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Collagen Type II (collagen + type_ii)
Selected AbstractsPrimary mouse embryonic fibroblasts: A model of mesenchymal cartilage formation,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Christopher J. Lengner Cartilage formation is an intricate process that requires temporal and spatial organization of regulatory factors in order for a mesenchymal progenitor cell to differentiate through the distinct stages of chondrogenesis. Gene function during this process has best been studied by analysis of in vivo cartilage formation in genetically altered mouse models. Mouse embryonic fibroblasts (MEFs) isolated from such mouse models have been widely used for the study of growth control and DNA damage response. Here, we address the potential of MEFs to undergo chondrogenic differentiation. We demonstrate for the first time that MEFs can enter and complete the program of chondrogenic differentiation ex vivo, from undifferentiated progenitor cells to mature, hypertrophic chondrocytes. We show that chondrogenic differentiation can be induced by cell,cell contact or BMP-2 treatment, while in combination, these conditions synergistically enhance chondrocyte differentiation resulting in the formation of 3-dimensional (3-D) cartilaginous tissue ex vivo. Temporal expression profiles of pro-chondrogenic transcription factors Bapx1 and Sox9 and cartilaginous extracellular matrix (ECM) proteins Collagen Type II and X (Coll II and Coll X) demonstrate that the in vivo progression of chondrocyte maturation is recapitulated in the MEF model system. Our findings establish the MEF as a powerful tool for the generation of cartilaginous tissue ex vivo and for the study of gene function during chondrogenesis. © 2004 Wiley-Liss, Inc. [source] Cartilage Tissue Engineering With Demineralized Bone Matrix Gelatin and Fibrin Glue Hybrid Scaffold: An In Vitro StudyARTIFICIAL ORGANS, Issue 2 2010Zheng-Hui Wang Abstract To develop a cartilage-like tissue with hybrid scaffolds of demineralized bone matrix gelatin (BMG) and fibrin, rabbit chondrocytes were cultured on hybrid fibrin/BMG scaffolds in vitro. BMG scaffolds were carefully soaked in a chondrocyte,fibrin suspension, which was polymerized by submerging the constructs into thrombin,calcium chloride solution. Engineered cartilage-like tissue grown on the scaffolds was characterized by histology, immunolocalization, scanning electron microscopy, biochemical assays, and analysis of gene expression at different time points of the in vitro culture. The presence of proteoglycan in the fibrin/BMG hybrid constructs was confirmed by positive toluidine blue and alcian blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene-encoded cartilage-specific markers, collagen type II, and aggrecan core protein. The glycosaminoglycan production and hydroxyproline content of tissue grown on the fibrin/BMG hybrid scaffolds were higher than that of the BMG group. In conclusion, the fibrin/BMG hybrid scaffolds may serve as a potential cell delivery vehicle and a structural basis for cartilage tissue engineering. [source] Analysis of N-cadherin function in limb mesenchymal chondrogenesis in vitro,DEVELOPMENTAL DYNAMICS, Issue 2 2002Anthony M. Delise Abstract During embryonic limb development, cartilage formation is presaged by a crucial mesenchymal cell condensation phase. N-Cadherin, a Ca2+ -dependent cell,cell adhesion molecule, is expressed in embryonic chick limb buds in a spatiotemporal pattern suggestive of its involvement during cellular condensation; functional blocking of N-cadherin homotypic binding, by using a neutralizing monoclonal antibody, results in perturbed chondrogenesis in vitro and in vivo. In high-density micromass cultures of embryonic limb mesenchymal cells, N-cadherin expression level is high during days 1 and 2, coincident with active cellular condensation, and decreases upon overt chondrogenic differentiation from day 3 on. In this study, we have used a transfection approach to evaluate the effects of gain- and loss-of-function expression of N-cadherin constructs on mesenchymal condensation and chondrogenesis in vitro. Chick limb mesenchymal cells were transfected by electroporation with recombinant expression plasmids encoding wild-type or two mutant extracellular/cytoplasmic deletion forms of N-cadherin. Expression of the transfected N-cadherin forms showed a transient profile, being high on days 1,2 of culture, and decreasing by day 3, fortuitously coincident with the temporal profile of endogenous N-cadherin gene expression. Examined by means of peanut agglutinin (PNA) staining for condensing precartilage mesenchymal cells, cultures overexpressing wild-type N-cadherin showed enhanced cellular condensation on culture days 2 and 3, whereas expression of the deletion mutant forms (extracellular/cytoplasmic) of N-cadherin resulted in a decrease in PNA staining, suggesting that a complete N-cadherin protein is required for normal cellular condensation to occur. Subsequent chondrogenesis was also affected. Cultures overexpressing the wild-type N-cadherin protein showed enhanced chondrogenesis, indicated by increased production of cartilage matrix (sulfated proteoglycans, collagen type II, and cartilage proteoglycan link protein), as well as increased cartilage nodule number and size of individual nodules, compared with control cultures and cultures transfected with either of the two mutant N-cadherin constructs. These results demonstrate that complete N-cadherin function, at the levels of both extracellular homotypic binding and cytoplasmic linkage to the cytoskeleton by means of the catenin complex, is required for chondrogenesis by mediating functional mesenchymal cell condensation. © 2002 Wiley-Liss, Inc. [source] Hypertrophy and physiological death of equine chondrocytes in vitroEQUINE VETERINARY JOURNAL, Issue 6 2007Y. A. Ahmed Summary Reasons for performing study: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. Objective: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. Methods: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. Results: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. Conclusions: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. Potential relevance: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis. [source] Fibroblast growth factor (FGF)-23 and fetuin-A in calcified carotid atheromaHISTOPATHOLOGY, Issue 6 2010Mathias Voigt Voigt M, Fischer D-C, Rimpau M, Schareck W & Haffner D (2010) Histopathology56, 775,788 Fibroblast growth factor (FGF)-23 and fetuin-A in calcified carotid atheroma Aims:, Human atheroma calcification occurs secondary to repetitive injury/remodelling of the vessel wall and might be initiated by adherence of mineral-loaded fetuin-A whether or not professional matrix mineralizing cells are present. The aim was to investigate the contribution of fibroblast growth factor (FGF)-23 to ectopic mineralization. Methods and results:, Serial sections of formalin-fixed paraffin-embedded human carotid atheroma (n = 54) were investigated with respect to (i) size and distribution of calcific deposits, (ii) indicators of chondrogenic/osteogenic transformation, and (iii) expression of fetuin-A and FGF-23. All specimens were calcified and SOX-9, collagen type II, cathepsin-K, fetuin-A and FGF-23 expression was seen in 46, 53, 53, 54 and 48 specimens, respectively. The intracellular detection of FGF-23 (45/48) indicates local synthesis. Whereas fetuin-A expression was seen also within areas of vascular smooth muscle actin-positive cells adjacent to calcific deposits, FGF-23 expression was apparently restricted to the mineralization-prone areas. Both local expression and FGF-23 serum concentrations were significantly associated with the degree of atheroma calcification. Conclusions:, Besides the induction of bone islets and subsequent mineral deposition, severe remodelling of the vessel wall is sufficient to create a mineralizable fetuin-A-attracting microenvironment. FGF-23 might contribute to the formation of proper mineral, i.e. control local phosphate concentration. [source] Quantification of expression levels of cellular differentiation markers does not support a general shift in the cellular phenotype of osteoarthritic chondrocytesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2003Pia Margarethe Gebhard Abstract Many studies have shown increased anabolic activity in osteoarthritic cartilage and have suggested changes in the cellular phenotypes of articular chondrocytes. Most of these studies relied on non-quantitative technologies, which did not allow the estimation of the relative importance of the different differentiation phenomena. In the present study, we developed and used quantitative PCR assays for collagen types I, II(total), IIA, III, and X as marker genes indicating cellular synthetic activity (collagen type II) as well as differentiation pattern of chondrocytes (collagen types I, IIA, III, and X) and quantified these genes in normal, early degenerative, and late stage osteoarthritic cartilage in parallel. At first sight, our results confirmed previously published data showing hardly any expression of collagen genes in normal and significantly enhanced expression in osteoarthritic cartilage. This included collagen types II, III, and IIA, but also collagen types I(,1) and X. However, if one considers the ratios of the various markers of chondrocytic differentiation in comparison to collagen type II, the main synthetic product of differentiated chondrocytes, no shift in the cellular phenotype was detectable. In fact, expression ratios remained constant or were even decreased in osteoarthritic cartilage. Our results confirm that normal adult human articular chondrocytes display hardly any expression activity of the collagen types investigated, whereas osteoarthritic chondrocytes show very increased synthetic activity. The largely unchanged ratios of collagen subtypes investigated indicate that no general shift in the cellular phenotype does occur in osteoarthritic cartilage as suggested by previous investigations. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Multilineage mesenchymal differentiation potential of human trabecular bone-derived cellsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2002Ulrich Nöth Abstract Explant cultures of adult human trabecular bone fragments give rise to osteoblastic cells, that are known to express osteoblast-related genes and mineralize extracellular matrix. These osteoblastic cells have also been shown to undergo adipogenesis in vitro and chondrogenesis in vivo. Here we report the in vitro developmental potential of adult human osteoblastic cells (hOB) derived from explant cultures of collagenase-pretreated trabecular bone fragments. In addition to osteogenic and adipogenic differentiation, these cells are capable of chondrogenic differentiation in vitro in a manner similar to adult human bone marrow-derived mesenchymal progenitor cells. High-density pellet cultures of hOB maintained in chemically defined serum-free medium, supplemented with transforming growth factor-,1, were composed of morphologically distinct, chondrocyte-like cells expressing mRNA transcripts of collagen types II, IX and X, and aggrecan. The cells within the high-density pellet cultures were surrounded by a sulfated prote-oglycan-rich extracellular matrix that immunostained for collagen type II and proteoglycan link protein. Osteogenic differentiation of hOB was verified by an increased number of alkaline phosphatase-positive cells, that expressed osteoblast-related transcripts such as alkaline phosphatase, collagen type I, osteopontin and osteocalcin, and formed mineralized matrix in monolayer cultures treated with ascorbate, ,-glycerophosphate, and bone morphogenetic protein-2. Adipogenic differentiation of hOB was determined by the appearance of intracellular lipid droplets, and expression of adipocyte-specific genes, such as lipoprotein lipase and peroxisome proliferator-activated receptor ,2, in monolayer cultures treated with dexamethasone, indomethacin, insulin and 3-isobutyl-l-methylxanthine. Taken together, these results show that cells derived from collagenase-treated adult human trabecular bone fragments have the potential to differentiate into multiple mesenchymal lineages in vitro, indicating their developmental plasticity and suggesting their mesenchymal progenitor nature. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Compressive compared with tensile loading of medial collateral ligament scar in vitro uniquely influences mRNA levels for aggrecan, collagen type II, and collagenaseJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2000Tokifumi Majima To test the hypothesis that loading conditions can be used to engineer early ligament scar behaviors, we used an in vitro system to examine the effect that cyclic hydrostatic compression and cyclic tension applied to 6-week rabbit medial collateral ligament scars had on mRNA levels for matrix molecules, collagenase, and the proto-oncogenes c-fos and c-jun. Our specific hypothesis was that tensile stress would promote more normal mRNA expression in ligament whereas compression would lead to higher levels of mRNA for cartilage-like molecules. Femur (injured medial collateral ligament)-tibia complexes were subjected to a hydrostatic pressure of 1 MPa or a tensile stress of 1 MPa of 0.5 Hz for 1 minute followed by 14 minutes of rest. On the basis of a preliminary optimization experiment, this 15-minute testing cycle was repeated for 4 hours. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed for mechanically treated medial collateral ligament scars with use of rabbit specific primer sets for types I, II, and III collagen, decorin, biglycan, fibromodulin, versican, aggrecan, collagenase, c-fos, c-jun, and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. Cyclic hydrostatic compression resulted in a statistically significant increase in mRNA levels of type-II collagen (171% of nonloaded values) and aggrecan (313% of nonloaded values) but statistically significant decreases in collagenase mRNA levels (35% of nonloaded values). Cyclic tension also resulted in a statistically significant decrease in collagenase mRNA levels (66% of nonloaded values) and an increase in aggrecan mRNA levels (458% of nonloaded values) but no significant change in the mRNA levels for the other molecules. The results show that it is possible to alter mRNA levels for a subset of genes in scar tissue by supplying unique mechanical stimuli in vitro and thus that further investigation of scar engineering for potential reimplantation appears feasible. [source] Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage regeneration when subcutaneously implanted in nude miceJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 7 2009J. T. Oliveira Abstract Gellan gum is a polysaccharide that has been recently proposed by our group for cartilage tissue-engineering applications. It is commonly used in the food and pharmaceutical industry and has the ability to form stable gels without the use of harsh reagents. Gellan gum can function as a minimally invasive injectable system, gelling inside the body in situ under physiological conditions and efficiently adapting to the defect site. In this work, gellan gum hydrogels were combined with human articular chondrocytes (hACs) and were subcutaneously implanted in nude mice for 4 weeks. The implants were collected for histological (haematoxylin and eosin and Alcian blue staining), biochemical [dimethylmethylene blue (GAG) assay], molecular (real-time PCR analyses for collagen types I, II and X, aggrecan) and immunological analyses (immunolocalization of collagen types I and II). The results showed a homogeneous cell distribution and the typical round-shaped morphology of the chondrocytes within the matrix upon implantation. Proteoglycans synthesis was detected by Alcian blue staining and a statistically significant increase of proteoglycans content was measured with the GAG assay quantified from 1 to 4 weeks of implantation. Real-time PCR analyses showed a statistically significant upregulation of collagen type II and aggrecan levels in the same periods. The immunological assays suggest deposition of collagen type II along with some collagen type I. The overall data shows that gellan gum hydrogels adequately support the growth and ECM deposition of human articular chondrocytes when implanted subcutaneously in nude mice. Copyright © 2009 John Wiley & Sons, Ltd. [source] Sheep embryonic stem-like cells transplanted in full-thickness cartilage defectsJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 3 2009Maria Dattena Abstract Articular cartilage regeneration is limited. Embryonic stem (ES) cell lines provide a source of totipotent cells for regenerating cartilage. Anatomical, biomechanical, physiological and immunological similarities between humans and sheep make this animal an optimal experimental model. This study examines the repair process of articular cartilage in sheep after transplantation of ES-like cells isolated from inner cell masses (ICMs) derived from in vitro -produced (IVP) vitrified embryos. Thirty-five ES-like colonies from 40 IVP embryos, positive for stage-specific embryonic antigens (SSEAs), were pooled in groups of two or three, embedded in fibrin glue and transplanted into osteochondral defects in the medial femoral condyles of 14 ewes. Empty defect (ED) and cell-free glue (G) in the controlateral stifle joint served as controls. The Y gene sequence was used to detect ES-like cells in the repair tissue by in situ hybridization (ISH). Two ewes were euthanized at 1 month post-operatively, three each at 2 and 6 months and four at 12 months. Repairing tissue was examined by biomechanical, macroscopic, histological, immunohistochemical (collagen type II) and ISH assays. Scores of all treatments showed no statistical significant differences among treatment groups at a given time period, although ES-like grafts showed a tendency toward a better healing process. ISH was positive in all ES-like specimens. This study demonstrates that ES-like cells transplanted into cartilage defects stimulate the repair process to promote better organization and tissue bulk. However, the small number of cells applied and the short interval between surgery and euthanasia might have negatively affected the results. Copyright © 2009 John Wiley & Sons, Ltd. [source] Two epithelial cell invasion-related loci of the oral pathogen Actinobacillus actinomycetemcomitansMOLECULAR ORAL MICROBIOLOGY, Issue 1 2004L. Li Two invasion-related loci, apiA and the two-gene operon apiBC, were isolated from the oral pathogen Actinobacillus actinomycetemcomitans UT32. apiA encodes a 32.5 kDa protein that migrates on SDS-PAGE as a 101 kDa protein as detected by Western blot analysis or silver staining of an outer membrane-enriched fraction of Escherichia coli transformants. E. coli expressing ApiA have a different phenotype than the host vector, in broth and on solid media, and a colony morphology that resembles that of fresh A. actinomycetemcomitans isolates. These E. coli transformants bound to chicken collagen type II, human collagen type II, III, V and fibronectin. apiB and apiC encode proteins of 130.1 and 70.6 kDa, respectively. ApiBC conferred on E. coli a slightly enhanced ability to bind to collagen type III. ApiA- and ApiB-deficient mutants were constructed in A. actinomycetemcomitans. The ApiB-mutant had 4-fold diminished invasion of KB cells; the ApiA-mutant had increased invasion. Both loci were found in all A. actinomycetemcomitans strains, although polymorphism was detected only for apiBC. The deduced sequences of these invasion-related proteins are homologous to members of the YadA adhesin/invasin family. [source] Cartilage Tissue Engineering With Demineralized Bone Matrix Gelatin and Fibrin Glue Hybrid Scaffold: An In Vitro StudyARTIFICIAL ORGANS, Issue 2 2010Zheng-Hui Wang Abstract To develop a cartilage-like tissue with hybrid scaffolds of demineralized bone matrix gelatin (BMG) and fibrin, rabbit chondrocytes were cultured on hybrid fibrin/BMG scaffolds in vitro. BMG scaffolds were carefully soaked in a chondrocyte,fibrin suspension, which was polymerized by submerging the constructs into thrombin,calcium chloride solution. Engineered cartilage-like tissue grown on the scaffolds was characterized by histology, immunolocalization, scanning electron microscopy, biochemical assays, and analysis of gene expression at different time points of the in vitro culture. The presence of proteoglycan in the fibrin/BMG hybrid constructs was confirmed by positive toluidine blue and alcian blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene-encoded cartilage-specific markers, collagen type II, and aggrecan core protein. The glycosaminoglycan production and hydroxyproline content of tissue grown on the fibrin/BMG hybrid scaffolds were higher than that of the BMG group. In conclusion, the fibrin/BMG hybrid scaffolds may serve as a potential cell delivery vehicle and a structural basis for cartilage tissue engineering. [source] Extent of cell differentiation and capacity for cartilage synthesis in human adult adipose-derived stem cells: Comparison with fetal chondrocytesBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Nastaran Mahmoudifar Abstract This study evaluated the extent of differentiation and cartilage biosynthetic capacity of human adult adipose-derived stem cells relative to human fetal chondrocytes. Both types of cell were seeded into nonwoven-mesh polyglycolic acid (PGA) scaffolds and cultured under dynamic conditions with and without addition of TGF-,1 and insulin. Gene expression for aggrecan and collagen type II was upregulated in the stem cells in the presence of growth factors, and key components of articular cartilage such as glycosaminoglycan (GAG) and collagen type II were synthesized in cultured tissue constructs. However, on a per cell basis and in the presence of growth factors, accumulation of GAG and collagen type II were, respectively, 3.4- and 6.1-fold lower in the stem cell cultures than in the chondrocyte cultures. Although the stem cells synthesized significantly higher levels of total collagen than the chondrocytes, only about 2.4% of this collagen was collagen type II. Relative to cultures without added growth factors, treatment of the stem cells with TGF-,1 and insulin resulted in a 59% increase in GAG synthesis, but there was no significant change in collagen production even though collagen type II gene expression was upregulated 530-fold. In contrast, in the chondrocyte cultures, synthesis of collagen type II and levels of collagen type II as a percentage of total collagen more than doubled after growth factors were applied. Although considerable progress has been achieved to develop differentiation strategies and scaffold-based culture techniques for adult mesenchymal stem cells, the extent of differentiation of human adipose-derived stem cells in this study and their capacity for cartilage synthesis fell considerably short of those of fetal chondrocytes. Biotechnol. Bioeng. 2010;107: 393,401. © 2010 Wiley Periodicals, Inc. [source] Seeding density modulates migration and morphology of rabbit chondrocytes cultured in collagen gelsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Ali Baradar Khoshfetrat Abstract The cultures of rabbit chondrocytes embedded in collagen gels were conducted to investigate the cell behaviors and consequent architectures of cell aggregation in an early culture phase. The chondrocyte cells seeded at 1.0,×,105 cells/cm3 underwent a transition to spindle-shaped morphology, and formed the loose aggregates with a starburst shape by means of possible migration and gathering. These aggregates accompanied the poor production of collagen type II, while the cells seeded at 1.6,×,106 cells/cm3 exhibited active proliferation to form the dense aggregates rich in collagen type II. Stereoscopic observation was performed at 5 days to define the migrating cells in terms of a morphology-relating parameter of sphericity determined for individual cells in the gels. The frequency of migrating cells decreased with increasing seeding density, while the frequency of dividing cells showed the counter trend. The culture seeded at 1.0,×,105 cells/cm3 gave the migrating cell frequency of 0.25, the value of which was 25 times higher than that at 1.6,×,106 cells/cm3. In addition, the analysis of mRNA expression revealed that the chondrocyte cells seeded at 1.0,×,105 cells/cm3 showed appreciable down-regulation in collagen type II relating to differentiation and up-regulation in matrix metalloproteinases relating to migration, as compared to the cells seeded at 1.6,×,106 cells/cm3. These data supports the morphological analyses concerning the cell migration and aggregate formation in the cultures with varied seeding densities. It is concluded that the seeding density is an important factor to affect the cell behaviors and architecture of aggregates and thereby to modulate the quality of cultured cartilage. © 2008 Wiley Periodicals, Inc. Biotechnol. Bioeng. 2009;102: 294,302. © 2008 Wiley Periodicals, Inc. [source] Diminished Bone Formation During Diabetic Fracture Healing is Related to the Premature Resorption of Cartilage Associated With Increased Osteoclast Activity,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2007Rayyan A Kayal Abstract Histological and molecular analysis of fracture healing in normal and diabetic animals showed significantly enhanced removal of cartilage in diabetic animals. Increased cartilage turnover was associated with elevated osteoclast numbers, a higher expression of genes that promote osteoclastogenesis, and diminished primary bone formation. Introduction: Diminished bone formation, an increased incidence of nonunions, and delayed fracture healing have been observed in animal models and in patients with diabetes. Fracture healing is characterized by the formation of a stabilizing callus in which cartilage is formed and then resorbed and replaced by bone. To gain insight into how diabetes affects fracture healing, studies were carried out focusing on the impact of diabetes on the transition from cartilage to bone. Materials and Methods: A low-dose treatment protocol of streptozotocin in CD-1 mice was used to induce a type 1 diabetic condition. After mice were hyperglycemic for 3 weeks, controlled closed simple transverse fractures of the tibia were induced and fixed by intramedullary pins. Histomorphometric analysis of the tibias obtained 12, 16, and 22 days after fracture was performed across the fracture callus at 0.5 mm proximal and distal increments using computer-assisted image analysis. Another group of 16-day samples were examined by ,CT. RNA was isolated from a separate set of animals, and the expression of genes that reflect the formation and removal of cartilage and bone was measured by real-time PCR. Results: Molecular analysis of collagen types II and × mRNA expression showed that cartilage formation was the same during the initial period of callus formation. Histomorphometric analysis of day 12 fracture calluses showed that callus size and cartilage area were also similar in normoglycemic and diabetic mice. In contrast, on day 16, callus size, cartilage tissue, and new bone area were 2.0-, 4.4-, and 1.5-fold larger, respectively, in the normoglycemic compared with the diabetic group (p < 0.05). Analysis of ,CT images indicated that the bone volume in the normoglycemic animals was 38% larger than in diabetic animals. There were 78% more osteoclasts in the diabetic group compared with the normoglycemic group (p < 0.05) on day 16, consistent with the reduction in cartilage. Real-time PCR showed significantly elevated levels of mRNA expression for TNF-,, macrophage-colony stimulating factor, RANKL, and vascular endothelial growth factor-A in the diabetic group. Similarly, the mRNA encoding ADAMTS 4 and 5, major aggrecanases that degrade cartilage, was also elevated in diabetic animals. Conclusions: These results suggest that impaired fracture healing in diabetes is characterized by increased rates of cartilage resorption. This premature loss of cartilage leads to a reduction in callus size and contributes to decreased bone formation and mechanical strength frequently reported in diabetic fracture healing. [source] Quantification of expression levels of cellular differentiation markers does not support a general shift in the cellular phenotype of osteoarthritic chondrocytesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2003Pia Margarethe Gebhard Abstract Many studies have shown increased anabolic activity in osteoarthritic cartilage and have suggested changes in the cellular phenotypes of articular chondrocytes. Most of these studies relied on non-quantitative technologies, which did not allow the estimation of the relative importance of the different differentiation phenomena. In the present study, we developed and used quantitative PCR assays for collagen types I, II(total), IIA, III, and X as marker genes indicating cellular synthetic activity (collagen type II) as well as differentiation pattern of chondrocytes (collagen types I, IIA, III, and X) and quantified these genes in normal, early degenerative, and late stage osteoarthritic cartilage in parallel. At first sight, our results confirmed previously published data showing hardly any expression of collagen genes in normal and significantly enhanced expression in osteoarthritic cartilage. This included collagen types II, III, and IIA, but also collagen types I(,1) and X. However, if one considers the ratios of the various markers of chondrocytic differentiation in comparison to collagen type II, the main synthetic product of differentiated chondrocytes, no shift in the cellular phenotype was detectable. In fact, expression ratios remained constant or were even decreased in osteoarthritic cartilage. Our results confirm that normal adult human articular chondrocytes display hardly any expression activity of the collagen types investigated, whereas osteoarthritic chondrocytes show very increased synthetic activity. The largely unchanged ratios of collagen subtypes investigated indicate that no general shift in the cellular phenotype does occur in osteoarthritic cartilage as suggested by previous investigations. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Multilineage mesenchymal differentiation potential of human trabecular bone-derived cellsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2002Ulrich Nöth Abstract Explant cultures of adult human trabecular bone fragments give rise to osteoblastic cells, that are known to express osteoblast-related genes and mineralize extracellular matrix. These osteoblastic cells have also been shown to undergo adipogenesis in vitro and chondrogenesis in vivo. Here we report the in vitro developmental potential of adult human osteoblastic cells (hOB) derived from explant cultures of collagenase-pretreated trabecular bone fragments. In addition to osteogenic and adipogenic differentiation, these cells are capable of chondrogenic differentiation in vitro in a manner similar to adult human bone marrow-derived mesenchymal progenitor cells. High-density pellet cultures of hOB maintained in chemically defined serum-free medium, supplemented with transforming growth factor-,1, were composed of morphologically distinct, chondrocyte-like cells expressing mRNA transcripts of collagen types II, IX and X, and aggrecan. The cells within the high-density pellet cultures were surrounded by a sulfated prote-oglycan-rich extracellular matrix that immunostained for collagen type II and proteoglycan link protein. Osteogenic differentiation of hOB was verified by an increased number of alkaline phosphatase-positive cells, that expressed osteoblast-related transcripts such as alkaline phosphatase, collagen type I, osteopontin and osteocalcin, and formed mineralized matrix in monolayer cultures treated with ascorbate, ,-glycerophosphate, and bone morphogenetic protein-2. Adipogenic differentiation of hOB was determined by the appearance of intracellular lipid droplets, and expression of adipocyte-specific genes, such as lipoprotein lipase and peroxisome proliferator-activated receptor ,2, in monolayer cultures treated with dexamethasone, indomethacin, insulin and 3-isobutyl-l-methylxanthine. Taken together, these results show that cells derived from collagenase-treated adult human trabecular bone fragments have the potential to differentiate into multiple mesenchymal lineages in vitro, indicating their developmental plasticity and suggesting their mesenchymal progenitor nature. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Differential effects of BMP-2 and TGF-,1 on chondrogenic differentiation of adipose derived stem cellsCELL PROLIFERATION, Issue 6 2007A. T. Mehlhorn Objectives: This article addresses the interaction of transforming growth factor ,1 (TGF-,1) and bone morphogenic protein 2 (BMP-2) during osteo-chondrogenic differentiation of adipose-derived adult stem cells (ASC). TGF-,1 was expected to modulate the BMP-2-induced effects through transcriptional regulation of Dlx-5, Msx-2 and Runx-2. Materials and Methods: Encapsulated ASC were cultured for 14 days in medium containing TGF-,1 and/or BMP-2. mRNA expression of the extracellular matrix molecules col2a1, cartilage oligomeric matrix protein, col10a1, alkaline phosphatase (AP) and transcription factors Msx-2, Dlx-5 and Runx-2 was analysed. Release of glycosaminoglycans, collagen types II and X into the extracellular matrix was demonstrated. Results: BMP-2 and TGF-,1 induced a chondrogenic phenotype in ASC. Combined growth factor treatment had a synergistic effect on col10a1 and an additive effect on col2a1 mRNA expression. Synthesis of glycosaminoglycans was enhanced by combined growth factor treatment. Addition of TGF-,1 inhibited BMP-2 induced AP expression and activity and both proteins promoted chondrogenic maturation. Conclusions: Prevention of BMP-2-induced osteogenic transdifferentiation by TGF-,1 seemed not to be mediated by transcriptional regulation of Dlx-5. Due to these findings, simultaneous stimulation of ASC with BMP-2 and TGF-,1 seemed to be beneficial for complete differentiation of ASC into chondrocytes. [source] | |||||||||||||