Effect of dynamic compressive loading and its combination with a growth factor on the chondrocytic phenotype of 3-dimensional scaffold-embedded chondrocytes

Background and purpose Three-dimensionally (3D-) embedded chondrocytes have been suggested to maintain the chondrocytic phenotype. Furthermore, mechanical stress and growth factors have been found to be capable of enhancing cell proliferation and ECM synthesis. We investigated the effect of mechanical loading and growth factors on reactivation of the 3D-embedded chondrocytes.Methods Freshly isolated chondrocytes from rat articular cartilage were grown in monolayer cultures and then in collagen gel. Real-time RT-PCR and histological analysis for aggrecan and type II and type I collagen was performed to evaluate their chondrocytic activity. Then, the 3D-embedded chondrocytes were cultured under either mechanical loading alone or in combination with growth factor. The dynamic compression (5% compression, 0.33 Hz) was loaded for 4 durations: 0, 10, 60, and 120 min/day. The growth factor administered was either basic fibroblast growth factor (bFGF) or bone morphogenetic protein-2 (BMP-2).Results Mechanical loading statistically significantly reactivated the aggrecan and type II collagen expression with loading of 60 min/day as compared to the other durations. The presence of BMP-2 and bFGF clearly enhanced the aggrecan and type II collagen expression of 3D-embedded chondrocytes. Unlike previous reports using monolayer chondrocytes, however, BMP-2 or bFGF did not augment the chondrocytic phenotype when applied together with mechanical loading.Interpretation Dynamic compression effectively reactivated the dedifferentiated chondrocytes in 3D culture. However, the growth factors did not play any synergistic role when applied with dynamic compressive loading, suggesting that growth factors should be administered at different time points during regeneration of the transplantation-ready cartilage.     

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.     

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.     

Intermittent sub-ambient interstitial hydrostatic pressure as a potential mechanical stimulator for chondrocyte metabolism.

OBJECTIVE: Experimental findings have suggested that the metabolic activities of articular cartilage can be influenced by mechanical stimuli. Our mathematical analysis predicted that cyclic compressive loading may create periods of intermittent sub-ambient hydrostatic pressure within the cartilage extracellular matrix. Based on this mathematical analysis, the present study was aimed to investigate whether the intermittent sub-ambient hydrostatic pressure, created in the cartilage extracellular matrix during cyclic compression, has a stimulative effect on the biosynthesis of chondrocytes. METHOD: In order to test this hypothesis, the present study developed a custom-designed sub-ambient pressure generator to subject a monolayer culture of chondrocytes to an intermittent sub-ambient pressure. Using this pressure generator, the monolayer chondrocyte culture system was analyzed for 35S-sulfate and 3H-proline incorporation rates for biosynthesis of proteoglycan and collagenous/noncollagenous protein molecules, respectively. Northern analyses for aggrecan and type II collagen mRNAs were also performed. RESULTS: It was found that the intermittent sub-ambient pressure produced a 40% increase in proteoglycan and a 17% increase in non-collagenous protein synthesis during the pressurization period (P < 0.05). The collagenous protein synthesis was not affected by the intermittent sub-ambient pressure regimen used in this study. After the intermittent sub-ambient pressurization, the metabolic activities of the chondrocytes returned to normal (control level). The intermittent sub-ambient pressure also produced an increase in the mRNA signals for aggrecan. Therefore, we conclude that intermittent sub-ambient pressure may be one of the potential mechanical stimulators of chondrocytes in articular cartilage during dynamic compression.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Regulation of articular chondrocyte aggrecan and collagen gene expression by multiple growth factor gene transfer

Gene transfer is a promising approach to the delivery of chondrotrophic growth factors to promote cartilage repair. It is unlikely that a single growth factor transgene will optimally regulate these cells. The aim of this study was to identify those growth factor transgene combinations that optimally regulate aggrecan, collagen type II and collagen type I gene expression by articular chondrocytes. We delivered combinations of the transgenes encoding fibroblast growth factor-2, insulin-like growth factor I, transforming growth factor beta1, bone morphogenetic protein-2, and/or bone morphogenetic protein-7 and assessed chondrocyte responses by measuring changes in the expression of aggrecan, type II collagen and type I collagen genes. These growth factor transgenes differentially regulated the magnitude and time course of all three-matrix protein genes. In concert, the transgenes regulated matrix gene expression in an interactive fashion that ranged from synergistic to inhibitory. Maximum stimulation of aggrecan (16-fold) and type II collagen (35-fold) expression was with the combination of IGF-I, BMP-2, and BMP-7 transgenes. The results indicate that the optimal choice of growth factor genes for cell-based cartilage repair cannot be predicted from observations of individual transgenes. Rather, such gene therapy will require an empirically based selection of growth factor gene combinations.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

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.     

Enhanced matrix synthesis and in vitro formation of cartilage-like tissue by genetically modified chondrocytes expressing BMP-7.

Bone morphogenic protein-7 (BMP-7) supports ectopic cartilage and bone formation, is expressed in normal articular cartilage, and increases matrix synthesis in chondrocytes. Based on this knowledge, we hypothesized that an adenovirus (Ad) vector encoding human BMP-7 could be used to modify chondrocytes genetically to improve their capacity for cartilage repair. An adenovirus vector encoding BMP-7 (AdBMP-7) was constructed and its bioactivity confirmed by ectopic bone formation assay. AdBMP-7 modification of bovine chondrocytes induced expression of BMP-7 mRNA and bioactive protein, resulting in an increase in incorporation of 35SO4- into proteoglycan, 3H-proline uptake into protein, and the expression of the cartilage-specific matrix genes, aggrecan and type II collagen. An in vitro model of chondrocyte transplantation was used to demonstrate the feasibility of using genetically modified chondrocytes to enhance formation of cartilage-like tissue. When transplanted onto cartilage explants and maintained in vitro for 3 weeks, chondrocytes modified with AdBMP-7 formed 1.9-fold thicker tissue than chondrocytes modified with a control vector (P < 0.001). This tissue was positive for type II collagen and proteoglycan but negative for type X collagen and demonstrated a cartilage-like morphology. These observations suggest that Ad-mediated transfer of BMP-7 gene to chondrocytes enhances the chondrocyte-specific matrix synthesis and their capacity to form cartilage-like tissue, thus representing a strategy that may improve cell-based cartilage repair.     

Rapid phenotypic changes in passaged articular chondrocyte subpopulations.

Articular chondrocytes are often expanded in vitro and then used to assist in healing articular cartilage defects. We investigated the extent of dedifferentiation in monolayer-passaged, zonal articular chondrocytes by using quantitative, real-time PCR. The relative gene expressions for collagen type I and II, aggrecan, and superficial zone protein were analyzed for relevant passage numbers (P0-P4) to determine how the expansion of chondrocytes affects the expression of cartilage extracellular matrix proteins. Results reveal that dramatic changes occur as early as first passage. Furthermore, these changes are shown to persist even when the expanded cells are encapsulated in 3D, alginate beads. Successful tissue engineering and autologous cell transplantation procedures rely heavily on having a cell source that expresses the chondrocytic phenotype. The results of this study suggest that major problems exist at the front-end of cartilage regeneration efforts.     

Phenotypic stability of articular chondrocytes in vitro: the effects of culture models, bone morphogenetic protein 2, and serum supplementation.

Numerous in vitro culture models have been developed for the investigation of chondrocyte and cartilage biology. In this study, we investigated the stability of the chondrocytic phenotype in monolayer, aggregate, pellet, and explant culture models and assessed the effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) and serum supplementation on the phenotype in each model. Phenotypic effects were assessed by analyses of procollagen type II, aggrecan, (V + C)- fibronectin, and procollagen type I messenger RNA expression. In monolayer cultures, we noted a characteristic loss of procollagen type II and induction of procollagen type I expression. The aggregate and pellet culture models supported matrix protein gene expression profiles more reflective of in vivo levels. In explant cultures, expression of matrix protein genes was consistently depressed. Treatment with rhBMP-2 significantly increased the expression of procollagen type II and aggrecan in monolayer cultures; however, other models showed comparatively little response. Similarly, serum supplementation significantly down-regulated procollagen type II and aggrecan expression in monolayer cultures but had less effect on gene expression in the other models. Serum supplementation increased procollagen type I expression in monolayer and aggregate cultures. These results suggest that the influence of exogenous BMP-2 and serum on expression of chondrocyte-specific matrix protein genes is influenced by aspects of substrate attachments, cellular morphology, and/or cytoskeletal organization. Finally, the analyses of fibronectin expression suggest that V and C region alternative splicing in chondrocytes is linked to the establishment of a three-dimensional multicellular complex.     

Gene expression and proliferation analysis in young, aged, and osteoarthritic sheep chondrocytes effect of growth factor treatment.

Cartilage is a support tissue with a poor capacity to self-repair. Its cells, chondrocytes, are responsible for synthesizing and renewing the matrix that surrounds them in a constant turnover mechanism. Autologous chondrocyte implantation (ACI) is one of the techniques that promises to be an alternative to common strategies for chondral lesions. To apply this technique, a large amount of cells must be obtained. In our work, we studied the state of cells from different cartilage (young, aged, and osteoarthritic sheep) cultured in monolayer by analyzing their proliferation rate using bromodeoxyuridine and their gene expression profile by RT-PCR. A decrease was found in expression of type II collagen and aggrecan in aged, osteoarthritic, and passaged chondrocytes. Treatment of cells with growth factors aFGF, IGF-I, TGF-beta, and OP-1 improved the proliferation rate in all cells studied and stimulated gene expression of type II collagen, aggrecan, and TGF-beta. Osteoarthritic cells showed a poor response according to matrix gene expression, while young cells responded properly, and aged chondrocytes showed a moderate response. These results suggest that the state of cartilage may affect the behavior of cultured chondrocytes.     

去分化关节软骨细胞生物反应器培养反分化的实验研究

目的 观察体外经传代培养去分化的成人关节软骨细胞，在生物反应器培养后生物学性状的变化，探索去分化软骨细胞反分化的手段，为软骨细胞移植修复关节软骨缺损建立合适的体外培养方法。方法 无菌条件下取成人关节软骨组织，Ⅱ型胶原酶消化法（0．2％，37C，3h）分离软骨细胞，分成两组：一组常规单层传代培养，另一组添加重组人的生长因子（1ng／ml转化生长因子β1＋5ng／ml成纤维细胞生长因子2）体外培养传代大量扩增后，无微载体生物反应器内培养3周。血小板计数器行细胞计数，计算各代细胞倍增时间；细胞爬片和石蜡、冰冻切片进行HE、蕃红O、阿利新蓝染色，Ⅰ、Ⅱ型胶原和aggrecan免疫组织化学检测，观察细胞表型变化。结果 成人关节软骨细胞体外培养3代后迅速去分化，增殖缓慢。添加生长因子培养细胞去分化速度减缓；传10代，细胞扩增2000倍以上，部分去分化，但细胞扩增增殖能力仍很强；传20代软骨细胞表型基本丢失，但仍有增殖能力；置于生物反应器继续培养3周，细胞番红O染色强阳性、aggrecan和Ⅱ型胶原阳性，Ⅰ型胶原阴性，表型恢复良好。结论 软骨细胞在体外大量扩增后，在生物反应器培养，可恢复其表型，可望用于在体外培养时去分化软骨细胞的再分化。     

Down-regulation of chondrocyte aggrecan and type-II collagen gene expression correlates with increases in static compression magnitude and duration.

The goal of this study was to examine the simultaneous effects of mechanical compression of chondrocytes on mRNA expression and macromolecular synthesis of aggrecan and type-II collagen. Bovine cartilage explants were exposed to different magnitudes and durations of applied mechanical compression, and levels of aggrecan and type-IIa collagen mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase were measured and quantified by Northern blot analysis. Synthesis of aggrecan and type-II collagen protein was measured by radiolabel incorporation of [35S]sulfate and [3H]proline into macromolecules. The results showed a dose-dependent decrease in mRNA levels for aggrecan and type-II collagen, with increasing compression relative to physiological cut thickness applied for 24 hours. Radiolabel incorporation into glycosaminoglycans and collagen also decreased with increasing compression in a dose-related manner similar to the changes seen in mRNA expression. The modulation of aggrecan and type-II collagen mRNA and protein synthesis were dependent on the duration of the compression. Aggrecan and type-II collagen mRNA expression increased during the initial 0.5 hours of static compression; however, 4-24 hours after compression was applied total mRNA levels had significantly decreased. The synthesis of aggrecan and collagen protein decreased more rapidly than did mRNA levels after the application of a step compression. Together, these results suggest that mechanical compression rapidly alters chondrocyte aggrecan and type-II collagen gene expression on application of load. However, our results indicate that the observed decreases in biosynthesis may not be related solely to changes in mRNA expression. The mechanisms by which mechanical forces affect different segments of the biosynthetic pathways remain to be determined.     

Statin stimulates bone morphogenetic protein-2, aggrecan,and type 2 collagen gene expression and proteoglycan synthesis in rat chondrocytes

Statins increase bone morphogenetic protein-2 (BMP-2) mRNA expression and subsequently increase new bone formation in vitro. However, the action of statins on the BMP-2 mRNA regulation of cartilage matrix synthesis by chondrocytes is unknown. We evaluated regulation of BMP-2, aggrecan, and type II collagen (COL2) mRNA and ³⁵S-labeled proteoglycan (PG) synthesis by mevastatin using cultured chondrocytes obtained from articular cartilage of fetal rats. Expression of BMP-2, aggrecan, and COL2 mRNAs were increased in the presence of 2µM mevastatin on day 2. However, longer (10 day) culture in the presence of the drug decreased the expression of these mRNAs. PG synthesis was increased 3 days after treating the cells with mevastatin, which was also decreased with longer (10 day) mevastatin treatment. These results suggest that mevastatin increases mRNA expression of BMP-2, aggrecan, and COL2 as well as PG synthesis by fetal rat chondrocytes early in the treatment period. We suggest that statins have implications for fracture and cartilage repair.     

BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel

OBJECTIVE: According to recent reports, the synovial membrane may contain mesenchymal stem cells with the potential to differentiate into chondrocytes under appropriate conditions. In order to assess the usefulness of synovium-derived progenitor cells for the purposes of cartilage tissue engineering, we explored their requirements for the expression of chondrocyte-specific genes after expansion in vitro. DESIGN: Mesenchymal progenitor cells were isolated from the synovial membranes of bovine shoulder joints and expanded in two-dimensions on plastic surfaces. They were then seeded either as micromass cultures or as single cells within alginate gels, which were cultured in serum-free medium. Under these three-dimensional conditions, chondrogenesis is known to be supported and maintained. Cell cultures were exposed either to bone morphogenetic protein-2 (BMP-2) or to isoforms of transforming growth factor-beta (TGF-beta). The levels of mRNA for Sox9, collagen types I and II and aggrecan were determined by RT-PCR. RESULTS: When transferred to alginate gel cultures, the fibroblast-like synovial cells assumed a rounded form. BMP-2, but not isoforms of TGF-beta, stimulated, in a dose-dependent manner, the production of messenger RNAs (mRNAs) for Sox9, type II collagen and aggrecan. Under optimal conditions, the expression levels of cartilage-specific genes were comparable to those within cultured articular cartilage chondrocytes. However, in contrast to cultured articular cartilage chondrocytes, synovial cells exposed to BMP-2 continued to express the mRNA for alpha1(I) collagen. CONCLUSIONS: This study demonstrates that bovine synovium-derived mesenchymal progenitor cells can be induced to express chondrocyte-specific genes. However, the differentiation process is not complete under the chosen conditions. The stimulation conditions required for full transformation must now be delineated.