruction of bone usually depends on substitute transplantation, which has drawbacks including the limited bone substitutes available, comorbidity, immune rejection, and limited endogenous bone regeneration. Here, we constructed a functionalized bone substitute by combining application of the demineralized bone matrix (DBM) and collagen-binding stromal-cell-derived factor-1α (CBD-SDF-1α). DBM was a poriferous and biodegradable bone substitute, derived from bovine bone and consisting mainly of collagen. CBD-SDF-1α could bind to collagen and be controllably released from the DBM to mobilize stem cells. In a rat femur defect model, CBD-SDF-1α-modified DBM scaffolds could efficiently mobilize CD34 + and c-kit + endogenous stem cells homing to the injured site at 3 days after implantation. According to the data from micro-CT, CBD-SDF-1α-modified DBM scaffolds could help the bone defects rejoin with mineralization accumulated and bone volume expanded. Interestingly, osteoprotegerin (OPG) and osteopontin (OPN) were highly expressed in CBD-SDF-1α group at an early time after implantation, while osteocalcin (OCN) was more expanded. H&E and Masson's trichrome staining showed that the CBD-SDF-1α-modified DBM scaffold group had more osteoblasts and that the bone defect rejoined earlier. The ultimate strength of the regenerated bone was investigated by three-point bending, showing that the CBD-SDF-1α group had superior strength. In conclusion, CBD-SDF-1α-modified DBM scaffolds could promote bone regeneration by recruiting endogenous stem cells.

Preservation of ancient DNA in thermally damaged archaeological bone

NASA Astrophysics Data System (ADS)

Ottoni, Claudio; Koon, Hannah E. C.; Collins, Matthew J.; Penkman, Kirsty E. H.; Rickards, Olga; Craig, Oliver E.

2009-02-01

Evolutionary biologists are increasingly relying on ancient DNA from archaeological animal bones to study processes such as domestication and population dispersals. As many animal bones found on archaeological sites are likely to have been cooked, the potential for DNA preservation must be carefully considered to maximise the chance of amplification success. Here, we assess the preservation of mitochondrial DNA in a medieval cattle bone assemblage from Coppergate, York, UK. These bones have variable degrees of thermal alterations to bone collagen fibrils, indicative of cooking. Our results show that DNA preservation is not reliant on the presence of intact collagen fibrils. In fact, a greater number of template molecules could be extracted from bones with damaged collagen. We conclude that moderate heating of bone may enhance the retention of DNA fragments. Our results also indicate that ancient DNA preservation is highly variable, even within a relatively recent assemblage from contexts conducive to organic preservation, and that diagenetic parameters based on protein diagenesis are not always useful for predicting ancient DNA survival.

Complex Meniscus Tears Treated with Collagen Matrix Wrapping and Bone Marrow Blood Injection

PubMed Central

Piontek, Tomasz; Ciemniewska-Gorzela, Kinga; Naczk, Jakub; Jakob, Roland; Szulc, Andrzej; Grygorowicz, Monika; Slomczykowski, Michal

2015-01-01

Objective To collect and analyze the 2-year follow-up clinical and MRI results of patients treated with an arthroscopic technique of collagen membrane-based meniscus repair. Design 53 consecutive patients with combined (horizontal and radial or longitudinal component) and complex meniscal tears (tear extended through avascular zones or/and composed with two or more morphological tear pattern) were treated with an “all-inside” arthroscopic suture of meniscus and wrapping with a collagen membrane (Chondro-Gide) technique with bone marrow blood injection. The IKDC 2000 subjective score, IKDC 2000 clinical evaluation score, Lysholm score and Barret clinical criteria of meniscal healing were recorded. All patients were examinated by MRI 2 years postoperatively, using modified WORMS criteria for meniscal integrity. Results The 2 year follow-up was achieved in 50 cases. Of these, 2 patients were excluded from the evaluation due to incomplete data and 2 patients underwent partial meniscectomy and were classified as failures. In 46 patients (86.8% of the intended to treat cases), a statistically significant improvement in IKDC 2000 subjective, Lysholm scores and IKDC 2000 clinical assessment between preoperative and the 2-year follow-up time points were obsereved. Barret criteria demonstrated an improved clinical outcome between pre- and post-operative values. MRI revealed a non-homogeneous signal without meniscal tear (WORMS grade 1) in 76% of the operated menisci (13% WORMS grade 2). Conclusions The 2-year follow-up data demonstrate that this technique is safe and can offer an additional tool to save the meniscus in the patients otherwise scheduled for meniscal removal. Level of evidence IV PMID:27047635

Preparation and characterization of collagen-hydroxyapatite/pectin composite.

PubMed

Wenpo, Feng; Gaofeng, Liang; Shuying, Feng; Yuanming, Qi; Keyong, Tang

2015-03-01

Pectin, a kind of plant polysaccharide, was introduced into collagen-hydroxyapatite composite system, and prepared collagen-hydroxyapatite/pectin (Col-HA/pectin) composite in situ. The structure of the composite was investigated by XRD, SEM, and FT-IR. The mechanical properties, water absorption, enzyme degradation, and cytotoxicity of the composite were investigated as well. The results show that the inorganic substance in the composite materials is hydroxyapatite in relatively low crystallinity. A new interface appeared by the interaction among hydroxyapatite and collagen-pectin, and formed smooth fine particles. The mechanical properties, water absorption, enzyme degradation, and cytotoxicity indicate a potential use in bone replacement for the new composite. Copyright © 2014 Elsevier B.V. All rights reserved.

Infrared spectroscopy reveals both qualitative and quantitative differences in equine subchondral bone during maturation

NASA Astrophysics Data System (ADS)

Kobrina, Yevgeniya; Isaksson, Hanna; Sinisaari, Miikka; Rieppo, Lassi; Brama, Pieter A.; van Weeren, René; Helminen, Heikki J.; Jurvelin, Jukka S.; Saarakkala, Simo

2010-11-01

The collagen phase in bone is known to undergo major changes during growth and maturation. The objective of this study is to clarify whether Fourier transform infrared (FTIR) microspectroscopy, coupled with cluster analysis, can detect quantitative and qualitative changes in the collagen matrix of subchondral bone in horses during maturation and growth. Equine subchondral bone samples (n = 29) from the proximal joint surface of the first phalanx are prepared from two sites subjected to different loading conditions. Three age groups are studied: newborn (0 days old), immature (5 to 11 months old), and adult (6 to 10 years old) horses. Spatial collagen content and collagen cross-link ratio are quantified from the spectra. Additionally, normalized second derivative spectra of samples are clustered using the k-means clustering algorithm. In quantitative analysis, collagen content in the subchondral bone increases rapidly between the newborn and immature horses. The collagen cross-link ratio increases significantly with age. In qualitative analysis, clustering is able to separate newborn and adult samples into two different groups. The immature samples display some nonhomogeneity. In conclusion, this is the first study showing that FTIR spectral imaging combined with clustering techniques can detect quantitative and qualitative changes in the collagen matrix of subchondral bone during growth and maturation.

Multiscale imaging of bone microdamage

PubMed Central

Poundarik, Atharva A.; Vashishth, Deepak

2015-01-01

Bone is a structural and hierarchical composite that exhibits remarkable ability to sustain complex mechanical loading and resist fracture. Bone quality encompasses various attributes of bone matrix from the quality of its material components (type-I collagen, mineral and non-collagenous matrix proteins) and cancellous microarchitecture, to the nature and extent of bone microdamage. Microdamage, produced during loading, manifests in multiple forms across the scales of hierarchy in bone and functions to dissipate energy and avert fracture. Microdamage formation is a key determinant of bone quality, and through a range of biological and physical mechanisms, accumulates with age and disease. Accumulated microdamage in bone decreases bone strength and increases bone’s propensity to fracture. Thus, a thorough assessment of microdamage, across the hierarchical levels of bone, is crucial to better understand bone quality and bone fracture. This review article details multiple imaging modalities that have been used to study and characterize microdamage; from bulk staining techniques originally developed by Harold Frost to assess linear microcracks, to atomic force microscopy, a modality that revealed mechanistic insights into the formation diffuse damage at the ultrastructural level in bone. New automated techniques using imaging modalities such as microcomputed tomography are also presented for a comprehensive overview. PMID:25664772

Polarization Raman spectroscopy to explain rodent models of brittle bone

NASA Astrophysics Data System (ADS)

Makowski, Alexander J.; Nyman, Jeffry S.; Mahadevan-Jansen, Anita

2013-03-01

Activation Transcription Factor 4 (Atf-4) is essential for osteoblast maturation and proper collagen synthesis. We recently found that these bones demonstrate a rare brittleness phenotype, which is independent of bone strength. We utilized a confocal Renishaw Raman microscope (50x objective; NA=.75) to evaluate embedded, polished cross-sections of mouse tibia from both wild-type and knockout mice at 8 weeks of age (24 mice, n<=8 per group). Analysis of peak ratios indicated statistically significant changes in both mineral and collagen; however, compositional changes did not fully encompass biomechanical differences. To investigate the impact of material organization, we acquired colocalized spectra aligning the polarization angle parallel and perpendicular to the long bone axis from wet intact femurs. To validate our results, we used MMP9-/- mice, which have a brittleness phenotype that is not explained by compositional Raman measures. Polarization angle difference spectra show marked significant changes in orientation of these compositional differences when comparing wild type to knockout bones. Relative to wild-type, Atf4 -/- and MMP9 -/- bones show significant differences (t-test; p<0.05) in prominent collagen peaks. Further investigation of known peak ratios illustrates that this physical anisotropy of molecular organization is tightly clustered in brittle knockout bones. Such findings could have alternate interpretations about net collagen orientation or the angular distribution of collagen molecules. Use of polarization specific Raman measurements has implicated a structural profile that furthers our understanding of models of bone brittleness. Polarization content of Raman spectra may prove significant in future studies of brittle fracture and human fracture risk.

Etanercept Promotes Bone Formation via Suppression of Dickkopf-1 Expression in Rats with Collagen-Induced Arthritis

PubMed Central

Tanida, Atsushi; Kishimoto, Yuji; Okano, Toru; Hagino, Hiroshi

2013-01-01

Background Various clinical reports suggest etanercept (ETN) has some efficacy in bone formation in rheumatoid arthritis (RA). To examine this effect, we investigated the gene expression of cytokines relevant to osteoblast/osteoclast differentiation, and evaluated histomorphometric findings in mature rats with collagen-induced arthritis (CIA). Methods Total RNA was extracted from knee joints with CIA after ETN or placebo administration. Subsequently, realtime-PCR was carried out to quantify the mRNAs encoding Wnt-1, Dickkopf-1 (DKK-1), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegelin (OPG) and TNF (tumor necrosis factor)-alpha. In histomorphometric analysis, the infiltrating pannus volume and pannus surface, and the following items in contact with pannus surface were measured: osteoclast number, osteoid surface, osteoid volume and labeling surface. These were evaluated in the distal femur with CIA with or without ETN administration. Results TNF-alpha, RANKL and OPG mRNA expressions, linked to osteoclastogenesis, were not significantly different with or without ETN administration. ETN administration significantly increased Wnt-1 mRNA expression, the osteoblast promoter, and decreased DKK-1 mRNA expression, the Wnt signal inhibitor. In histomorphometric analysis, pannus volume, pannus surface and osteoclast number, parameters of bone destruction, were not significantly different among groups. Osteoid volume, osteoid surface and labeling surface, parameters of bone formation, increased significantly with ETN administration. Conclusion Our results suggest that ETN suppresses DDK-1 expression, and, as a result, Wnt expression is promoted and osteoblastogenesis becomes more active, independent of the regulation of osteoclast activity. Marked bone formation is attributed to the fact that ETN directly promotes osteoblastogenesis, not as a result of suppressing osteoclastogenesis. PMID:24031147

One-step cartilage repair in the knee: collagen-covered microfracture and autologous bone marrow concentrate. A pilot study.

PubMed

Enea, D; Cecconi, S; Calcagno, S; Busilacchi, A; Manzotti, S; Gigante, A

2015-01-01

Different single-stage surgical approaches are currently under evaluation to repair cartilage focal lesions. To date, only little is known on even short-term clinical follow-up and almost no knowledge exists on histological results of such treatments. The present paper aims to analyze the clinical and histological results of the collagen-covered microfracture and bone marrow concentrate (C-CMBMC) technique in the treatment of focal condylar lesions of knee articular cartilage. Nine patients with focal lesions of the condylar articular cartilage were consecutively treated with arthroscopic microfractures (MFX) covered with a collagen membrane immersed in autologous bone marrow concentrate (BMC) from the iliac crest. Patients were retrospectively assessed using several standardized outcome assessment tools and MRI scans. Four patients consented to undergo second look arthroscopy and biopsy harvest. Every patient was arthroscopically treated for a focal condylar lesion (mean area 2.5 SD(0.4) cm(2)). All the patients (mean age 43 SD(9) years) but one experienced a significant clinical improvement from the pre-operative condition to the latest follow-up (mean 29 SD(11) months). Cartilage macroscopic assessment at 12 months revealed that all the repairs appeared almost normal. Histological analysis showed a hyaline-like cartilage repair in one lesion, a fibrocartilaginous repair in two lesions and a mixture of both in one lesion. The first clinical experience with single-stage C-CMBMC for focal cartilage defects in the knee suggests that it is safe, it improves the short-term knee function and that it has the potential to recreate hyaline-like cartilage. Copyright © 2014. Published by Elsevier B.V.

Microstructural and Photoacoustic Infrared Spectroscopic Studies of Human Cortical Bone with Osteogenesis Imperfecta

NASA Astrophysics Data System (ADS)

Gu, Chunju; Katti, Dinesh R.; Katti, Kalpana S.

2016-04-01

The molecular basis of bone disease osteogenesis imperfecta (OI) and the mineralization of hydroxyapatite in OI bone have been of significant research interest. To further investigate the mechanism of OI disease and bone mineralization, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy, and x-ray diffraction (XRD) are used in the present study to describe the structural and compositional differences between OI and healthy bone. OI bone exhibits more porous, fibrous features, abnormal collagen fibrils, and abnormal mineral deposits. Likewise, photoacoustic-FTIR experiments indicate an aberrant collagen structure and an altered mineral structure in OI. In contrast, there is neither significant difference in the non-collagenous proteins (NCPs) composition observed nor apparent change in the crystal structure between OI and healthy bone minerals as shown in XRD and energy-dispersive x-ray spectroscopy (EDS) results. This observation indicates that the biomineralization process is more controlled by the bone cells and non-collagenous phosphorylated proteins. The present study also confirms that there is an orientational influence on the stoichiometry of the mineral in OI bone. Also, a larger volume of the hydrated layer in the transverse plane than the longitudinal plane of the mineral crystal structure is proposed. The appearance of a new C-S band in the FTIR spectra in OI bone suggests the substitution of glycine by cysteine in collagen molecules or/and an increased amount of cysteine-rich osteonectin that relates to mineral nucleation and mineral crystal formation.

Injectable collagen/RGD systems for bone tissue engineering applications.

PubMed

Kung, Fu-Chen

2018-01-01

Imbalance crosslink density and polymer concentration gradient is formed within the traditional alginate hydrogel using calcium chloride as a crosslinking agent in external gelation for instantaneously process. In this studying, type I collagen (Col I) blended calcium salt form of poly(γ-glutamic acid) (γCaPGA) was mixing with RGD-modified alginate with convenient gelation process and suitable for practical use. The hydrophilicity of the resulting hydrogels was evaluated through swelling tests, water retention capacity tests, and water vapor permeation tests. Mineralization was qualitatively evaluated by alizarin red dyeing at day 14, verifying the deposition of calcium. The in vitro osteogenic differentiation is monitored by determining the early and late osteocalcin (OCN) and osteopontin (OPN) markers with MG63 cells. Obtained results demonstrated that no extremely changes in mechanical properties. After 14 days of culture, hydrogels significantly stimulated OCN/OPN gene expressions and MG63 cell proliferation. Unusually, γCaPGA with RGD-modified alginate appeared better calcium deposition in 14 days than the other. However, addition of Col I can counterpoise RGD effect in blood coagulation and platelet adhesion made the hydrogel more flexibility and selectively in use. This studying provided that non-covalently crosslinked hydrogel by γCaPGA with alginate can be upgrading by RGD and Col I in water uptake capability, obviously effective for MG63 cells and are remarkably biocompatible and exhibited no cytotoxicity. Moreover, results also displayed the injectable process without complicated procedure, have high cost/performance ratio and have great potential for bone regeneration.

Alteration of the bone tissue material properties in type 1 diabetes mellitus: A Fourier transform infrared microspectroscopy study.

PubMed

Mieczkowska, Aleksandra; Mansur, Sity Aishah; Irwin, Nigel; Flatt, Peter R; Chappard, Daniel; Mabilleau, Guillaume

2015-07-01

Type 1 diabetes mellitus (T1DM) is a severe disorder characterized by hyperglycemia and hypoinsulinemia. A higher occurrence of bone fractures has been reported in T1DM, and although bone mineral density is reduced in this disorder, it is also thought that bone quality may be altered in this chronic pathology. Vibrational microscopies such as Fourier transform infrared microspectroscopy (FTIRM) represent an interesting approach to study bone quality as they allow investigation of the collagen and mineral compartment of the extracellular matrix in a specific bone location. However, as spectral feature arising from the mineral may overlap with those of the organic component, the demineralization of bone sections should be performed for a full investigation of the organic matrix. The aims of the present study were to (i) develop a new approach, based on the demineralization of thin bone tissue section to allow a better characterization of the bone organic component by FTIRM, (ii) to validate collagen glycation and collagen integrity in bone tissue and (iii) to better understand what alterations of tissue material properties in newly forming bone occur in T1DM. The streptozotocin-injected mouse (150 mg/kg body weight, injected at 8 weeks old) was used as T1DM model. Animals were randomly allocated to control (n = 8) or diabetic (n = 10) groups and were sacrificed 4 weeks post-STZ injection. Bones were collected at necropsy, embedded in polymethylmethacrylate and sectioned prior to examination by FTIRM. FTIRM collagen parameters were collagen maturity (area ratio between 1660 and 1690 cm(-1) subbands), collagen glycation (area ratio between the 1032 cm(-1) subband and amide I) and collagen integrity (area ratio between the 1338 cm(-1) subband and amide II). No significant differences in the mineral compartment of the bone matrix could be observed between controls and STZ-injected animals. On the other hand, as compared with controls, STZ-injected animals presented with

A paradigm shift for bone quality in dentistry: A literature review.

PubMed

Kuroshima, Shinichiro; Kaku, Masaru; Ishimoto, Takuya; Sasaki, Muneteru; Nakano, Takayoshi; Sawase, Takashi

2017-10-01

The aim of this study was to present the current concept of bone quality based on the proposal by the National Institutes of Health (NIH) and some of the cellular and molecular factors that affect bone quality. This is a literature review which focuses on collagen, biological apatite (BAp), and bone cells such as osteoblasts and osteocytes. In dentistry, the term "bone quality" has long been considered to be synonymous with bone mineral density (BMD) based on radiographic and sensible evaluations. In 2000, the NIH proposed the concept of bone quality as "the sum of all characteristics of bone that influence the bone's resistance to fracture," which is completely independent of BMD. The NIH defines bone quality as comprising bone architecture, bone turnover, bone mineralization, and micro-damage accumulation. Moreover, our investigations have demonstrated that BAp, collagen, and bone cells such as osteoblasts and osteocytes play essential roles in controlling the current concept of bone quality in bone around hip and dental implants. The current concept of bone quality is crucial for understanding bone mechanical functions. BAp, collagen and osteocytes are the main factors affecting bone quality. Moreover, mechanical loading dynamically adapts bone quality. Understanding the current concept of bone quality is required in dentistry. Copyright © 2017 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.

Collagen: A review on its sources and potential cosmetic applications.

PubMed

Avila Rodríguez, María Isabela; Rodríguez Barroso, Laura G; Sánchez, Mirna Lorena

2018-02-01

Collagen is a fibrillar protein that conforms the conjunctive and connective tissues in the human body, essentially skin, joints, and bones. This molecule is one of the most abundant in many of the living organisms due to its connective role in biological structures. Due to its abundance, strength and its directly proportional relation with skin aging, collagen has gained great interest in the cosmetic industry. It has been established that the collagen fibers are damaged with the pass of time, losing thickness and strength which has been strongly related with skin aging phenomena [Colágeno para todo. 60 y más. 2016. http://www.revista60ymas.es/InterPresent1/groups/revistas/documents/binario/ses330informe.pdf.]. As a solution, the cosmetic industry incorporated collagen as an ingredient of different treatments to enhance the user youth and well-being, and some common presentations are creams, nutritional supplement for bone and cartilage regeneration, vascular and cardiac reconstruction, skin replacement, and augmentation of soft skin among others [J App Pharm Sci. 2015;5:123-127]. Nowadays, the biomolecule can be obtained by extraction from natural sources such as plants and animals or by recombinant protein production systems including yeast, bacteria, mammalian cells, insects or plants, or artificial fibrils that mimic collagen characteristics like the artificial polymer commercially named as KOD. Because of its increased use, its market size is valued over USD 6.63 billion by 2025 [Collagen Market By Source (Bovine, Porcine, Poultry, Marine), Product (Gelatin, Hydrolyzed Collagen), Application (Food & Beverages, Healthcare, Cosmetics), By Region, And Segment Forecasts, 2014 - 2025. Grand View Research. http://www.grandviewresearch.com/industry-analysis/collagen-market. Published 2017.]. Nevertheless, there has been little effort on identifying which collagen types are the most suitable for cosmetic purposes, for which the present review will try to enlighten

Loss of Type I Collagen Telopeptide Lysyl Hydroxylation Causes Musculoskeletal Abnormalities in a Zebrafish Model of Bruck Syndrome.

PubMed

Gistelinck, Charlotte; Witten, Paul Eckhard; Huysseune, Ann; Symoens, Sofie; Malfait, Fransiska; Larionova, Daria; Simoens, Pascal; Dierick, Manuel; Van Hoorebeke, Luc; De Paepe, Anne; Kwon, Ronald Y; Weis, MaryAnn; Eyre, David R; Willaert, Andy; Coucke, Paul J

2016-11-01

Bruck syndrome (BS) is a disorder characterized by joint flexion contractures and skeletal dysplasia that shows strong clinical overlap with the brittle bone disease osteogenesis imperfecta (OI). BS is caused by biallelic mutations in either the FKBP10 or the PLOD2 gene. PLOD2 encodes the lysyl hydroxylase 2 (LH2) enzyme, which is responsible for the hydroxylation of lysine residues in fibrillar collagen telopeptides. This hydroxylation directs crosslinking of collagen fibrils in the extracellular matrix, which is necessary to provide stability and tensile integrity to the collagen fibrils. To further elucidate the function of LH2 in vertebrate skeletal development, we created a zebrafish model harboring a homozygous plod2 nonsense mutation resulting in reduced telopeptide hydroxylation and crosslinking of bone type I collagen. Adult plod2 mutants present with a shortened body axis and severe skeletal abnormalities with evidence of bone fragility and fractures. The vertebral column of plod2 mutants is short and scoliotic with compressed vertebrae that show excessive bone formation at the vertebral end plates, and increased tissue mineral density in the vertebral centra. The muscle fibers of mutant zebrafish have a reduced diameter near the horizontal myoseptum. The endomysium, a layer of connective tissue ensheathing the individual muscle fibers, is enlarged. Transmission electron microscopy of mutant vertebral bone shows type I collagen fibrils that are less organized with loss of the typical plywood-like structure. In conclusion, plod2 mutant zebrafish show molecular and tissue abnormalities in the musculoskeletal system that are concordant with clinical findings in BS patients. Therefore, the plod2 zebrafish mutant is a promising model for the elucidation of the underlying pathogenetic mechanisms leading to BS and the development of novel therapeutic avenues in this syndrome. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for

Engineering bone grafts with enhanced bone marrow and native scaffolds.

PubMed

Hung, Ben P; Salter, Erin K; Temple, Josh; Mundinger, Gerhard S; Brown, Emile N; Brazio, Philip; Rodriguez, Eduardo D; Grayson, Warren L

2013-01-01

The translation of tissue engineering approaches to the clinic has been hampered by the inability to find suitable multipotent cell sources requiring minimal in vitro expansion. Enhanced bone marrow (eBM), which is obtained by reaming long bone medullary canals and isolating the solid marrow putty, has large quantities of stem cells and demonstrates significant potential to regenerate bone tissues. eBM, however, cannot impart immediate load-bearing mechanical integrity or maintain the gross anatomical structure to guide bone healing. Yet, its putty-like consistency creates a challenge for obtaining the uniform seeding necessary to effectively combine it with porous scaffolds. In this study, we examined the potential for combining eBM with mechanically strong, osteoinductive trabecular bone scaffolds for bone regeneration by creating channels into scaffolds for seeding the eBM. eBM was extracted from the femurs of adult Yorkshire pigs using a Synthes reamer-irrigator-aspirator device, analyzed histologically, and digested to extract cells and characterize their differentiation potential. To evaluate bone tissue formation, eBM was seeded into the channels in collagen-coated or noncoated scaffolds, cultured in osteogenic conditions for 4 weeks, harvested and assessed for tissue distribution and bone formation. Our data demonstrates that eBM is a heterogenous tissue containing multipotent cell populations. Furthermore, coating scaffolds with a collagen hydrogel significantly enhanced cellular migration, promoted uniform tissue development and increased bone mineral deposition. These findings suggest the potential for generating customized autologous bone grafts for treating critical-sized bone defects by combining a readily available eBM cell source with decellularized trabecular bone scaffolds. © 2013 S. Karger AG, Basel

Techniques for Type I Collagen Organization

NASA Astrophysics Data System (ADS)

Anderson-Jackson, LaTecia Diamond

Tissue Engineering is a process in which cells, engineering, and material methods are used in amalgamation to improve biological functions. The purpose of tissue engineering is to develop alternative solutions to treat or cure tissues and organs that have been severely altered or damaged by diseases, congenital defects, trauma, or cancer. One of the most common and most promising biological materials for tissue engineering to develop scaffolds is Type I collagen. A major challenge in biomedical research is aligning Type I collagen to mimic biological structures, such as ligaments, tendons, bones, and other hierarchal aligned structures within the human body. The intent of this research is to examine possible techniques for organizing Type I collagen and to assess which of the techniques is effective for potential biological applications. The techniques used in this research to organize collagen are soft lithography with solution-assisted sonication embossing, directional freezing, and direct poling. The final concentration used for both soft lithography with solution-assisted sonication embossing and direct poling was 1 mg/ml, whereas for directional freezing the final concentration varied between 4mg/ml, 2mg/ml, and 1 mg/ml. These techniques were characterized using the Atomic Force Microscope (AFM) and Helium Ion Microscope (HIM). In this study, we have found that out of the three techniques, the soft lithography and directional freezing techniques have been successful in organizing collagen in a particular pattern, but not alignment. We concluded alignment may be dependent on the pH of collagen and the amount of acetic acid used in collagen solution. However, experiments are still being conducted to optimize all three techniques to align collagen in a unidirectional arrangement.

Three-dimensional collagenous niche and azacytidine selectively promote time-dependent cardiomyogenesis from human bone marrow-derived MSC spheroids.

PubMed

Joshi, Jyotsna; Mahajan, Gautam; Kothapalli, Chandrasekhar R

2018-04-17

Endogenous adult cardiac regenerative machinery is not capable of replacing the lost cells following myocardial infarction, often leading to permanent alterations in structure-function-mechanical properties. Regenerative therapies based on delivering autologous stem cells within an appropriate 3D milieu could meet such demand, by enabling homing and directed differentiation of the transplanted cells into lost specialized cell populations. Since type I collagen is the predominant cardiac tissue matrix protein, we here optimized the 3D niche which could promote time-dependent evolution of cardiomyogenesis from human bone marrow-derived mesenchymal stem cells (BM-MSC). 3D collagen gel physical and mechanical characteristics were assessed using SEM and AFM, respectively, while the standalone and combined effects of collagen concentration, culture duration, and 5-azacytidine (aza) dose on the phenotype and genotype of MSC spheroids were quantified using immunofluorescence labeling and RT-PCR analysis. Increasing collagen concentration led to a significant increase in Young's modulus (p < 0.01) but simultaneous decrease in the mean pore size, resulting in stiffer gels. Spheroid formation significantly modulated MSC differentiation and genotype, mostly due to better cell-cell interactions. Among the aza dosages tested, 10 μM appears to be optimal, while 3 mg/ml gels resulted in significantly lower cell viability compared to 1 or 2 mg/ml gels. Stiffer gels (2 and 3 mg/ml) and exposure to 10 μM aza upregulated early and late cardiac marker expressions in a time-dependent fashion. On the other hand, cell-cell signaling within the MSC spheroids seem to have a strong role in influencing mature cardiac markers expression, since neither aza nor gel stiffness seem to significantly improve their expression. Western blot analysis suggested that canonical Wnt/β-catenin signaling pathway might be primarily mediating the observed benefits of aza on cardiac

Mechanical Properties of Single Collagen Fibrils Revealed by Force Spectroscopy

NASA Astrophysics Data System (ADS)

Graham, John; Phillips, Charlotte; Grandbois, Michel

2004-03-01

In the field of biomechanics, collagen fibrils are believed to be robust mechanical structures characterized by a low extensibility. Until very recently, information on the mechanical properties of collagen fibrils could only be derived from ensemble measurements performed on complete tissues such as bone, skin and tendon. Here we measure force-elongation/relaxation profiles of single collagen fibrils using atomic force microscopy-based force spectroscopy. The elongation profiles indicate that in vitro assembled heterotrimeric type I collagen fibrils are characterized by a large extensibility. Numerous discontinuities and a plateau in the force profile indicate major reorganization occurs within the fibrils in the 1.5 -- 4.5 nN range. Our study demonstrates that newly assembled collagen fibrils are robust structures with a significant reserve of elasticity that could play a determinant role in cellular motion in the context of tissue growth and morphogenesis. In contrast, homotrimeric collagen fibrils corresponding to osteogenesis imperfecta pathology exhibit a marked difference in their elasticity profile.

Osmotic pressure induced tensile forces in tendon collagen

NASA Astrophysics Data System (ADS)

Masic, Admir; Bertinetti, Luca; Schuetz, Roman; Chang, Shu-Wei; Metzger, Till Hartmut; Buehler, Markus J.; Fratzl, Peter

2015-01-01

Water is an important component of collagen in tendons, but its role for the function of this load-carrying protein structure is poorly understood. Here we use a combination of multi-scale experimentation and computation to show that water is an integral part of the collagen molecule, which changes conformation upon water removal. The consequence is a shortening of the molecule that translates into tensile stresses in the range of several to almost 100 MPa, largely surpassing those of about 0.3 MPa generated by contractile muscles. Although a complete drying of collagen would be relevant for technical applications, such as the fabrication of leather or parchment, stresses comparable to muscle contraction already occur at small osmotic pressures common in biological environments. We suggest, therefore, that water-generated tensile stresses may play a role in living collagen-based materials such as tendon or bone.

Increasing the pore sizes of bone-mimetic electrospun scaffolds comprised of polycaprolactone, collagen I and hydroxyapatite to enhance cell infiltration

PubMed Central

Phipps, Matthew C.; Clem, William C.; Grunda, Jessica M.; Clines, Gregory A.; Bellis, Susan L.

2012-01-01

Bone-mimetic electrospun scaffolds consisting of polycaprolactone (PCL), collagen I and nanoparticulate hydroxyapatite (HA) have previously been shown to support the adhesion, integrin-related signaling and proliferation of mesenchymal stem cells (MSCs), suggesting these matrices serve as promising degradable substrates for osteoregeneration. However, the small pore sizes in electrospun scaffolds hinder cell infiltration in vitro and tissue-ingrowth into the scaffold in vivo, limiting their clinical potential. In this study, three separate techniques were evaluated for their capability to increase the pore size of the PCL/col I/nanoHA scaffolds: limited protease digestion, decreasing the fiber packing density during electro-spinning, and inclusion of sacrificial fibers of the water-soluble polymer PEO. The PEO sacrificial fiber approach was found to be the most effective in increasing scaffold pore size. Furthermore, the use of sacrificial fibers promoted increased MSC infiltration into the scaffolds, as well as greater infiltration of endogenous cells within bone upon placement of scaffolds within calvarial organ cultures. These collective findings support the use of sacrificial PEO fibers as a means to increase the porosity of complex, bone-mimicking electrospun scaffolds, thereby enhancing tissue regenerative processes that depend upon cell infiltration, such as vascularization and replacement of the scaffold with native bone tissue. PMID:22014462

In vitro and in vivo evaluation of bone morphogenetic protein-2 (BMP-2) immobilized collagen-coated polyetheretherketone (PEEK)

NASA Astrophysics Data System (ADS)

Du, Ya-Wei; Zhang, Li-Nan; Ye, Xin; Nie, He-Min; Hou, Zeng-Tao; Zeng, Teng-Hui; Yan, Guo-Ping; Shang, Peng

2015-03-01

Polyetheretherketone (PEEK) is regarded as one of the most potential candidates of biomaterials in spinal implant applications. However, as a bioinert material, PEEK plays a limited role in osteoconduction and osseointegration. In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) was immobilized onto the surface of collagen-coated PEEK in order to prepare a multi-functional material. After adsorbed onto the PEEK surface by hydrophobic interaction, collagen was cross-linked with N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). EDC/NHS system also contributed to the immobilization of rhBMP-2. Water contact angle tests, XPS and SEM clearly demonstrated the surface changes. ELISA tests quantified the amount of rhBMP-2 immobilized and the release over a period of 30 d. In vitro evaluation proved that the osteogenesis differentiation rate was higher when cells were cultured on modified PEEK discs than on regular ones. In vivo tests were conducted and positive changes of major parameters were presented. This report demonstrates that the rhBMP-2 immobilized method for PEEK modification increase bioactivity in vitro and in vivo, suggesting its practicability in orthopedic and spinal clinical applications.

Collagen scaffolds derived from a marine source and their biocompatibility.

PubMed

Song, Eun; Yeon Kim, So; Chun, Taehoon; Byun, Hyun-Jung; Lee, Young Moo

2006-05-01

The primary sources of industrial collagens are calf skin and bone. However, these carry a high risk of bovine spongiform encephalopathy or transmissible spongiform encephalopathy. In this study, a novel form of acid-soluble collagen was extracted from jellyfish in an effort to obtain an alternative and safer collagen. Porous scaffolds composed of jellyfish collagen were prepared by freeze-drying and cross-linking with 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide to be used in tissue engineering applications. Enzymatic degradation kinetics of jellyfish collagen scaffolds were controlled by EDC/NHS-cross-linking density. Results from an MTT assay indicated that jellyfish collagen exhibited higher cell viability than other naturally derived biomaterials, including bovine collagen, gelatin, hyaluronic acid, and glucan. Jellyfish collagen scaffolds also had a highly porous and interconnected pore structure, which is useful for an high-density cell seeding, an efficient nutrient and an oxygen supply to the cells cultured in the three-dimensional matrices. To determine whether jellyfish collagen evokes any specific inflammatory response compared to that induced by bovine collagen or gelatin, we measured the levels of pro-inflammatory cytokines and antibody secretions and monitored the population changes of immune cells after in vivo implantation. Jellyfish collagen was found to induce an immune response at least comparable to those caused by bovine collagen and gelatin.

Characterization of collagen II fibrils containing biglycan and their effect as a coating on osteoblast adhesion and proliferation.

PubMed

Douglas, Timothy; Heinemann, Sascha; Hempel, Ute; Mietrach, Carolin; Knieb, Christiane; Bierbaum, Susanne; Scharnweber, Dieter; Worch, Hartmut

2008-04-01

Collagen has been used as a coating material for titanium-based implants for bone contact and as a component of scaffolds for bone tissue engineering. In general collagen type I has been used, however very little attention has been focussed on collagen type II. Collagen-based coatings and scaffolds have been enhanced by the incorporation of the glycosaminoglycan chondroitin sulphate (CS), however the proteglycan biglycan, which is found in bone and contains glycosaminoglycan chains consisting of CS, has not been used as a biomaterial component. The study had the following aims: firstly, five different collagen II preparations were compared with regard to their ability to bind CS and biglycan and the changes in fibril morphology thereby induced. Secondly, the effects of biglycan on the adhesion of primary rat osteoblasts (rO) as well as the proliferation of rO, primary human osteoblasts (hO) and the osteoblast-like cell line 7F2 were studied by culturing the cells on surfaces coated with collagen II fibrils containing biglycan. Fibrils of the collagen II preparation which bound the most biglycan were used to coat titanium surfaces. Bare titanium, titanium coated with collagen II fibrils and titanium coated with collagen II fibrils containing biglycan were compared. It was found that different collagen II preparations showed different affinities for CS and biglycan. In four of the five preparations tested, biglycan reduced fibril diameter, however the ability of a preparation to bind more biglycan did not appear to lead to a greater reduction in fibril diameter. Fibrils containing biglycan promoted the formation of focal adhesions by rO and significantly enhanced the proliferation of hO but not of rO or 7F2 cells. These results should encourage further investigation of biglycan as a component of collagen-based scaffolds and/or coatings.

Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast.

PubMed

Seol, Ja Young; Yoon, Ji Young; Jeong, Hee Sun; Joo, Nami; Choi, Soon Young

2016-01-01

Many researchers revealed that collagen contribute to maintaining the skin's elasticity and inhibit wrinkling of skin. Korean native cattle (Hanwoo) bone (leg bone, foot and tail) infusion contains the various inorganic materials, collagen and chondroitin sulfate. All of this, a large quantity of collagen is included in Hanwoo infusion. Therefore, this study emphasized on the effects of collagen in the Hanwoo bone infusion. For the first time, Hanwoo bone infusions were directly added to the media of Human Dermal Fibroblast (NHDF-c) to test anti-aging effects. First, it was identified that growth rate of skin fibroblast was increased. Furthermore, the Hanwoo bone infusion increased a 50% of fibroblast collagen synthesis. Also, suppression of skin fibroblast aging was confirmed by treatment Hanwoo bone infusion. In conclusion, this study demonstrates the effects of infusion made from Hanwoo leg bone, foot and tail on anti-aging, wrinkle inhibiting and skin fibroblast elasticity maintaining. Therefore, this study identified that traditional infusion has effects that are good for skin elasticity.

The Great Irish Famine: Identifying Starvation in the Tissues of Victims Using Stable Isotope Analysis of Bone and Incremental Dentine Collagen

PubMed Central

Montgomery, Janet

2016-01-01

The major components of human diet both past and present may be estimated by measuring the carbon and nitrogen isotope ratios (δ13C and δ15N) of the collagenous proteins in bone and tooth dentine. However, the results from these two tissues differ substantially: bone collagen records a multi-year average whilst primary dentine records and retains time-bound isotope ratios deriving from the period of tooth development. Recent studies harnessing a sub-annual temporal sampling resolution have shed new light on the individual dietary histories of our ancestors by identifying unexpected radical short-term dietary changes, the duration of breastfeeding and migration where dietary change occurs, and by raising questions regarding factors other than diet that may impact on δ13C and δ15N values. Here we show that the dentine δ13C and δ15N profiles of workhouse inmates dating from the Great Irish Famine of the 19th century not only record the expected dietary change from C3 potatoes to C4 maize, but when used together they also document prolonged nutritional and other physiological stress resulting from insufficient sustenance. In the adults, the influence of the maize-based diet is seen in the δ13C difference between dentine (formed in childhood) and rib (representing an average from the last few years of life). The demonstrated effects of stress on the δ13C and δ15N values will have an impact on the interpretations of diet in past populations even in slow-turnover tissues such as compact bone. This technique also has applicability in the investigation of modern children subject to nutritional distress where hair and nails are unavailable or do not record an adequate period of time. PMID:27508412

Immunohistochemical response in rats of beta-tricalcium phosphate (TCP) with or without BMP-2 in the production of collagen matrix critical defects.

PubMed

Luvizuto, Eloá Rodrigues; de Oliveira, Júlio César Silva; Gomes-Ferreira, Pedro Henrique Silva; Pereira, Cassiano Costa Silva; Faverani, Leonardo Perez; Antoniali, Cristina; Okamoto, Roberta

2017-04-01

This study aimed to assess the biological response of BMP-2 (bone morphogenetic protein-2) in supplementation with β-tricalcium phosphate (TCP) as a carrier in the bone healing of surgical defects in rats' calvaria. A critical-size defect (5mm in diameter) was filled with β-TCP alone or added with that plus 5mg of BMP-2 at 5, 15, and 30 postoperative days. Histomorphometric and immunohistochemical (osteocalcin, collagen type I, and metalloproteinase-9) analysis was performed to assess the features of bone healing. Histological behavior and collagen type I labeling showed increased formation of the collagen matrix, leading to a higher percentage of newly formed bone and biomaterial for tissue and more total mineralization of pure TCP when compared to the other groups. The supplementation with BMP-2 promoted faster TCP remodeling; however, there was no statistically significant difference for the bone formed in both groups (P>0.05). Collagen-matrix formation and new bone formation reached maximum levels when the defects were filled with pure TCP, even exceeding the levels from BMP-2 supplementation. Copyright © 2017 Elsevier GmbH. All rights reserved.

In vitro non-enzymatic ribation reduces post-yield strain accommodation in cortical bone.

PubMed

Willett, Thomas L; Sutty, Sibi; Gaspar, Anne; Avery, Nick; Grynpas, Marc

2013-02-01

Non-enzymatic glycation (NEG) and advanced glycation endproducts (AGEs) may contribute to bone fragility in various diseases, ageing, and other conditions by modifying bone collagen and causing degraded mechanical properties. In this study, we sought to further understand how collagen modification in an in vitro non-enzymatic ribation model leads to loss of cortical bone toughness. Previous in vitro studies using non-enzymatic ribation reported loss of ductility in the cortical bone. Increased crosslinking is most commonly blamed for these changes; however, some studies report positive correlations between measures of total collagen crosslinking and work-to-fracture/toughness measurements whilst correlations between general NEG and measures of ductility are often negative. Fifteen bone beam triplets were cut from bovine metatarsi. Each provided one native non-incubated control, one incubated control and one ribated specimen. Incubation involved simulated body fluid±ribose for fourteen days at 37°C. Pentosidine and pyridinoline crosslinks were measured using HPLC. Three-point bending tests quantified mechanical properties. Fracture surfaces were examined using scanning electron microscopy. The effects of ribation on bone collagen molecular stability and intermolecular connectivity were investigated using differential scanning calorimetry and hydrothermal isometric tension testing. Ribation caused increased non-enzymatic collagen modification and pentosidine content (16mmol/mol collagen) and inferior post-yield mechanical behaviour, especially post-yield strain and flexural toughness. Fracture surfaces were smoother with less collagen fibril deformation or tearing than observed in controls. In the ribated group only, pentosidine content and thermomechanical measures of crosslinking were positively correlated with measures of strain accommodation and energy absorption before failure. Non-enzymatic ribation and the resulting modifications reduce cortical bone pseudo

Doses effects of zoledronic acid on mineral apatite and collagen quality of newly-formed bone in the rat's calvaria defect.

PubMed

Olejnik, Cécile; Falgayrac, Guillaume; During, Alexandrine; Cortet, Bernard; Penel, Guillaume

2016-08-01

Due to their inhibitory effects on resorption, bisphosphonates are widely used in the treatment of diseases associated to an extensive bone loss. Yet, little is known about bisphosphonates effects on newly-formed bone quality. In the present study, adult male Sprague-Dawley rats (n=80) with a bone defect calvaria area were used and short-term effects of zoledronic acid (ZA) were studied on the healing bone area. Three ZA treatments were tested by using either: 1°) a low single dose (120μgZA/kg, n=10; equivalent to human osteoporosis treatment), 2°) a low fractionated doses (20μgZA/kg daily for 6days either a total of 120μg/kg, n=15), and 3°) a high fractionated doses, (100μgZA/kg weekly for 6weeks, n=15; equivalent to 6months of human bone metastasis treatment). For each treatment, a control "vehicle" treatment was performed (with an identical number of rats). After ZA administration, the intrinsic bone material properties were evaluated by quantitative backscattered electron imaging (qBEI) and Raman microspectroscopy. Neither single nor fractionated low ZA doses modify the intrinsic bone material properties of the newly-formed bone compared to their respective control animals. On the opposite, the high ZA treatment resulted in a significant decrease of the crystallinity (-25%, P< 0.05) and of the hydroxyproline-to-proline ratio (-30%, P<0.05) in newly-formed bones. Moreover, with the high ZA treatment, the crystallinity was positively correlated with the hydroxyproline-to-proline ratio (ρ=0.78, P<0.0001). The present data highlight new properties for ZA on bone formation in a craniofacial defect model. As such, ZA at high doses disrupted the apatite crystal organization. In addition, we report here for the first time that high ZA doses decreased the hydroxyproline-to-proline ratio suggesting that ZA may affect the early collagen organization during the bone healing. Copyright © 2016 Elsevier Inc. All rights reserved.

Collagen based polyurethanes—A review of recent advances and perspective.

PubMed

Zuber, Mohammad; Zia, Fatima; Zia, Khalid Mahmood; Tabasum, Shazia; Salman, Mahwish; Sultan, Neelam

2015-09-01

Collagen is mostly found in fibrous tissues such as tendons, ligaments and skin. Collagen makes up approximately 30% of the proteins within the body. These are tough and strong structures found all over the body: in bones, tendons and ligaments. Collagen being the most abundant protein provides tensile strength via cell matrix interactions to tissue architecture. Biomimetic materials of collagen origin gained wide spread acceptance in clinical applications. Vitamin C deficiency causes scurvy a serious and painful disease in which defective collagen prevents the formation of strong connective tissue, gums deteriorate and bleed, with loss of teeth; skin discolors, and wounds do not heal. Effective collagens prevent the manifestation of such disorders. Polyurethanes on the other hand are frequently used for various applications as they offered in wide-ranging of compositions, properties and complex structures. Collagen/PU bio-composites have potential array for biomedical applications. Considering versatile properties of the elongated fibrils and wide industrial and biomedical applications including biocompatibility of polyurethane, this review shed a light on collagen based polyurethane materials with their potential applications especially focusing the bio-medical field. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of Concentration of Collagen Gel on Functional Activity of Bone Marrow Mesenchymal Stromal Cells.

PubMed

Nashchekina, Yu A; Yudintceva, N M; Nikonov, P O; Ivanova, E A; Smagina, L V; Voronkina, I V

2017-05-01

Collagen I gels with protein concentrations of 1, 2, and 3.5 mg/ml were prepared and embedded in a porous polylactide scaffold to reduce their contraction. Concentration of the gel did not affect its degradation. Collagen gels promoted the formation of cell networks. The cells in the collagen gel with a concentration of 1 mg/ml embedded in polylactide scaffold had elongated spindle-like shape, in contrast to flattened cells in collagen gel of the same concentration not embedded in the scaffold. Stabilization of the collagen gel in the polylactide scaffold promoted active synthesis of laminin and fibronectin by cells as soon as on day 5 of culturing in comparison with that in free collagen substrate.

Influence of collagen-fibril-based coatings containing decorin and biglycan on osteoblast behavior.

PubMed

Douglas, Timothy; Hempel, Ute; Mietrach, Carolin; Viola, Manuela; Vigetti, Davide; Heinemann, Sascha; Bierbaum, Susanne; Scharnweber, Dieter; Worch, Hartmut

2008-03-01

Collagen is used as a scaffold material for tissue engineering as well as a coating material for implants with a view to enhancing osseointegration by mimicry of the bone extracellular matrix in vivo. The biomimicry strategy can be taken further by incorporating the small leucine-rich proteoglycans (SLRPs) decorin and biglycan, which are expressed in bone. Both bind to fibrils during fibrillogenesis in vitro. In this study, the ability of collagen types I, II, and III to bind decorin and biglycan was compared. Collagen type II bound significantly more SLRPs in fibrils than collagen I and III, with more biglycan than decorin bound by all three collagen types. Therefore, type II fibrils with bound decorin or biglycan or neither were used to coat titanium surfaces. Bioavailability of SLRPs was confirmed by direct ELISA after SLRP biotinilation. The in vitro behavior of osteoblasts from rat calvaria (rOs) and human knee (hOs) cultured on different surfaces was compared. Proliferation and collagen synthesis were determined. Also, the influence of SLRPs on the formation of focal adhesions by rO was investigated. Biglycan enhanced the formation of focal adhesions after 2 and 24 h. Decorin and biglycan affected rO and hO proliferation and collagen synthesis differently. Biglycan stimulated hO proliferation significantly but had no effect on rO proliferation, and also inhibited rO collagen synthesis significantly while not affecting hO collagen synthesis. Decorin promoted hO proliferation slightly but did not influence rO proliferation. The results could be relevant when designing implant coatings or tissue engineering scaffolds. (c) 2007 Wiley Periodicals, Inc.

Differences in collagen distribution of healthy and regenerated periodontium. Histomorphometric study in dogs.

PubMed

Souza, Sérgio L S; Macedo, Guilherme O; Silveira E Souza, Adriana M M; Taba, Mário; Novaes, Arthur B; Oliver, Constance; Jamur, Maria C; Correa, Vani M A

2013-10-01

Previous studies have shown that there is a relationship between periodontal disease and the distribution of collagen fibers. This study evaluated the distribution of collagen types I and III in regenerated bone and periodontal ligament, comparing them to the tissues near the regenerated area and to the healthy periodontium. In the third (P3) and fourth (P4) mandibular premolars of 5 healthy mongrel dogs, bilaterally, buccal class 2 furcation lesions were surgically created and chronified for 3 weeks. After that, full flaps were elevated and expanded polytetrafluoroethylene (e-PTFE) membranes were adapted, sutured and recovered by the flaps. Two weeks after surgery, two membranes on the same side were removed and the other membranes were removed four weeks after surgery. The dogs were euthanized at 12 weeks following placement of the e-PTFE membranes. P3 and P4 teeth as well as the second premolars (healthy control teeth) and their periodontal tissues were removed and histologically processed for Collagen Quantification (COLQ). The amount of type III collagen was higher in native bone compared to the regenerated area. For periodontal ligament, COLQ for type I collagen showed statistically significant differences (Tukeys's Multiple Comparison, p⟨0.05) between the regenerated groups and the control group. These differences were not found for type III COLQ. There are significant differences in collagen distribution among the regenerated, native and control tissues. Membrane removal 2 or 4 weeks postoperatively did not influence the collagen composition.

Bone Marrow Stromal Cells Contribute to Bone Formation Following Infusion into Femoral Cavities of a Mouse Model of Osteogenesis Imperfecta

PubMed Central

Li, Feng; Wang, Xujun; Niyibizi, Christopher

2010-01-01

Currently, there are conflicting data in literature regarding contribution of bone marrow stromal cells (BMSCs) to bone formation when the cells are systemically delivered in recipient animals. To understand if BMSCs contribute to bone cell phenotype and bone formation in osteogenesis imperfecta bones (OI), MSCs marked with GFP were directly infused into the femurs of a mouse model of OI (oim). The contribution of the cells to the cell phenotype and bone formation was assessed by histology, immunohistochemistry and biomechanical loading of recipient bones. Two weeks following infusion of BMSCs, histological examination of the recipient femurs demonstrated presence of new bone when compared to femurs injected with saline which showed little or no bone formation. The new bone contained few donor cells as demonstrated by GFP fluorescence. At six weeks following cell injection, new bone was still detectable in the recipient femurs but was enhanced by injection of the cells suspended in pepsin solublized type I collagen. Immunofluorescence and immunohistochemical staining showed that donor GFP positive cells in the new bone were localized with osteocalcin expressing cells suggesting that the cells differentiated into osteoblasts in vivo. Biomechanical loading to failure in thee point bending, revealed that, femurs infused with BMSCs in PBS or in soluble type I collagen were biomechanically stronger than those injected with PBS or type I collagen alone. Taken together, the results indicate that transplanted cells differentiated into osteoblasts in vivo and contributed to bone formation in vivo; we also speculate that donor cells induced differentiation or recruitment of endogenous cells to initiate reparative process at early stages following transplantation. PMID:20570757

Comparing membranes and bone substitutes in a one-stage procedure for horizontal bone augmentation. A double-blind randomised controlled trial.

PubMed

Merli, Mauro; Moscatelli, Marco; Mariotti, Giorgia; Pagliaro, Umberto; Raffaelli, Eugenia; Nieri, Michele

2015-01-01

The objective of this parallel randomised controlled trial is to compare two bone substitutes and collagen membranes in a one-stage procedure for horizontal bone augmentation: anorganic bovine bone (Bio-Oss) and collagen porcine membranes (Bio-Gide) (BB group) versus a synthetic resorbable bone graft substitute made of pure β-tricalcium phosphate (Ceros TCP) and porcine pericardium collagen membranes (Jason) (CJ group). Patients in need of implant treatment having at least one site with horizontal osseous defects at a private clinic in Rimini (Italy) were included in this study. Patients were randomised to receive either the BB or CJ treatment. Randomisation was computer-generated with allocation concealment by opaque sequentially numbered sealed envelopes. Patients and the outcome assessor were blinded to group assignment. The main outcome measures were implant failure, complications, clinical bone gain at augmented sites, and complete filling of the bone defect. Secondary outcome measures were chair-time, postoperative pain and peri-implant marginal bone level changes. Twenty-five patients with 32 implants were allocated to the BB group and 25 patients with 29 implants to the CJ group. All 50 randomised patients received the treatment as allocated and there were no dropouts up to 6-months post-loading (12 months post-surgery). There were no failures and there were three complications in the BB group and three complications in the CJ group (relative risk: 1.00, 95% CI from 0.22 to 4.49, P = 1.00). The estimated difference between treatments in the vertical defect bone gain was -0.15 mm (95% CI from -0.65 to 0.35, P = 0.5504) favouring the BB group, and the estimated difference between treatments in the horizontal defect bone gain was -0.27 mm (95%CI from -0.73 to 0.19, P = 0.3851) favouring the BB group. There was no difference in the complete filling of the defect (relative risk: 0.88, 95%CI from 0.58 to 1.34, P = 0.7688). No significant differences were

Two dimensional alveolar ridge augmentation using particulate hydroxyapatite and collagen membrane: A case report

PubMed Central

Singh, Aparna; Daing, Anika; Anand, Vishal; Dixit, Jaya

2014-01-01

Background Ridge augmentation procedures require bone regeneration outside of the existing bony walls or housing and are therefore often considered to be the most challenging surgical procedures. The bony deficiencies can be managed with GBR techniques involving bone grafting material and membrane while vertical augmentation may require the use of space-creating support mechanisms. Non-degradable membranes have been used for ridge augmentation with encouraging results however; requirement of second surgery for its removal and associated infection on exposure may compromise the desired results. These problems can be overcome by employing resorbable collagen membranes. Different bone graft materials are also used in combination with resorbable membranes, for prevention of membrane collapse and maintenance of space, as they lack sufficient rigidity. Particulate hydroxyapatite bone graft may be better alternative, because it treats the underlying bone defect to restore the natural support of the tissue architecture. Moreover, its use avoids potential donor site complications associated with autogenous block grafts. Method Patient described in this report presented with missing right maxillary incisor with ridge deficiency. A treatment approach involving localised ridge augmentation with particulate hydroxyapatite and collagen membrane was used. Result Six month post-operative periapical radiograph demonstrated a significant vertical bone fill. Conclusion The clinical and radiographic findings of the present case suggests that HA in conjunction with a resorbable collagen membrane may be an acceptable alternative to the autogenous block graft and non-resorbable membrane in the treatment of compromised alveolar ridge deficiencies. PMID:25737935

Biological Safety of Fish (Tilapia) Collagen

PubMed Central

Yamamoto, Kohei; Sugimoto, Kouji; Yoshizawa, Yuu; Yanagiguchi, Kajiro

2014-01-01

Marine collagen derived from fish scales, skin, and bone has been widely investigated for application as a scaffold and carrier due to its bioactive properties, including excellent biocompatibility, low antigenicity, and high biodegradability and cell growth potential. Fish type I collagen is an effective material as a biodegradable scaffold or spacer replicating the natural extracellular matrix, which serves to spatially organize cells, providing them with environmental signals and directing site-specific cellular regulation. This study was conducted to confirm the safety of fish (tilapia) atelocollagen for use in clinical application. We performed in vitro and in vivo biological studies of medical materials to investigate the safety of fish collagen. The extract of fish collagen gel was examined to clarify its sterility. All present sterility tests concerning bacteria and viruses (including endotoxin) yielded negative results, and all evaluations of cell toxicity, sensitization, chromosomal aberrations, intracutaneous reactions, acute systemic toxicity, pyrogenic reactions, and hemolysis were negative according to the criteria of the ISO and the Ministry of Health, Labour and Welfare of Japan. The present study demonstrated that atelocollagen prepared from tilapia is a promising biomaterial for use as a scaffold in regenerative medicine. PMID:24809058

Biological safety of fish (tilapia) collagen.

PubMed

Yamamoto, Kohei; Igawa, Kazunari; Sugimoto, Kouji; Yoshizawa, Yuu; Yanagiguchi, Kajiro; Ikeda, Takeshi; Yamada, Shizuka; Hayashi, Yoshihiko

2014-01-01

Marine collagen derived from fish scales, skin, and bone has been widely investigated for application as a scaffold and carrier due to its bioactive properties, including excellent biocompatibility, low antigenicity, and high biodegradability and cell growth potential. Fish type I collagen is an effective material as a biodegradable scaffold or spacer replicating the natural extracellular matrix, which serves to spatially organize cells, providing them with environmental signals and directing site-specific cellular regulation. This study was conducted to confirm the safety of fish (tilapia) atelocollagen for use in clinical application. We performed in vitro and in vivo biological studies of medical materials to investigate the safety of fish collagen. The extract of fish collagen gel was examined to clarify its sterility. All present sterility tests concerning bacteria and viruses (including endotoxin) yielded negative results, and all evaluations of cell toxicity, sensitization, chromosomal aberrations, intracutaneous reactions, acute systemic toxicity, pyrogenic reactions, and hemolysis were negative according to the criteria of the ISO and the Ministry of Health, Labour and Welfare of Japan. The present study demonstrated that atelocollagen prepared from tilapia is a promising biomaterial for use as a scaffold in regenerative medicine.

Early effects of zoledronic acid and teriparatide on bone microarchitecture, remodeling and collagen crosslinks: comparison between iliac crest and lumbar vertebra in ewes.

PubMed

Portero-Muzy, N R; Chavassieux, P M; Bouxsein, M L; Gineyts, E; Garnero, P; Chapurlat, R D

2012-10-01

Iliac crest bone biopsies are used to assess the mechanism of action of drug treatments, yet there are little data comparing this site to sites prone to fracture. The purpose of this study was to compare the delay and the amplitude of responses to treatment in two different bone sites. The short-term effects of zoledronic acid and teriparatide on microarchitecture, collagen crosslinks and bone remodeling were evaluated in iliac crest and lumbar vertebrae. Aged ewes (n=8/gr) received either vehicle (CTRL) or a single injection of zoledronic acid (ZOL, 10mg) or daily injections of teriparatide (TPTD, 20 μg/d) for 3 months. Blood samples were collected monthly for assessing bone turnover markers. At the end of the study, a transiliac bone biopsy (IC) and L1 lumbar vertebrae (LV1) were collected to assess bone microarchitecture; pyridinoline (PYD), deoxypyridinoline (DPD), pentosidine (PEN) content, static and dynamic parameters of bone remodeling. In CTRL, Tb-BV/TV was significantly higher in LV1 than IC (p<0.0001). This was associated with a trend of higher Tb.N, Tb.Th, DA, an inferior Conn.D and a lower bone turnover as shown by the decreases of osteoid parameters, MS/BS, Ac.f in LV1 when compared to IC. In addition, the ratio PYD/DPD was 4 times higher in LV1 than IC. After 3 months, significant decreases of sALP (p<0.001) and sCTX (p<0.001) were observed in the ZOL-group whereas in TPTD-group, after transient increases, they returned to baseline values. When compared to their respective CTRL, ZOL induced significant increases in Tb.BV/TV, Conn.D, Tb.N and Tb.Sp, in IC but not in LV1. Regardless of the site, ZOL markedly depressed the bone turnover: The static parameters of bone formation significantly decreased and the diminution of MS/BS, BFR/BS and Ac.f varied from -94 to -98% vs CTRL (p<0.01 to 0.001). It was associated with a diminution of the DPD content and the PYD/DPD ratio mainly in IC cortices. In contrast, after 3 months, TPTD did not modify the 3D

Differential effects of collagen prolyl 3-hydroxylation on skeletal tissues.

PubMed

Homan, Erica P; Lietman, Caressa; Grafe, Ingo; Lennington, Jennifer; Morello, Roy; Napierala, Dobrawa; Jiang, Ming-Ming; Munivez, Elda M; Dawson, Brian; Bertin, Terry K; Chen, Yuqing; Lua, Rhonald; Lichtarge, Olivier; Hicks, John; Weis, Mary Ann; Eyre, David; Lee, Brendan H L

2014-01-01

Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1(H662A) ). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity

In Situ Imaging of Tissue Remodeling with Collagen Hybridizing Peptides

PubMed Central

2017-01-01

Collagen, the major structural component of nearly all mammalian tissues, undergoes extensive proteolytic remodeling during developmental states and a variety of life-threatening diseases such as cancer, myocardial infarction, and fibrosis. While degraded collagen could be an important marker of tissue damage, it is difficult to detect and target using conventional tools. Here, we show that a designed peptide (collagen hybridizing peptide: CHP), which specifically hybridizes to the degraded, unfolded collagen chains, can be used to image degraded collagen and inform tissue remodeling activity in various tissues: labeled with 5-carboxyfluorescein and biotin, CHPs enabled direct localization and quantification of collagen degradation in isolated tissues within pathologic states ranging from osteoarthritis and myocardial infarction to glomerulonephritis and pulmonary fibrosis, as well as in normal tissues during developmental programs associated with embryonic bone formation and skin aging. The results indicate the general correlation between the level of collagen remodeling and the amount of denatured collagen in tissue and show that the CHP probes can be used across species and collagen types, providing a versatile tool for not only pathology and developmental biology research but also histology-based disease diagnosis, staging, and therapeutic screening. This study lays the foundation for further testing CHP as a targeting moiety for theranostic delivery in various animal models. PMID:28877431

Clinical evaluations of mineralized collagen in the extraction sites preservation

PubMed Central

Feng, Lu; Zhang, Liang; Cui, Yun; Song, Tian-Xi; Qiu, Zhi-Ye; Wang, Xiu-Mei; Tan, Bao-Sheng

2016-01-01

The purpose of this study was to explore the different effects between biomimetic mineralized collagen (MC) and ordinary physically blended hydroxyapatite/collagen (HA/Col) composite in evaluating new bone formation and regenerated bone height in human extraction sockets. Thirty-four patients who cannot retain teeth caused by trauma or decay were randomly selected from Department of Stomatology of Dongzhimen Hospital from December 2013 to December 2014. The patients were randomly divided into two groups. After the operation of tooth extraction, 17 patients were implanted with biomimetic MC (MC group), and other 17 patients were implanted with ordinary physically blended nHA/Col composite (nHA/Col group). X-ray positioning projection by auto-photographing was taken to test the distance between the lowest position and the neighboring CEJm-CEJd immediately, 1 month and 3 months after the operation. The height of new bone formation of the MC group was significantly higher than the nHA/Col group. Biomimetic MC showed better clinical outcomes in the bone formation for extraction site preservation and would have broad application prospect in the field of oral and maxillofacial surgeries. PMID:26815224

Architecturally defined scaffolds from synthetic collagen and elastin analogues for the fabrication of bioengineered tissues

NASA Astrophysics Data System (ADS)

Caves, Jeffrey Morris

The microstructure and mechanics of collagen and elastin protein fiber networks dictate the mechanical responses of all soft tissues and related organ systems. In this project, we endeavored to meet or exceed native tissue biomechanical properties through mimicry of these extracellular matrix components with synthetic collagen fiber and a recombinant elastin-like protein polymer. Significantly, this work led to the development of a framework for the design and fabrication of protein-based tissue substitutes with enhanced strength, resilience, anisotropy, and more. We began with the development of a spinning process for scalable production of synthetic collagen fiber. Fiber with an elliptical cross-section of 53 +/- 14 by 21 +/- 3 mum and an ultimate tensile strength of 90 +/- 19 MPa was continuously produced at 60 meters per hour from an ultrafiltered collagen solution. The starting collagen concentration, flowrate, and needle size could be adjusted to control fiber size. The fiber was characterized with mechanical analysis, micro-differential scanning calorimetry, transmission electron microscopy, second harmonic generation analysis, and subcutaneous murine implant. We subsequently describe the scalable, semi-automated fabrication of elastin-like protein sheets reinforced with synthetic collagen fibers that can be positioned in a precisely defined three-dimensional hierarchical pattern. Multilamellar, fiber-reinforced elastic protein sheets were constructed with controlled fiber orientation and volume fraction. Structures were analyzed with scanning electron microscopy, transmission electron microscopy, and digital volumetric imaging. The effect of fiber orientation and volume fraction on Young's Modulus, yield stress, ultimate tensile stress, strain-to-failure, and resilience was evaluated in uniaxial tension. Increased fiber volume fraction and alignment with applied deformation significantly increased Young's Modulus, resilience, and yield stress. Highly

Anterior Cruciate Ligament Reconstruction in a Rabbit Model Using Silk-Collagen Scaffold and Comparison with Autograft

PubMed Central

Bi, Fanggang; Shi, Zhongli; Liu, An; Guo, Peng; Yan, Shigui

2015-01-01

The objective of the present study was to perform an in vivo assessment of a novel silk-collagen scaffold for anterior cruciate ligament (ACL) reconstruction. First, a silk-collagen scaffold was fabricated by combining sericin-extracted knitted silk fibroin mesh and type I collagen to mimic the components of the ligament. Scaffolds were electron-beam sterilized and rolled up to replace the ACL in 20 rabbits in the scaffold group, and autologous semitendinosus tendons were used to reconstruct the ACL in the autograft control group. At 4 and 16 weeks after surgery, grafts were retrieved and analyzed for neoligament regeneration and tendon-bone healing. To evaluate neoligament regeneration, H&E and immunohistochemical staining was performed, and to assess tendon-bone healing, micro-CT, biomechanical test, H&E and Russell-Movat pentachrome staining were performed. Cell infiltration increased over time in the scaffold group, and abundant fibroblast-like cells were found in the core of the scaffold graft at 16 weeks postoperatively. Tenascin-C was strongly positive in newly regenerated tissue at 4 and 16 weeks postoperatively in the scaffold group, similar to observations in the autograft group. Compared with the autograft group, tendon-bone healing was better in the scaffold group with trabecular bone growth into the scaffold. The results indicate that the silk-collagen scaffold has considerable potential for clinical application. PMID:25938408

Chondrogenic differentiation of human mesenchymal stem cells on fish scale collagen.

PubMed

Hsu, Han-Hsiu; Uemura, Toshimasa; Yamaguchi, Isamu; Ikoma, Toshiyuki; Tanaka, Junzo

2016-08-01

Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

In Vivo Osteogenic Potential of Biomimetic Hydroxyapatite/Collagen Microspheres: Comparison with Injectable Cement Pastes

PubMed Central

Manzanares, Maria-Cristina; Ginebra, Maria-Pau; Franch, Jordi

2015-01-01

The osteogenic capacity of biomimetic calcium deficient hydroxyapatite microspheres with and without collagen obtained by emulsification of a calcium phosphate cement paste has been evaluated in an in vivo model, and compared with an injectable calcium phosphate cement with the same composition. The materials were implanted into a 5 mm defect in the femur condyle of rabbits, and bone formation was assessed after 1 and 3 months. The histological analysis revealed that the cements presented cellular activity only in the margins of the material, whereas each one of the individual microspheres was covered with osteogenic cells. Consequently, bone ingrowth was enhanced by the microspheres, with a tenfold increase compared to the cement, which was associated to the higher accessibility for the cells provided by the macroporous network between the microspheres, and the larger surface area available for osteoconduction. No significant differences were found in terms of bone formation associated with the presence of collagen in the materials, although a more extensive erosion of the collagen-containing microspheres was observed. PMID:26132468

Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding.

PubMed

Barnes, Aileen M; Carter, Erin M; Cabral, Wayne A; Weis, MaryAnn; Chang, Weizhong; Makareeva, Elena; Leikin, Sergey; Rotimi, Charles N; Eyre, David R; Raggio, Cathleen L; Marini, Joan C

2010-02-11

Osteogenesis imperfecta is a heritable disorder that causes bone fragility. Mutations in type I collagen result in autosomal dominant osteogenesis imperfecta, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (cartilage-associated protein [CRTAP] and prolyl 3-hydroxylase 1 [P3H1]) cause autosomal recessive osteogenesis imperfecta with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. We identified two siblings who had recessive osteogenesis imperfecta without rhizomelia. They had a homozygous start-codon mutation in the peptidyl-prolyl isomerase B gene (PPIB), which results in a lack of cyclophilin B (CyPB), the third component of the complex. The proband's collagen had normal collagen folding and normal prolyl 3-hydroxylation, suggesting that CyPB is not the exclusive peptidyl-prolyl cis-trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought. 2010 Massachusetts Medical Society

Lack of Cyclophilin B in Osteogenesis Imperfecta with Normal Collagen Folding

PubMed Central

Barnes, Aileen M.; Carter, Erin M.; Cabral, Wayne A.; Weis, MaryAnn; Chang, Weizhong; Makareeva, Elena; Leikin, Sergey; Rotimi, Charles N.; Eyre, David R.; Raggio, Cathleen L.; Marini, Joan C.

2011-01-01

SUMMARY Osteogenesis imperfecta is a heritable disorder that causes bone fragility. Mutations in type I collagen result in autosomal dominant osteogenesis imperfecta, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (cartilage-associated protein [CRTAP] and prolyl 3-hydroxylase 1 [P3H1]) cause autosomal recessive osteogenesis imperfecta with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. We identified two siblings who had recessive osteogenesis imperfecta without rhizomelia. They had a homozygous start-codon mutation in the peptidyl-prolyl isomerase B gene (PPIB), which results in a lack of cyclophilin B (CyPB), the third component of the complex. The proband’s collagen had normal collagen folding and normal prolyl 3-hydroxylation, suggesting that CyPB is not the exclusive peptidyl-prolyl cis–trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought. PMID:20089953

Correlating the nanoscale mechanical and chemical properties of knockout mice bones

NASA Astrophysics Data System (ADS)

Kavukcuoglu, Nadire Beril

Bone is a mineral-organic composite where the organic matrix is mainly type I collagen plus small amounts of non-collagenous proteins including osteopontin (OPN), osteocalcin (OC) and fibrillin 2 (Fbn2). Mature bone undergoes remodeling continually so new bone is formed and old bone resorbed. Uncoupling between the bone resorption and bone formation causes an overall loss of bone mass and leads to diseases like osteoporosis and osteopenia. These are characterized by structural deterioration of the bone tissue and an increased risk of fracture. The non-collagenous bone proteins are known to have a role in regulating bone turnover and to affect the structural integrity of bone. OPN and OC play a key role in bone resorption and formation, while absence of Fbn-2 causes a connective tissue disorder (congenital contractural arachnodactyly) and has been associated with decreased bone mass. In this thesis nanoindentation and Raman-microspectroscopy techniques were used to investigate and correlate the mechanical and chemical properties of cortical femoral bones from OPN deficient (OPN-/-), OC deficient (OC-/-) and Fbn-2 deficient (Fbn2-/-) mice and their age, sex and background matched wild-type controls (OPN+/+, OC+/+ and Fbn2+/+). For OPN the hardness (H) and elastic modulus (E) of under 12 week OPN-/- bones were significantly lower than for OPN+/+ bones, but Raman showed no significant difference. Mechanical properties of bones from mice older than 12 weeks were not significantly different with genotype. However, mineralization and crystallinity from >50 week OPN-/- bones were significantly higher than for OPN+/+ bones. Mechanical properties of OPN-/- bones showed no variation with age, but mineralization, crystallinity and type-B carbonate substitution increased for both genotypes. For OC-/- intra-bone analyses showed that the hardness and crystallinity of the bones were significantly higher, especially in the mid-cortical sections, compared to OC+/+ bones. Fbn2

'Universal' microstructural patterns in cortical and trabecular, extracellular and extravascular bone materials: micromechanics-based prediction of anisotropic elasticity.

PubMed

Fritsch, Andreas; Hellmich, Christian

2007-02-21

Bone materials are characterized by an astonishing variability and diversity. Still, because of 'architectural constraints' due to once chosen material constituents and their physical interaction, the fundamental hierarchical organization or basic building plans of bone materials remain largely unchanged during biological evolution. Such universal patterns of microstructural organization govern the mechanical interaction of the elementary components of bone (hydroxyapatite, collagen, water; with directly measurable tissue-independent elastic properties), which are here quantified through a multiscale homogenization scheme delivering effective elastic properties of bone materials: at a scale of 10nm, long cylindrical collagen molecules, attached to each other at their ends by approximately 1.5nm long crosslinks and hosting intermolecular water inbetween, form a contiguous matrix called wet collagen. At a scale of several hundred nanometers, wet collagen and mineral crystal agglomerations interpenetrate each other, forming the mineralized fibril. At a scale of 5-10microm, the extracellular solid bone matrix is represented as collagen fibril inclusions embedded in a foam of largely disordered (extrafibrillar) mineral crystals. At a scale above the ultrastructure, where lacunae are embedded in extracellular bone matrix, the extravascular bone material is observed. Model estimates predicted from tissue-specific composition data gained from a multitude of chemical and physical tests agree remarkably well with corresponding acoustic stiffness experiments across a variety of cortical and trabecular, extracellular and extravascular materials. Besides from reconciling the well-documented, seemingly opposed concepts of 'mineral-reinforced collagen matrix' and 'collagen-reinforced mineral matrix' for bone ultrastructure, this approach opens new possibilities in the exploitation of computer tomographic data for nano-to-macro mechanics of bone organs.

Serum sclerostin levels associated with lumbar spine bone mineral density and bone turnover markers in patients with postmenopausal osteoporosis.

PubMed

Xu, Xiao-juan; Shen, Lin; Yang, Yan-ping; Lu, Fu-rong; Zhu, Rui; Shuai, Bo; Li, Cheng-gang; Wu, Man-xiang

2013-07-01

Sclerostin, expressed exclusively by osteocytes, is a negative regulator of bone formation. To gain insights into the action of sclerostin in postmenopausal osteoporosis, we evaluated serum sclerostin levels in postmenopausal women and investigated its possible associations with bone turnover markers in patients with postmenopausal osteoporosis. We detected serum sclerostin, and measured lumbar spine bone mineral density in 650 Chinese postmenopausal women. We also assessed serum levels of β-isomerized C-terminal crosslinking of type I collagen, intact N-terminal propeptide of type I collagen, N-mid fragment of osteocalcin, 25-hydroxyvitamin D, and estradiol. Serum sclerostin levels were lower in postmenopausal osteoporotic women compared with non-osteoporotic postmenopausal women ((38.79 ± 7.43) vs. (52.86 ± 6.69) pmol/L, P < 0.001). Serum sclerostin was positively correlated with lumbar spine bone mineral density (r = 0.391, P < 0.001) and weakly negatively correlated with β-isomerized C-terminal crosslinking of type I collagen, intact N-terminal propeptide of type I collagen, N-mid fragment of osteocalcin (r = -0.225, P < 0.001; r = -0.091, P = 0.046; r = -0.108, P = 0.018; respectively) in postmenopausal osteoporosis. There was no significant association of serum sclerostin with age, body mass index, 25-hydroxyvitamin D, and estradiol (r = -0.004, P = 0.926; r = 0.067, P = 0.143; r = 0.063, P = 0.165; r = -0.045, P = 0.324; respectively). Sclerostin may be involved in the pathogenesis of postmenopausal osteoporosis and may play a role in bone turnover.

The role of carboxy-terminal cross-linking telopeptide of type I collagen, dual x-ray absorptiometry bone strain and Romberg test in a new osteoporotic fracture risk evaluation: A proposal from an observational study.

PubMed

Ulivieri, Fabio M; Piodi, Luca P; Grossi, Enzo; Rinaudo, Luca; Messina, Carmelo; Tassi, Anna P; Filopanti, Marcello; Tirelli, Anna; Sardanelli, Francesco

2018-01-01

The consolidated way of diagnosing and treating osteoporosis in order to prevent fragility fractures has recently been questioned by some papers, which complained of overdiagnosis and consequent overtreatment of this pathology with underestimating other causes of the fragility fractures, like falls. A new clinical approach is proposed for identifying the subgroup of patients prone to fragility fractures. This retrospective observational study was conducted from January to June 2015 at the Nuclear Medicine-Bone Metabolic Unit of the of the Fondazione IRCCS Ca' Granda, Milan, Italy. An Italian population of 125 consecutive postmenopausal women was investigated for bone quantity and bone quality. Patients with neurological diseases regarding balance and vestibular dysfunction, sarcopenia, past or current history of diseases and use of drugs known to affect bone metabolism were excluded. Dual X-ray absorptiometry was used to assess bone quantity (bone mineral density) and bone quality (trabecular bone score and bone strain). Biochemical markers of bone turnover (type I collagen carboxy-terminal telopeptide, alkaline phosphatase, vitamin D) have been measured. Morphometric fractures have been searched by spine radiography. Balance was evaluated by the Romberg test. The data were evaluated with the neural network analysis using the Auto Contractive Map algorithm. The resulting semantic map shows the Minimal Spanning Tree and the Maximally Regular Graph of the interrelations between bone status parameters, balance conditions and fractures of the studied population. A low fracture risk seems to be related to a low carboxy-terminal cross-linking telopeptide of type I collagen level, whereas a positive Romberg test, together with compromised bone trabecular microarchitecture DXA parameters, appears to be strictly connected with fragility fractures. A simple assessment of the risk of fragility fracture is proposed in order to identify those frail patients at risk for

Viscoelastic properties of model segments of collagen molecules.

PubMed

Gautieri, Alfonso; Vesentini, Simone; Redaelli, Alberto; Buehler, Markus J

2012-03-01

Collagen is the prime construction material in vertebrate biology, determining the mechanical behavior of connective tissues such as tendon, bone and skin. Despite extensive efforts in the investigation of the origin of collagen unique mechanical properties, a deep understanding of the relationship between molecular structure and mechanical properties remains elusive, hindered by the complex hierarchical structure of collagen-based tissues. In particular, although extensive studies of viscoelastic properties have been pursued at the macroscopic (fiber/tissue) level, fewer investigations have been performed at the smaller scales, including in particular collagen molecules and fibrils. These scales are, however, important for a complete understanding of the role of collagen as an important constituent in the extracellular matrix. Here, using an atomistic modeling approach, we perform in silico creep tests of a collagen-like peptide, monitoring the strain-time response for different values of applied external load. The results show that individual collagen molecules exhibit a nonlinear viscoelastic behavior, with a Young's modulus increasing from 6 to 16GPa (for strains up to 20%), a viscosity of 3.84.±0.38Pa·s, and a relaxation time in the range of 0.24-0.64ns. The single molecule viscosity, for the first time reported here, is several orders of magnitude lower than the viscosity found for larger-scale single collagen fibrils, suggesting that the viscous behavior of collagen fibrils and fibers involves additional mechanisms, such as molecular sliding between collagen molecules within the fibril or the effect of relaxation of larger volumes of solvent. Based on our molecular modeling results we propose a simple structural model that describes collagen tissue as a hierarchical structure, providing a bottom-up description of elastic and viscous properties form the properties of the tissue basic building blocks. Copyright © 2011 International Society of Matrix Biology

Preparation of hydroxyapatite/collagen injectable bone paste with an anti-washout property utilizing sodium alginate. Part 1: influences of excess supplementation of calcium compounds.

PubMed

Sato, Taira; Kikuchi, Masanori; Aizawa, Mamoru

2017-03-01

The anti-washout property, viscosity, and cytocompatibility to an osteoblastic cell line, MG-63, of anti-washout pastes were investigated. Mixing a hydroxyapatite/collagen bone-like nanocomposite (HAp/Col), an aqueous solution of sodium alginate (Na-Alg), which is a paste hardening and lubricant agent, and supplementation of calcium carbonate or calcium citrate (Ca-Cit) as a calcium resource for the hardening reaction realized an injectable bone paste. Adding Ca-Cit at a concentration greater than eight times the Ca 2+ ion concentration to Na-Alg improved the anti-washout property. Although the viscosity test indicated a gradual increase in the paste viscosity as the calcium compounds increased, pastes with excess supplementation of calcium compounds exhibited injectability through a syringe with a 1.8 mm inner diameter, realizing an injectable bone filler. Furthermore, the anti-washout pastes with Ca-Cit had almost the same cell proliferation rate as that of the HAp/Col dense body. Therefore, HAp/Col injectable anti-washout pastes composed of the HAp/Col, Na-Alg, and Ca-Cit are potential candidates for bioresorbable bone filler pastes.

Stabilization and Anomalous Hydration of Collagen Fibril under Heating

PubMed Central

Gevorkian, Sasun G.; Allahverdyan, Armen E.; Gevorgyan, David S.; Simonian, Aleksandr L.; Hu, Chin-Kun

2013-01-01

Background Type I collagen is the most common protein among higher vertebrates. It forms the basis of fibrous connective tissues (tendon, chord, skin, bones) and ensures mechanical stability and strength of these tissues. It is known, however, that separate triple-helical collagen macromolecules are unstable at physiological temperatures. We want to understand the mechanism of collagen stability at the intermolecular level. To this end, we study the collagen fibril, an intermediate level in the collagen hierarchy between triple-helical macromolecule and tendon. Methodology/Principal Finding When heating a native fibril sample, its Young’s modulus decreases in temperature range 20–58°C due to partial denaturation of triple-helices, but it is approximately constant at 58–75°C, because of stabilization by inter-molecular interactions. The stabilization temperature range 58–75°C has two further important features: here the fibril absorbs water under heating and the internal friction displays a peak. We relate these experimental findings to restructuring of collagen triple-helices in fibril. A theoretical description of the experimental results is provided via a generalization of the standard Zimm-Bragg model for the helix-coil transition. It takes into account intermolecular interactions of collagen triple-helices in fibril and describes water adsorption via the Langmuir mechanism. Conclusion/Significance We uncovered an inter-molecular mechanism that stabilizes the fibril made of unstable collagen macromolecules. This mechanism can be relevant for explaining stability of collagen. PMID:24244320

Maintenance of a bone collagen phenotype by osteoblast-like cells in 3D periodic porous titanium (Ti-6Al-4 V) structures fabricated by selective electron beam melting

PubMed Central

Hrabe, Nikolas W.; Heinl, Peter; Bordia, Rajendra K.; Körner, Carolin; Fernandes, Russell J.

2013-01-01

Regular 3D periodic porous Ti-6Al-4 V structures were fabricated by the selective electron beam melting method (EBM) over a range of relative densities (0.17–0.40) and pore sizes (500–1500 μm). Structures were seeded with human osteoblast-like cells (SAOS-2) and cultured for four weeks. Cells multiplied within these structures and extracellular matrix collagen content increased. Type I and type V collagens typically synthesized by osteoblasts were deposited in the newly formed matrix with time in culture. High magnification scanning electron microscopy revealed cells attached to surfaces on the interior of the structures with an increasingly fibrous matrix. The in-vitro results demonstrate that the novel EBM-processed porous structures, designed to address the effect of stress-shielding, are conducive to osteoblast attachment, proliferation and deposition of a collagenous matrix characteristic of bone. PMID:23869614

A Spontaneous 3D Bone-On-a-Chip for Bone Metastasis Study of Breast Cancer Cells.

PubMed

Hao, Sijie; Ha, Laura; Cheng, Gong; Wan, Yuan; Xia, Yiqiu; Sosnoski, Donna M; Mastro, Andrea M; Zheng, Si-Yang

2018-03-01

Bone metastasis occurs at ≈70% frequency in metastatic breast cancer. The mechanisms used by tumors to hijack the skeleton, promote bone metastases, and confer therapeutic resistance are poorly understood. This has led to the development of various bone models to investigate the interactions between cancer cells and host bone marrow cells and related physiological changes. However, it is challenging to perform bone studies due to the difficulty in periodic sampling. Herein, a bone-on-a-chip (BC) is reported for spontaneous growth of a 3D, mineralized, collagenous bone tissue. Mature osteoblastic tissue of up to 85 µm thickness containing heavily mineralized collagen fibers naturally formed in 720 h without the aid of differentiation agents. Moreover, co-culture of metastatic breast cancer cells is examined with osteoblastic tissues. The new bone-on-a-chip design not only increases experimental throughput by miniaturization, but also maximizes the chances of cancer cell interaction with bone matrix of a concentrated surface area and facilitates easy, frequent observation. As a result, unique hallmarks of breast cancer bone colonization, previously confirmed only in vivo, are observed. The spontaneous 3D BC keeps the promise as a physiologically relevant model for the in vitro study of breast cancer bone metastasis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Calorie restriction and bone health in young, overweight individuals.

PubMed

Redman, Leanne M; Rood, Jennifer; Anton, Stephen D; Champagne, Catherine; Smith, Steven R; Ravussin, Eric

2008-09-22

Calorie restriction (CR) is promoted to increase longevity, yet this regimen could lead to bone loss and fracture and therefore affect quality of life. Forty-six individuals were randomized to 4 groups for 6 months: (1) healthy diet (control group); (2) 25% CR from baseline energy requirements (CR group); (3) 25% energy deficit by a combination of CR and increased aerobic exercise (CR + EX group); and (4) low-calorie diet (890 kcal/d; goal, 15% weight loss) followed by weight maintenance (LCD group). Bone mineral density (total body and hip by dual-energy x-ray absorptiometry) and serum bone markers (bone-specific alkaline phosphatase, osteocalcin, cross-linked C-telopeptide of type I collagen, and cross-linked N-telopeptide of type I collagen) were measured at baseline and after 6 months. Mean +/- SE body weight was reduced by -1.0% +/- 1.1% (control), -10.4% +/- 0.9% (CR), -10.0% +/- 0.8% (CR + EX), and -13.9% +/- 0.7% (LCD). Compared with the control group, none of the groups showed any change in bone mineral density for total body or hip. Bone resorption by serum cross-linked C-telopeptide of type I collagen was increased in all 3 intervention groups, with the largest change observed in the LCD group (CR, 23% +/- 10%; CR + EX, 22% +/- 9%; and LCD, 74% +/- 16% vs control, 4% +/- 10%). Serum levels of cross-linked N-telopeptide of type I collagen were also increased in the LCD group. With regard to bone formation, bone alkaline phosphatase levels were decreased in the CR group (-23% +/- 10%) but were unchanged in the CR + EX, LCD, and control groups. Moderate CR, with or without exercise, that preserves calcium intake for 6 months leads to large changes in body composition without significant bone loss in young adults. Longer studies with assessments of bone architecture are needed to confirm that CR nutrient-dense diets have no deleterious effect on bone health. clinicaltrials.gov Identifier: NCT00099151.