Recombinant human bone morphogenetic protein-2 maintains the articular chondrocyte phenotype in long-term culture

Bone morphogenetic proteins have been shown to increase matrix synthesis by articular chondrocytes in short-term cultures. Members of this family of proteins have also been shown to induce endochondral ossification in vivo. The present study was performed to determine if the addition of human recombinant bone morphogenetic protein-2 to a long-term monolayer articular chondrocyte cell culture system affected the ability of the chondrocytes to divide in vitro, whether the cytokine altered expression of the articular chondrocyte phenotype and synthesis of matrix proteoglycans, and whether the cytokine was capable of inducing differentiation to a hypertrophic chondrocyte. Human recombinant bone morphogenetic protein-2 did not alter cell proliferation. It caused 3.5–6.2 times more proteoglycan synthesis by articular chondrocytes during each of the time points tested after 4 days in culture. Total proteoglycan accumulation in the extracellular matrix after 28 days in culture was 6.7 times as great in the treated cultures as in the control. Treatment with human recombinant bone morphogenetic protein-2 maintained the articular chondrocyte phenotype of cells in culture as demonstrated by Northern blot analysis: the expression of type-I collagen genes was increased and that of type-II collagen and aggrecan mRNA was lost in untreated chondrocyte cultures after 14–21 days in culture. In contrast, exposure to 100 ng/ml human recombinant bone morphogenetic protein-2 maintained expression of type-II collagen and increased expression of aggrecan compared with controls during the 28-day culture period. Northern blot analysis of the expression of type-X collagen and osteocalcin by chondrocytes treated with human recombinant bone morphogenetic protein-2 showed a lack of expression of these genes, indicating no alteration in phenotype. These experiments demonstrated the ability of human recombinant bone morphogenetic protein-2 to promote the articular chondrocyte phenotype and matrix synthesis in long-term culture. Characteristics of cell growth were not affected, and the cytokine did not induce differentiation to a hypertrophic chondrocyte.     

Review and comparison of culture-techniques for articular chondrocytes

AIM: In-vitro techniques for articular chondrocytes allow the analysis of their metabolism in the presence and absence of mediators or drugs against osteoarthritis or rheumatoid arthritis, as well as the synthesis of de-novo cartilage tissue for implantation into articular defects in vivo. This review aims to give an overview about the basics of different methods of cultivation of articular chondrocytes and about several specific demands (e.g., phenotypical stability with synthesis of aggrecan and type-II collagen, no cell-to-cell contact, low proliferation rates, low matrix molecule turn-over) to such methods. METHOD: Current techniques for the cultivation of articular chondrocytes and their development were identified via "medline". Their evaluation was based on our own experience and on data from the literature. RESULTS: Two- and three-dimensional culture systems are employed to maintain articular chondrocytes in vitro. Two-dimensional cultures (monolayer) support the proliferation of articular chondrocytes, but lead to a de-differentiation to fibroblast-like cells. Three-dimensional set-ups (e.g., organ, alginate, agarose cultures) not only maintain the articular cartilage phenotype, but they also support the re-differentiation of de-differentiated chondrocytes. CONCLUSION: The choice of a culture system for in-vitro studies with articular chondrocytes should be adapted to the question asked.     

Quantitative analysis of gene expression in human articular chondrocytes assigned for autologous implantation

Autologous chondrocyte implantation (ACI) relies on the implantation of in vitro expanded cells. The aim was to study the dedifferentiation of human articular chondrocytes under different cultivating conditions [days 0–10 in the primary culture (P0); passages in a monolayer from P0 to P3; monolayer vs. alginate and monolayer vs. alginate/agarose hydrogels] using real‐time PCR analysis. The relative gene expressions for collagen type I and II, aggrecan and versican were quantified and the corresponding differentiation indexes (Col2/Col1, Agr/Ver) were calculated. The values of both differentiation indexes decreased exponentially with time in the P0 monolayer culture, and continued with a significant decrease over the subsequent monolayer passages. On the contrary, the chondrocytes seeded in either of the hydrogels significantly increased the indexes compared to their parallel monolayer cultures. These results indicate that alginate and alginate/agarose hydrogels offer an appropriate environment for human articular chondrocytes to redifferentiate after being expanded in vitro. Therefore the three‐dimensional (3D) hydrogel chondrocyte cultures present not only surgical, but also biological advantage over the classic suspension–periosteum chondrocyte implantation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:847–853, 2008     

Characterisation of human knee meniscus cell phenotype

OBJECTIVE: Studies on the biology of the human meniscus cell are scarce. The objective of our studies was to assess survival/proliferation of human meniscus cells in different culture conditions and to characterize the extracellular matrix (ECM) produced by these cells in these artificial environments. The composition of this ECM offers a variable to define the distinct meniscus cell phenotype. MATERIALS AND METHODS: Human meniscus cells were isolated enzymatically from visually intact lateral and medial knee menisci. Cells were cultured in monolayer conditions or in alginate gel. The composition of the cell-associated matrix (CAM) accumulated by the isolated cells during culture was investigated and compared to the CAM of articular chondrocytes cultured in alginate using flow cytometry with fluorescein isothiocyanate-conjugated monoclonal antibodies against type I collagen, type II collagen and aggrecan. Additional cell membrane markers analysis was performed to further identify the different meniscus cell populations in the alginate culture conditions and meniscus tissue sections. Proliferation was analyzed using the Hoechst 33258 dye method. In some experiments, the effect of TGFbeta1 on some of these variables was investigated. RESULTS: The CAM of monolayer cultured meniscus cells is composed of high amounts of type I and II collagen and low amounts of aggrecan. A major population of alginate cultured meniscus cells on the other hand synthesized a CAM containing high amounts of type I collagen, low amounts of type II collagen and high amounts of aggrecan. This population is CD44+CD105+CD34-CD31-. In contrast, a minor cell population in the alginate culture did not accumulate ECM and was mainly CD34+. The CAM of alginate cultured articular chondrocytes is composed of low amounts of type I collagen, high amounts of type II collagen and aggrecan. The expression of aggrecan and of type II collagen was increased by the addition of TGFbeta1 to the culture medium. The proliferation of meniscus cells is increased in the monolayer culture conditions. Cell numbers decrease slightly in the alginate culture, but can be increased after the addition of TGFbeta1. CONCLUSION: These results demonstrate that the human meniscus is populated by different cell types which can be identified by a distinct CAM composition and membrane marker expression. Unlike the monolayer culture conditions, the alginate culture conditions appear to favor a more fibrochondrocyte-like cell accumulating a CAM resembling the native tissue composition. This CAM composition is distinctly different from the CAM composition of phenotypically stable articular cartilage chondrocytes cultured in the same alginate matrix.     

Cartilage from the edge of a debrided articular defect is inferior to that from a standard donor site when used for autologous chondrocyte cultivation

The aim of this study was to evaluate the cultivation potential of cartilage taken from the debrided edge of a chronic lesion of the articular surface. A total of 14 patients underwent arthroscopy of the knee for a chronic lesion on the femoral condyles or trochlea. In addition to the routine cartilage biopsy, a second biopsy of cartilage was taken from the edge of the lesion. The cells isolated from both sources underwent parallel cultivation as monolayer and three-dimensional (3D) alginate culture. The cell yield, viability, capacity for proliferation, morphology and the expressions of typical cartilage genes (collagen I, COL1; collagen II, COL2; aggrecan, AGR; and versican, VER) were assessed. The cartilage differentiation indices (COL2/COL1, AGR/VER) were calculated. The control biopsies revealed a higher mean cell yield (1346 cells/mg vs 341 cells/mg), but similar cell proliferation, viability and morphology compared with the cells from the edge of the lesion. The cartilage differentiation indices were superior in control cells: COL2/COL1 (threefold in biopsies (non-significant)); sixfold in monolayer cultures (p = 0.012), and 7.5-fold in hydrogels (non-significant), AGR/VER (sevenfold in biopsies (p = 0.04), threefold (p = 0.003) in primary cultures and 3.5-fold in hydrogels (non-significant)). Our results suggest that the cultivation of chondrocytes solely from the edges of the lesion cannot be recommended for use in autologous chondrocyte implantation.     

Flow cytometric analysis of the human articular chondrocyte phenotype in vitro

OBJECTIVE: To develop flow cytometry for the study of human articular cartilage cell phenotype and to validate the method on chondrocytes cultured in different in-vitro systems. METHODS: Chondrocyte phenotype was modulated by culturing the cells under different in-vitro conditions: i.e. in monolayer and in suspension culture in gelled agarose. Monolayer cultured chondrocyte phenotype was assayed by immunohistochemical staining with monoclonal antibodies against chondrocyte-specific aggrecan, type II and I collagen. Flow cytometry was used to quantify the proportions of chondrocytes expressing these extracellular matrix molecules in both culture conditions. To exclude the effects of cell-harvesting methods on the presence of cell-bound ECM molecules, non-proteolytic isolation procedures were used to obtain the chondrocytes for flow cytometry. Subconfluent cells from monolayer cultures were detached with EDTA. Chondrocytes cultured in gelled agarose were obtained after the agarose was enzymatically digested with agarase. RESULTS: Immunohistochemical staining showed that monolayer-cultured chondrocytes, in the presence of serum, gradually lost the expression of chondrocyte-specific aggrecan and type II collagen, while type I collagen was increasingly expressed. Flow cytometry allowed monolayer cultured chondrocyte phenotype to be assessed reproducibly. Chondrocyte phenotype was characterized through the cell membrane-associated extracellular matrix antigens. EDTA, used to obtain single cells from monolayer cultures, did not affect the cell-associated matrix. Where the chondrocytes had been cultured in gelled agarose, flow cytometry allowed quantification of the percentages of chondrocytes maintaining or reexpressing their original phenotype. The agarase digestion procedure used to isolate the cells from the agarose gel did not affect the plasma membrane-associated extracellular matrix antigens. CONCLUSION: Flow cytometry allows quantification of cells expressing aggrecan, type II and I collagen in their cell-associated extracellular matrix. A continuously increasing number of specific monoclonal antibodies will broaden the range of applications offered by this method.     

Gene expression during redifferentiation of human articular chondrocytes

OBJECTIVE: The aim of the present study was to investigate gene expression during the in vitro redifferentiation process of human articular chondrocytes isolated from clinical samples from patient undergoing an autologous chondrocyte transplantation therapy (ACT). METHOD: Monolayer (ML) expanded human articular chondrocytes from four donors were cultured in a 3D pellet model and the redifferentiation was investigated by biochemistry, histology, immunohistochemistry and microarray analysis. RESULTS: The culture expanded chondrocytes redifferentiated in the pellet model as seen by an increase in collagen type II immunoreactivity between day 7 and 14. The gene expression from ML to pellet at day 7 included an increase in cartilage matrix proteins like collagen type XI, tenascin C, dermatopontin, COMP and fibronectin. The late phase consisted of a strong downregulation of extracellular signal-regulated protein kinase (ERK-1) and an upregulation of p38 kinase and SOX-9, suggesting that the late phase mimicked parts of the signaling processes involved in the early chondrogenesis in limb bud cells. Other genes, which indicated a transition from proliferation to tissue formation, were the downregulated cell cycle genes GSPT1 and the upregulated growth-arrest-specific protein (gas). The maturation of the pellets included no signs of hypertrophy or apoptosis as seen by downregulation of collagen type X, Matrix Gla protein and increased expression of caspase 3. CONCLUSION: Our data show that human articular chondrocytes taken from surplus cells of patient undergoing ACT treatment and expanded in ML, redifferentiate and form cartilage like matrix in vitro and that this dynamic process involves genes known to be expressed in early chondrogenesis.     

Enhanced proliferation and differentiation of human articular chondrocytes when seeded at low cell densities in alginate in vitro.

Dedifferentiated human articular chondrocytes exhibited a wide variation in their capacity to proliferate and redifferentiate in an alginate suspension culture system. The greatest extent of proliferation and redifferentiation was seen to be dependent on the formation of clonal populations of chondrocytes and correlated inversely with the initial cell seeding density. Redifferentiating chondrocytes seeded at low density (1 x 10(4) cells/ml alginate) compared with chondrocytes that were seeded at high density (1 x 10(6) cells/ml alginate) showed a nearly 3-fold higher median increase in cell number. a 19-fold greater level of type-II collagen mRNA expression, a 4-fold greater level of aggrecan mRNA expression, and a 6-fold greater level of sulfated glycosaminoglycan deposition at 4 weeks of culture. Matrix molecules from low-density cultures were assembled into chondrocyte-encapsulated, spherical extracellular matrices that were readily visualized in sections from 12-week cultures stained with antibodies against types I and II collagen and aggrecan. Ultrastructural analysis of 12-week low-density cultures confirmed the presence of thin collagen fibrils throughout the matrix.     

Comparison of articular and auricular cartilage as a cell source for the autologous chondrocyte implantation

Articular (medial femoral condyle) and auricular cartilage (anithelix) was compared as a cell source for the autologous joint repair. Cells isolated from five human cadaveric donors were cultured parallel in the monolayer cultures and in the 3D alginate hydrogel constructs for 1 week. Cell morphology was controlled by the fluorescent microscopy and gene expressions of type I collagen (COL1), type II collagen (COL2), aggrecan (AGR), versican (VER), and elastin (ELS) were analyzed by the real‐time polymerase chain reaction. COL1 and ELS, predominant in the phenotype of auricular biopsy, were statistically lower in the articular biopsies. Even though COL2 and AGR decreased in monolayers of both cell sources, the dedifferentiation process affected auricular cells intensely. Cells embedded in the alginate hydrogel directly after the isolation did not exhibit the dedifferentiated phenotype. Additionally, COL1, COL2, AGR, and VER were comparable between the two sources. ELS however, remained higher in the auricular cells regardless of the culture type. The study indicates that auricular chondrocytes cultured in a 3D environment immediately after the isolation have a neo‐cartilage potential for the articular surface reconstruction. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 943–948, 2009     

In vitro stimulation of articular chondrocyte mRNA and extracellular matrix synthesis by hydrostatic pressure

This study tested the effects of hydrostatic pressure (10 MPa) on adult articular chondrocyte mRNA and extracellular matrix synthesis in vitro. High density primary cultures of bovine chondrocytes were exposed to hydrostatic pressure applied intermittently at 1 Hz or constantly for 4 hours in serum-free medium or in medium containing 1% fetal bovine serum, mRNAs for aggrecan, types I and II collagen, and β-actin were analyzed by Northern blots and quantified by slot blots. Proteoglycan synthesis was quantified by ³⁵SO₄ uptake into cetylpyridinium chloride-precipitable glycosaminoglycans, and cell-associated aggrecan and type-II collagen were detected by immunohistochemical techniques. In serum-free medium, intermittent pressure increased aggrecan mRNA signal by 14% and constant pressure decreased type-II collagen mRNA signal by 16% (p < 0.05). In the presence of 1% fetal bovine serum, intermittent pressure increased aggrecan and type-II collagen mRNA signals by 31% (p < 0.01) and 36% (p < 0.001), respectively, whereas constant pressure had no effect on either mRNA. Intermittent and constant pressure stimulated glycosaminoglycan synthesis 65% (p < 0.001) and 32% (p < 0.05), respectively. Immunohistochemical detection of cell-associated aggrecan and type-II collagen was increased in response to both intermittent and constant pressure. These data support the hypothesis that physiologic hydrostatic pressure directly influences the extracellular matrix metabolism of articular chondrocytes.     

Coordinate upregulation of cartilage matrix synthesis in fibrin cultures supplemented with exogenous insulin-like growth factor-I.

The addition of insulin-like growth factor-I to cartilage cultures is known to stimulate the synthesis of proteoglycan and type-II collagen in explant and monolayer studies. The purpose of this study was to determine the effects of long-term supplementation with insulin-like growth factor-I in chondrocytes cultured in fibrin discs as a preliminary investigation to in vivo application of chondrocyte/insulin-like growth factor-I/fibrin grafts to articular-cartilage repair procedures. Chondrocyte-fibrin cultures were maintained for 14 days, with insulin-like growth factor-I added at varying concentrations of 0, 10, 50, or 100 ng/ml medium. Cultures supplemented with 50 or 100 ng of growth factor/ml had increased levels of aggrecan and type-IIB procollagen mRNA, and translation to aggrecan and type-IIB collagen was confirmed by dye-binding assay of total proteoglycan, type-II collagen immunohistochemistry, and determination of collagen content by high-performance liquid chromatography. Maintenance of the chondrocyte phenotype during the 14 days of culture was confirmed by round cell morphology on routine staining, expression of type-II procollagen mRNA on in situ hybridization, evidence of production of pericellular type-II collagen on immunocytochemistry, synthesis of large-molecular-size aggrecan monomer on CL-2B column chromatography, and lack of appreciable message expression for type I or IIA collagen on Northern blot hybridization. Dose-response effects of insulin-like growth factor-I on the expression of chondrocyte matrix constituents were most pronounced at 50 and 100 ng of growth factor per milliliter of medium. These data confirm that (a) culture of chondrocytes for extended periods in three-dimensional cultures of fibrin maintains the chondrocyte phenotype and (b) supplementation with increasing concentrations of insulin-like growth factor-I enhances chondrocyte matrix synthesis and may provide a means to enhance chondrocyte phenotypic stability and function during transplantation grafting procedures.     

Comparison of gene expression patterns in articular cartilage and dedifferentiated articular chondrocytes

During monolayer culture, articular chondrocytes dedifferentiate into fibroblast‐like cells. The mechanisms underlying this process are poorly understood. We sought to further characterize dedifferentiation by identifying an extended panel of genes that distinguish articular cartilage from dedifferentiated chondrocytes. Thirty‐nine candidate marker‐genes were identified from previous studies on articular‐cartilage gene‐expression. Real‐time PCR was used to evaluate the mRNA levels for these candidates in calf articular cartilage and dedifferentiated articular chondrocytes. Twenty‐two of the candidate marker genes exhibited at least a two‐fold difference in gene expression in the two cell types. Twelve of these genes had at least a ten‐fold difference in gene expression. Tenascin C (TNC), type I collagen (COL1A1), and hypoxia‐inducible factor 1 alpha (HIF1α) showed the highest relative expression levels in dedifferentiated chonodrocytes. Type II collagen (COL2A1), type XI collagen (COL11A2), and superficial zone protein (SZP) showed the highest relative expression levels in articular cartilage. In contrast to previous findings, fibromodulin mRNA, and protein levels were higher in dedifferentiated chondrocytes. Compared to smaller subsets of markers, this panel of 12 highly differentially expressed genes may more precisely distinguish articular cartilage from dedifferentiated chondrocytes. Since many of the genes up‐regulated in dedifferentiated chondrocytes are also expressed during cartilage development, dedifferentiated chondrocytes may possess features of cartilage precursor cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:234–245, 2012     

Comparison of meshes, gels and ceramic for cartilage tissue engineering in vitro

Full-thickness articular cartilage defects lack the capacity of healing because of lack of blood supply and lack of chondrocyte proliferation around the injury site. These factors contribute to the difficulty of getting good healing of cartilage defects. Autologous chondrocyte transplantation has been proposed as a method for treating cartilage defects using chondrocytes grown in vitro, which are then transplanted into the defects using a periosteal flap to retain the cells at the defect site. Studies that followed have attempted to refine this technique by using a cell matrix to support the chondrocytes. The reason for adding a resorbable cell matrix support that acts as a temporary scaffold until the chondrocytes are capable of producing extracellular matrix. Moreover, such a matrix may help in maintaining chondrocyte differentiation and phenotype. In this study, we have investigated the biocompatibility between human chondrocytes and biomaterials that could be used as matrix implants. It is a comparative study in vitro that involves assessing the proliferation and differentiation of human articular chondrocytes cultured on different resorbable biomaterials. Human chondrocytes were isolated from collagenase digest of articular cartilage provided by patients undergoing total knee replacements for osteoarthritis from the non-involved areas of the knee. The chondrocytes were then allowed to proliferate in vitro to increase the number of cells available for study. After adequate multiplication, the cells were seeded onto different biomaterials and allowed to from a cell biomaterial construct. The biomaterials used in this study were collagen I, calcium alginate, agarose, polyglycolic acid and Bioglass 45S5. The cell–biomaterial constructs were then collected at specific time points 3, 7, 14 and 21 days for histological and biochemical studies. The assessment includes studying proliferation, differentiation and extracellular matrix production. This was performed by immunostaining for collagen I and II production and histochemistry staining for glycosaminoglycans. Chondrocyte proliferation was more effective on 3D gels compared to ceramics and mesh. Cells on Bioglass expressed the same collagen type and at the same proportion as that expressed by freshly isolated cells. Moreover, Bioglass has induced cells to re-differentiate after they lost their differentiation in monolayer culture. Overall, however, there was no clear relationship between the cell morphology and type of collagen produced. Bioactive glass seems to behave as a suitable material for chondrocyte tissue engineering because it can maintain a chondrocyte phenotype.     

兔关节软骨细胞聚集培养的生物学性状观察

目的 观察聚集培养软骨细胞生物学性状的变化,为软骨细胞移植建立合适的体外培养方法。方法2001年11月至2004年6月酶消化法分离成年兔关节软骨细胞,分别低密度单层培养和高密度聚集培养,组化及免疫组化法观察细胞表型变化。结果低密度培养时,前3代细胞增殖迅速,但很快去分化,3代以后增殖缓慢,细胞表型大部丢失;聚集培养时,软骨细胞去分化速度减缓;传3代后细胞聚集培养,细胞表型部分恢复。结论聚集培养利于维持软骨细胞表型,原代细胞聚集培养或传代培养后聚集培养是较好的获取大量优良软骨细胞的培养方式。     

Development of articular cartilage grafts using organoid formation techniques

In order to develop articular cartilage grafts, one must control shape and safety. We have developed scaffold-free culture methods in which the cells form multicellular aggregates (organoids). In this study, we applied the organoid culture method to chondrocytes attempting to reconstitute articular cartilage grafts. Primary rat costal chondrocytes and subcultured human articular chondrocytes were immobilized in hollow fibers by centrifugation at a density of 3 x 10(8) cells/cm3 to induce the formation of cylindrical-shaped organoids. To improve convenience, we developed a culture device to form sheet-shaped organoids (organoid-sheet). Primary bovine articular chondrocytes were cultured in this device. These organoids were evaluated by histological and gene expression analyses. In the primary rat culture system, chondrocytes formed cylindrical organoids in hollow fibers. Histochemical analysis revealed the presence of extracellular matrix (collagen and proteoglycan). The organoid maintained cartilage-specific gene expression (type II collagen, aggrecan) for 1 month of culture. In the subcultured human chondrocyte system, the organoid regained the decreased cartilage-specific gene expression. In the primary bovine culture system, the cells formed a 300 microm thickness organoid-sheet including abundant extracellular matrix. In conclusion, our organoid formation method was effective to form cartilage-like tissue. This result suggested that the technique may be applicable for the development of an articular cartilage graft.     

Loss of chondrogenic potential in dedifferentiated chondrocytes correlates with deficient Shc-Erk interaction and apoptosis

OBJECTIVE: If dedifferentiated chondrocytes could be induced to redifferentiate in vitro, then we might thereby be furnished with a population of phenotypically stable cells for autologous implantation in reconstructive surgery. We therefore investigated the redifferentiation capabilities of chondrocytes which, having migrated from alginate beads to form a monolayer, were subsequently passaged. We also characterized the molecular traits of irreversibly dedifferentiated cells. METHODS: Human chondrocytes that had migrated from alginate beads to form a monolayer (passage 1) were passaged seven times (passages 2-8). Cells from each passage were then recultivated in alginate beads. We assessed the synthesis of type-II collagen, cartilage-specific proteoglycans, adhesion molecules (integrins), signaling proteins (Src-homology collagen [Shc] and extracellular-signal-regulated kinase [Erk]) and the apoptosis marker 'activated' caspase-3 in monolayer or secondary alginate cultures. RESULTS: The synthesis of cartilage-specific type-II collagen, alpha 3-integrin, Shc and activated Erk1/2 decreased rapidly after four passages in monolayer culture. Up to passage 4, cells redifferentiated in alginate culture. However, between passages 5 and 8, cells began to produce activated caspase-3; these cells not only failed to redifferentiate when recultivated in alginate, but underwent apoptosis. CONCLUSION: We conclude that the loss of chondrogenic potential by chondrocytes maintained in monolayer culture is associated with a decrease in the synthesis of cartilage markers and with a suppressed activation of key signaling proteins in the Ras-mitogen-activated protein kinase pathway (Shc and Erk1/2). These events lead to apoptosis. A decrease in Shc/Erk expression/interaction could serve as a recognition marker for irreversibly dedifferentiated chondrocytes in tissue engineering.     

In situ localization of collagen production by chondrocytes and osteoblasts in fracture callus

An experimental model of fracture-healing was used to study the production of types-I and II collagen by in situ hybridization. The distribution of cartilage matrix in callus was determined by histochemical staining. Messenger RNA (mRNA) for cartilage-specific type-II collagen was detectable as early as the fifth day in a small number of cells that had acquired a chondrocyte phenotype but that also contained type-I collagen mRNA, suggesting an ongoing change in the expression of collagen genes. The location of the first chondrocytes, which were adjacent to cortical bone, suggested that they originated from cells that had derived from the periosteum by differentiation. On the seventh day of callus formation, the presence of both type-I and type-II collagen mRNA in chondrocytes of expanding cartilage suggested that most growth occurred by differentiation of mesenchymal cells and less by proliferation of differentiated chondrocytes. Expansion continued until the tenth to fourteenth day, after which the cartilage was replaced by woven bone. This was characterized by the presence of osteoblasts that were active in the synthesis of type-I collagen.