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.     

The proteoglycan synthesis repertoire of rabbit chondrocytes maintained in type II collagen gels

Repair of experimental articular cartilage lesions employing cultured rabbit articular chondrocytes requires a detailed knowledge of the phenotypic stability of these cells. A suitable matrix vehicle for use in chondrocyte transplantation is a much sought-after component of any transplantation paradigm. We studied the proteoglycan synthesis repertoire of young immature rabbit articular chondrocytes maintained in chick type II collagen gels or collagen gels supplemented with recombinant human transforming growth factor-beta 1 (rhTGF beta 1). Maintenance of chondrocytes in type II collagen gels increased the percentage 35SO4-labeled proteoglycans reaching equilibrium in the A1D1 or D1 fraction of CsCl density gradient when compared to chondrocytes maintained in polystyrene microwell cultures. Although rhTGF beta 1 supplementation increased the percentage of A1D1/D1 proteoglycan by chondrocytes grown on polystyrene, rhTGF beta 1 did not augment this percentage increase in A1D1/D1 when added to collagen II gels. Rabbit chondrocytes synthesized two core proteins derived from the high-density aggregatable proteoglycans. LI and LII have apparent molecular sizes of 480 kDa and 390 kDa, respectively. Both core protein forms were found in the medium fraction, but the predominant core protein form associated with the cell fraction was LI. Maintenance of chondrocytes in collagen II gels increased synthesis of both core proteins. In addition to the large core proteins, three other core proteins with properties on SDS PAGE characteristic of the small dermatan sulfate proteoglycans, biglycan and decorin, were identified. Synthesis of these core proteins was stimulated by maintenance in collagen gels. Furthermore, they were preferentially retained in the gel matrix. Chondrocytes maintained on glass or in type II collagen gels stained with monoclonal antibodies specific for chondroitin-6-sulfate, chondroitin-4-sulfate and keratan sulfate. However, while chondrocytes grown on glass slides failed to stain with monoclonal antibody 3B3 in the absence of chondroitinase ABC digestion, chondrocytes grown in collagen II gels stained intensely in the absence of enzyme pretreatment. These results were confirmed by Western blots.     

Response of human chondrocytes prepared for autologous implantation to growth factors

This study investigated metabolism of autologous chondrocytes after initial expansion immediately before implantation. Chondrocytes cultured in either monolayers or alginate beads were treated with insulin-like growth factor-1 (IGF-1), osteogenic protein-1 (OP-1), or a combination. Proteoglycan synthesis and DNA content were tested in both cultures. Alginate beads also were analyzed with live/dead cell assay, safranin O/fast green stain for histology, and immunohistochemistry with antibodies against collagen type II and VI, aggrecan, decorin, and fibronectin. In monolayers, autologous chondrocytes changed their morphologic appearance. In alginate, they maintained chondrocytic phenotype. Growth factors, especially combined, promoted cell survival and induced chondrocyte proliferation. OP-1 stimulated the largest cartilage-specific matrix and the most accumulation of collagen type II and fibronectin, although the overall matrix synthesized by autologous chondrocyte implantation cells was smaller than that produced by normal chondrocytes. The clinical implications of this study suggest a significant promise for anabolic growth factors in cartilage repair as a potential modifying therapy for the enhancement of chondrocytic phenotype of autologous chondrocytes.     

Bone morphogenetic protein (BMP)-2 enhances the expression of type II collagen and aggrecan in chondrocytes embedded in alginate beads

OBJECTIVE: For autologous chondrocyte transplantation (ACT) chondrocytes are expanded in vitro. During expansion these cells may dedifferentiate. This change in phenotype is characterized by a raised expression of type I collagen and a decrease in type II collagen expression. Since high expression of type II collagen is of central importance for the properties of hyaline cartilage, we investigated if the growth factor bone morphogenetic protein-2 (BMP-2) may modulate the chondrogenic phenotype in monolayer cell cultures and in three-dimensional culture systems. DESIGN: Chondrocytes from articular knee cartilage of 11 individuals (average age: 39.8 years) with no history of joint disease were isolated and seeded either in monolayer cultures or embedded in alginate beads in presence or absence of human recombinant BMP-2 (hr-BMP-2). Then, cells were harvested and analysis of the chondrogenic phenotype was performed using quantitative RT-PCR, immunocytochemistry and ELISA. RESULTS: Addition of BMP-2 to chondrocytes expanded in two-dimensional (2D) cultures during the first subculture (P1) had no effect on mRNA amounts encoding type II collagen and interleukin-1beta (IL-1beta). In contrast, seeding chondrocytes in three-dimensional (3D) alginate cultures raised type II collagen expression significantly and addition of BMP-2 enhanced this effect. CONCLUSIONS: We conclude that chondrocytes during expansion for ACT may benefit from BMP-2 activation only when seeded in an appropriate 3D culture system.     

Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro

Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.     

The use of debrided human articular cartilage for autologous chondrocyte implantation: maintenance of chondrocyte differentiation and proliferation in type I collagen gels.

Autologous chondrocyte implantation (ACI) is the most promising surgical treatment for large full thickness knee joint articular cartilage (AC) defects where cells from healthy non-weight bearing area AC are multiplied in vitro and implanted into such defects. In the routine surgical procedure for symptomatic knee full thickness AC defects, damaged AC surrounding the edge and the base of such defects is usually debrided and discarded. The purpose of this study was to examine if chondrocytes from this 'debrided' AC can proliferate, synthesize a cartilage specific matrix and thus can be used for ACI. METHODS: Biopsies were retrieved from 12 patients (debrided articular cartilage: DAC, aged 35-61) and from two autopsies (normal articular cartilage: NAC, aged 21 and 25). Chondrocytes were isolated, seeded at low density in type I collagen gels and as monolayer cultures for 4 weeks without passage. RESULTS: After 4 weeks cultures in type I collagen gels, cell proliferation from DAC (18.34 +/- 1.95 fold) was similar to cells from NAC (11.24 +/- 1.02 fold). Syntheses of proteoglycan and collagen in DAC were also similar to NAC. Newly synthesized matrices in gel cultures consisted predominantly of type II collagen as shown by immuno-labelling and SDS-PAGE followed by fluorography. Chondrocytes from 'debrided human AC' cultured at low density in type I collagen gels may be used for the ACI procedure as they provide sufficient viable cell numbers for ACI and maintain their chondrocyte phenotype as they synthesize a cartilage-like matrix.     

Effects of cell density on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel

The effects of cell density on the proliferation and chondroitin sulfate synthesis of chondrocytes embedded in Atelocollagen gel were examined. Chondrocytes of 21 10-week-old Japanese white rabbits isolated by collagenase digestion were embedded in Atelocollagen gel and cultured in Dulbecco's modified Eagles medium at cell densities of 2 x 105 cells/ml (105 group), 2 x 106 cells/ml (106 group), and 2 x 107 cells/ml (107 group) for 4 weeks. Chondrocytes in the 105 group gradually proliferated more than the other two groups. In contrast, most chondrocytes in the 107 group showed increased capability to produce chondroitin 6-sulfate. Cartilage-like tissue was produced from high-density cultures (107 cells/ml), although a decrease in cell number was seen. Even in three-dimensional cultures, the proliferation and chondroitin sulfate synthesis of chondrocytes were influenced by the cell density. These results are informative for the clinical application of chondrocyte transplantation in three-dimensional cultures for cartilage repair.     

Repair of rabbit articular surfaces with allografts of chondrocytes embedded in collagen gels

In an attempt to restore articular cartilage, allogeneic articular chondrocytes embedded in collagen gels were transplanted onto full-thickness defects in rabbit articular cartilage. Within 24 weeks after the transplantation, the defects were filled with hyaline cartilage, specifically synthesizing type II collagen. These chondrocytes were autoradiographically proven to be originated from the originally transplanted chondrocytes. As histologically assessed, success rate was about 80%, a marked improvement over the results (40% success rate) in previous studies reporting chondrocyte transplantation without collagen gels. On the other hand, the defects without chondrocyte transplantation healed with fibrocartilaginous tissue more than 24 weeks after treatment. Immunological enhancement induced by transplanted allogeneic chondrocytes or collagen was not significant for eight weeks after treatment, so far as shown by both direct and indirect blastformation reactions. Thus, allogeneic transplantation of isolated chondrocytes embedded in collagen gels appears to be one of the most promising methods for the restoration of articular cartilage.     

Phenotypic differences in murine chondrocyte cell lines derived from mature articular cartilage

OBJECTIVE: To obtain well characterized immortalized murine chondrocyte cell lines. The cell lines were obtained from mature articular chondrocytes, instead of embryonal cells which are used in most other studies. METHODS: Pieces of articular cartilage were cut from murine patellae and femoral heads. Chondrocytes were isolated by digestion with collagenase. These cells were cultured in monolayer and immortalized by transfection of the SV40 large T antigen gene. To preserve the differentiated phenotype, the resulting clones were cultured in three-dimensional carriers, alginate beads. The phenotypes of the cells were characterized using the following parameters: Cell morphology (light microscopy), messenger RNA (RT-PCR) and protein (immunohistochemistry) levels of extracellular matrix molecules. Moreover, responsiveness to interleukin-1(IL-1) was determined by measuring production of proteoglycans ((35)S-sulfate incorporation) and of nitric oxide (Griess reaction). RESULTS: Sixteen clones were obtained, ten (P1 to P10) derived from patellar cartilage, and six (H1 to H6) from femoral head cartilage. In seven cell lines (P2, P5, H1, H3, H4, H5, H6) high production of type II collagen corresponded with high levels of mRNA of type II collagen (and prevalence of the IIB type) and with high IL-1-induced suppression of proteoglycan synthesis. Like intact murine articular cartilage, all cell lines produced type I and type X collagens, but mRNA levels of both types of collagen were never higher in the cell lines as compared with intact cartilage. CONCLUSION: Our results demonstrate that it is possible to immortalize mature murine articular chondrocytes. Each of the obtained chondrocyte cell lines appeared to have a stable phenotype. Both relatively differentiated and relatively dedifferentiated chondrocyte cell lines could be identified.     

The effects of hyaluronic acid on articular chondrocytes

The purpose of this study was to examine the effects of hyaluronic acid supplementation on chondrocyte metabolism in vitro. The clinical benefits of intra-articular hyaluronic acid injections are thought to occur through improved joint lubrication. Recent findings have shown that exogenous hyaluronic acid is incorporated into articular cartilage where it may have a direct biological effect on chondrocytes through CD44 receptors.Bovine articular chondrocytes were isolated and seeded into alginate constructs. These were cultured in medium containing hyaluronic acid at varying concentrations. Samples were assayed for biochemical and histological changes.There was a dose-dependent response to the exposure of hyaluronic acid to bovine articular chondrocytes in vitro. Low concentrations of hyaluronic acid (0.1 mg/mL and 1 mg/mL) significantly increase DNA, sulphated glycosaminoglycan and hydroxyproline synthesis. Immunohistology confirmed the maintenance of cell phenotype with increased matrix deposition of chondroitin-6-sulphate and collagen type II. These findings confirm a stimulatory effect of hyaluronic acid on chondrocyte metabolism.     

Articular chondrocyte tenascin-C production and assembly into de Novo extracellular matrix

Tenascin-C is an oligomeric glycoprotein of the extracellular matrix that is expressed in a variety of processes including development, tissue remodeling, wound healing, cell adhesion/antiadhesion, and cell/matrix interactions. Tenascin has recently been acknowledged as a component of the extracellular matrix of articular cartilage, but its function remains unclear. In this study, bovine articular chondrocytes were grown in alginate beads for 35 days to examine the kinetics of tenascin synthesis and incorporation into de novo extracellular matrix. During the culture period, 6 harvest days were established in which culture medium was recovered, alginate beads were dissociated with an EDTA solution, and chondrocytes were collected and lysed by sonication. Total DNA determination performed on the cell lysates demonstrated chondrocyte survival and proliferation. Western blotting performed on the medium, EDTA/alginate, and lysate samples demonstrated the production of both the 220 and 320 kDa tenascin size variants and their differential compartmentalization within the culture system. Tenascin was incorporated into the alginate bead matrix at a constant rate of 3.8 μg/day. The 320 kDa variant was produced in higher quantity, but the 220 kDa fragment was twice as likely to be incorporated into the de novo matrix. Methylene blue/acid fuchsin staining and tenascin immunohistochemistry demonstrated the incorporation of tenascin into a progressively expanding matrix surrounding the chondrocytes. The results suggest a role for tenascin in the assembly of the chondrocyte matrix and as a soluble mediator of chondrocytes with possible diverse functions for the tenascin size variants.     

Type II collagen modulates the composition of extracellular matrix synthesized by articular chondrocytes.

The articular cartilage extracellular matrix (ECM) interfaces with chondrocytes and influences many biological processes important to cartilage homeostasis and repair. The alginate bead culture system can be viewed as a model of cartilage repair in which the chondrocyte attempts to recreate the pericellular matrix while maintaining a differentiated phenotype. The purpose of this study was to evaluate the alteration in epitopes of proteoglycan and tenascin synthesized by chondrocytes in the presence of exogenous extracellular type II collagen. We evaluated the effects on four biomarkers associated with the creation of the denovo matrix using ELISA and immunohistochemistry: keratan sulfate epitope (5D4), 3B3(-) neoepitope of chondroitin-6- sulfate, 3B3(+) chondroitinase-generated epitope of chondroitin-6-sulfate, and tenascin-C expression. TGF-beta1 stimulated the production of 3B3(+), 5D4, and tenascin-C in a dose-dependent manner and decreased 3B3(-) levels. Following the addition of exogenous type II collagen, 3B3(-) increased and tenascin-C decreased but did not change the direction of TGF-beta1 effects. In contrast, 5D4 expression decreased in the presence of collagen II as TGF-beta1 increased to 10 ng/ml. Interestingly, the amount of 3B3(+) epitope was not affected by the incorporation of type II collagen. Immunohistochemistry found there was no significant difference in distribution of these biomarkers in the presence and absence of extracellular type II collagen incorporation. These results elucidate the subtle biochemical differences in ECM synthesized by chondrocytes in the presence of type II collagen and further characterize the role played by ECM in the TGF-beta1 regulation of the articular cartilage physiology.     

Variations in matrix composition and GAG fine structure among scaffolds for cartilage tissue engineering

OBJECTIVE: To compare matrix composition and glycosaminoglycan (GAG) fine structure among five scaffolds commonly used for in vitro chondrocyte culture and cartilage tissue engineering. DESIGN: Bovine articular chondrocytes were seeded into agarose, alginate, collagen I, fibrin and polyglycolic acid (PGA) constructs and cultured for 20 or 40 days. In addition to construct DNA and sulfated GAG (sGAG) contents, the delta-disaccharide compositions of the chondroitin/dermatan sulfate GAGs were determined for each scaffold group via fluorophore-assisted carbohydrate electrophoresis (FACE). RESULTS: Significant differences were found in cell proliferation and extracellular matrix accumulation among the five scaffold groups. Significant cell proliferation was observed for all scaffold types but occurred later (20-40 days) in PGA constructs compared to the other groups (0-20 days). By 40 days, agarose constructs had the highest sGAG to DNA ratio, while alginate and collagen I had the lowest levels. Quantitative differences in the Delta-disaccharide composition of the GAGs accumulated in the different scaffolds were also found, with the most striking variations in unsulfated and disulfated delta-disaccharides. Agarose constructs had the highest fraction of disulfated residues and the lowest fraction of unsulfated residues, with a 6-sulfated/4-sulfated disaccharide ratio most similar to that of native articular cartilage. CONCLUSIONS: The similarities and differences among scaffolds in proteoglycan accumulation and GAG composition suggest that the scaffold material directly or indirectly influences chondrocyte proteoglycan metabolism and may have an influence on the quality of tissue engineered cartilage.     

Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel

In an attempt to repair articular cartilage, allograft articular chondrocytes embedded in collagen gel, were transplanted into full-thickness defects in rabbit articular cartilage. Twenty-four weeks after the transplantation, the defects were filled with hyaline cartilage, specifically synthesising Type II collagen. These chondrocytes were autoradiographically proven to have originated from the transplanted grafts. Assessed histologically the success rate was about 80%, a marked improvement over the results reported in previous studies on chondrocyte transplantation without collagen gel. By contrast, the defects without chondrocyte transplantation healed with fibrocartilage. Immunological enhancement induced by transplanted allogenic chondrocytes or collagen was not significant at eight weeks after treatment, so far as shown by both direct and indirect blastformation reactions. Thus, allogenic transplantation of isolated chondrocytes embedded in collagen gel appears to be one of the most promising methods for the restoration of articular cartilage.     

Interaktion zwischen humanen Chondrozyten und extrazellulärer Matrix in vitro

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

Adult human chondrocytes in alginate culture. Preservation of the phenotype for further use in transplantation models

Successful and cartilage-specific cultivation of chondrocytes requires a stable phenotype during the in vitro culture period. This is based on a differentiated extracellular matrix synthesis. The alginate system as a three-dimensional support is a useful system to culture chondrocytes and to analyze the biochemical processes in this system. Talar cartilage from the talocrural joints of 40 different donors were obtained through the Regional Organ Bank of Illinois within 24 hours of death. In 65% of the tissues the cartilage was classified as being undamaged. In these studies we were interested in the results of short-term culture over 14 days. Cell proliferation, total collagen content and total proteoglycan content were measured in the different matrix compartments and were visualized by histology and immunohistochemistry. Already after 7 days in culture the adult human chondroctes looked intact and formed a stable and cell-associated cartilage-specific extracellular matrix in the presence of 10% calf serum. This could be also demonstrated in the presence of IGF-I. With regards to the collagen content IGF-I at a concentration of 50 ng/ml seemed to induce an equal effect to 10% serum; with regards to the proteoglycan content IGF-I at a concentration of 20 ng/ml was equivalent. These encouraging preliminary results may lead to a new approach in tissue engineering for chondrocyte transplantation in combination with their extracellular matrix.     

兔脊索细胞体外培养及生物学特点观察

目的建立体外兔椎间盘脊索细胞藻酸盐凝胶培养模型,观察脊索细胞形态及生物学特点。方法采用胶原酶消化法及Percoll不连续密度梯度沉淀法体外分离收集原代椎间盘脊索细胞,于1.2%藻酸盐凝胶(低密度)中培养。倒置相差显微镜下观察细胞形态,经Ⅱ型胶原免疫荧光染色对细胞表型初步鉴定,并分别以细胞增殖和细胞毒性试剂-8(CCK-8)检测细胞在藻酸盐凝胶中的存活和增殖能力。结果成功分离获得原代椎间盘脊索细胞,可稳定表达Ⅱ型胶原。原代脊索细胞在藻酸盐凝胶中生长良好,但增殖缓慢。结论初步了解兔椎间盘脊索细胞体外生物学特性,为椎间盘退变机制及组织工程学髓核种子细胞的研究提供一定的实验依据。     

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.     

Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension.

OBJECTIVE: To determine the influence of low oxygen tension on the redifferentiation and matrix production of dedifferentiated articular chondrocytes in monolayer and alginate bead culture. METHODS: Bovine articular chondrocytes were isolated enzymatically. After multiplication and dedifferentiation in a 2-week monolayer culture under 21% oxygen, the cells were subcultured in monolayer or alginate bead culture and subjected to 21% or 5% O(2)for 2 or 3 weeks in order to redifferentiate. Controls consisted of primary cultures in alginate. Matrix production was monitored immunocytochemically [collagen types I, II, IX, and GAGs (keratan sulfate, chondroitin-4- and -6-sulfate)] and collagen type II additionally assayed by Western blotting. Biosynthetic activity was measured by [(3)H]-proline incorporation and cell-viability by the trypan blue exclusion method. RESULTS: The cell number increased more than four-fold during dedifferentiation. Collagen type II was not produced by dedifferentiated chondrocytes under 5% or 21% oxygen in the monolayers or under 21% in alginate. However, dedifferentiated cells in alginate subjected to 5% oxygen exhibited a strong collagen type II expression indicating a redifferentiation. Additionally, collagen type IX and GAGs were also higher and [(3)H]-proline incorporation increased significantly. Primary cultures in alginate displayed a stronger collagen type II expression under 5% but no significant differences for other extracellular matrix components, or [(3)H]-proline incorporation. Viability was approximately 90% for all alginate cultures. CONCLUSION: A combination of alginate and high oxygen tension might not be suitable for redifferentiation or culturing of dedifferentiated chondrocytes. However, low oxygen tension promotes or induces a redifferentiation of dedifferentiated cells in alginate, stimulates their biosynthetic activity, and increases collagen type II production in primary alginate cultures.