Altered in vitro chondrogenic properties of chondrocytes harvested from unaffected cartilage in osteoarthritic joints (original) (raw)
Related papers
2018
Osteoarthritis (OA) is a joint disease involving cartilage degeneration. This study aimed 15 to compare properties of chondrocytes from less-affected (LA-Cartilage) and severely-affected (SA16 Cartilage) of human OA articular cartilage. Based on Dougados classification, OA cartilage was 17 classified into two groups; less-affected (Grade 0-1) and severely-affected (Grade 2-3). Chondrocytes 18 from each group were cultured until passage (P) 4. Growth, migration, stem cell properties and 19 chondrogenic properties under normal and inflammatory conditions, and the formation of in vitro 20 3D cartilage tissues were compared between groups. The growth and migratory properties of LA21 chondrocytes and SA-chondrocytes were similar, except that the migration rate of SA-chondrocytes 22 was significantly higher at P0 compared to LA-chondrocytes. Both LA-chondrocytes and SA23 chondrocytes expressed mesenchymal stem cell markers and tri-lineage differentiation, but the 24 expression of stem cel...
Chondrogenic Potential of Articular Chondrocytes Depends on Their Original Location
Tissue Engineering Part A, 2013
Objective: This study aimed to investigate the regenerative capacity of chondrocytes derived from debrided defect cartilage and healthy cartilage from different regions in the joint to determine the best cell source for regenerative cartilage therapies. Methods: Articular cartilage was obtained from Outerbridge grade III and IV cartilage lesions and from macroscopically healthy weight-bearing and nonweight-bearing (NWB) locations in the knee. Chondrocytes isolated from all locations were either pelleted directly (P0 pellets) or after expansion (P2 pellets) and analyzed for glycosaminoglycan (GAG), DNA, and cartilage-specific gene expression. Harvested cartilage samples and cultured pellets were also analyzed by Safranin O histology and immunohistochemistry for collagen I, II, and X. Immunohistochemical stainings were quantified using a computerized pixel-intensity staining segmentation method. Results: After 4 weeks of culture, the P0 pellets derived from grade III or healthy weight-bearing chondrocytes contained more ( p < 0.015) GAG and GAG normalized per DNA compared to those from grade IV and NWB locations. After expansion, these differences were lost. Cartilage-specific gene expression was higher ( p < 0.04) in P0 pellets from grade III chondrocytes compared to grade IV chondrocytes. Semiquantitative immunohistochemistry showed a more intense ( p < 0.033) collagen I and X staining for grade IV debrided cartilage compared to grade III and weight-bearing cartilage. Also, collagen type X staining intensity was higher ( p < 0.033) in NWB cartilage compared to grade III and weight-bearing regions. Conclusion: Chondrocytes derived from debrided cartilage perform better than cells from the NWB biopsy site, however, this difference is lost upon expansion. Based thereon, the debrided defect cartilage could be a viable donor site for regenerative cartilage surgery.
Effect of Collagen Type I or Type II on Chondrogenesis by Cultured Human Articular Chondrocytes
Tissue Engineering Part A, 2013
Introduction: Current cartilage repair procedures using autologous chondrocytes rely on a variety of carriers for implantation. Collagen types I and II are frequently used and valuable properties of both were shown earlier in vitro, although a preference for either was not demonstrated. Recently, however, fibrillar collagens were shown to promote cartilage degradation. The goal of this study was to evaluate the effects of collagen type I and type II coating on chondrogenic properties of in vitro cultured human chondrocytes, and to investigate if collagen-mediated cartilage degradation occurs. Methods: Human chondrocytes of eight healthy cartilage donors were isolated, expanded, and cultured on culture well inserts coated with either collagen type I, type II, or no coating (control). After 28 days of redifferentiation culture, safranin O and immunohistochemical staining for collagen types I, II, X, and Runx2/ Cbfa1 were performed and glycosaminoglycan (GAG) and DNA content and release were examined. Further, expression of collagen type I, type II, type X, MMP13, Runx2/Cbfa1, DDR2, a2 and b1 integrin were examined by reverse transcriptase-polymerase chain reaction. Results: The matrix, created by chondrocytes grown on collagen type I-and II-coated membranes, resembled cartilage more than when grown on noncoated membranes as reflected by histological scoring. Immunohistochemical staining did not differ between the conditions. GAG content as well as GAG/DNA were higher for collagen type II-coated cartilage constructs than control. GAG release was also higher on collagen type I-and IIcoated constructs. Expression of collagen type X was higher of chondrocytes grown on collagen type II compared to controls, but no collagen X protein could be demonstrated by immunohistochemistry. No effects of collagen coating on DDR2 nor MMP-13 gene expression were found. No differences were observed between collagen types I and II. Conclusion: Chondrocyte culture on collagen type I or II promotes more active matrix production and turnover. No significant differences between collagen types I and II were observed, nor were hypertrophic changes more evident in either condition. The use of collagen type I or II coating for in vitro models, thus, seems a sound basis for in vivo repair procedures.
Therapeutic advances in musculoskeletal disease, 2012
Chondrogenesis occurs as a result of mesenchymal cell condensation and chondroprogenitor cell differentiation. Following chondrogenesis, the chondrocytes remain as resting cells to form the articular cartilage or undergo proliferation, terminal differentiation to chondrocyte hypertrophy, and apoptosis in a process termed endochondral ossification, whereby the hypertrophic cartilage is replaced by bone. Human adult articular cartilage is a complex tissue of matrix proteins that varies from superficial to deep layers and from loaded to unloaded zones. A major challenge to efforts to repair cartilage by stem cell-based and other tissue-engineering strategies is the inability of the resident chondrocytes to lay down a new matrix with the same properties as it had when it was formed during development. Thus, understanding and comparing the mechanisms of cartilage remodeling during development, osteoarthritis (OA), and aging may lead to more effective strategies for preventing cartilage d...
Tissue Engineering, 2005
Autologous chondrocyte implantation is currently applied in clinics as an innovative tool for articular cartilage repair. Animal models have been and still are being used to validate and further improve the technique. However, in various species, the outcome varies from hyaline-like cartilage to fibrocartilage. This may be due partly to the spontaneous dedifferentiation of chondrocytes once cultured in vitro. Here we assessed whether the extent of dedifferentiation varies between species and we hypothesized that the level of chondrocyte phenotype stability during expansion may contribute to the maintenance of their chondrogenic commitment and redifferentiation potential. Condyle chondrocytes were harvested from sheep, dog, and human, and expanded for 1, 6, or 12 cell duplications. At each interval, cell phenotype was monitored (morphology and biosynthesis of cartilage markers) and redifferentiation was assessed by an in vitro assay of chondrogenesis in micromass pellet and an in vivo assay of ectopic cartilage formation in immunodeficient mice. Results indicate that, during culture, the sheep chondrocyte phenotype is maintained better than that of human chondrocytes, which in turn dedifferentiate to a lesser extent than dog chondrocytes Accordingly, after expansion, sheep chondrocytes spontaneously reform hyaline-like cartilage; human chondrocytes redifferentiate only under stimulation with chondrogenic inducers whereas, after a few passages, dog chondrocytes lose any capacity to redifferentiate regardless of the presence of inducers. Thus, conditions allowing cartilage formation in one species are not necessarily transposable to other species. Therefore, results with animal models should be cautiously applied to humans. In addition, for tissue-engineering purposes, the number of cell duplications must be, for each species, carefully monitored to remain in the range of amplification allowing redifferentiation and chondrogenesis.
Arthritis & Rheumatism, 2007
Objective. To identify markers associated with the chondrogenic capacity (CC) of expanded human articular chondrocytes and to use these markers for sorting of more chondrogenic subpopulations. Methods. The CC of chondrocyte populations derived from different donors (n=21) or different clonal strains from the same cartilage biopsy (n=21) was defined based on the glycosaminoglycan (GAG) content of tissues generated using a pellet culture model. Selected cell populations were analysed by microarray and cytofluorimetry. In some experiments, cells were sorted using antibodies against molecules found to be associated with differential CC and again assessed in pellet cultures. Results. Significance analysis of microarrays indicated that chondrocytes with low CC expressed greater levels of insulin-like growth factor-1 and of catabolic genes (e.g., metalloproteinase-2, aggrecanase-2), while chondrocytes with high CC expressed greater levels of genes involved in cell-cell or cell-matrix interactions (e.g., CD49c, CD49f). Cytofluorimetry analysis showed that CD44, CD151 and CD49c were expressed at significantly greater levels in chondrocytes with higher CC. Cytofluorimetrical analysis of clonal chondrocyte strains indicated that CD44 and CD151 can also identify more chondrogenic clones. Chondrocytes sorted for brighter CD49c or CD44 signal expression produced tissues with higher GAG/DNA (up to 1.4-fold) and collagen type II mRNA (up to 3.4-fold) than unsorted cells. Conclusion. We identified markers enabling to characterize the capacity of monolayer expanded chondrocytes to form in vitro cartilaginous tissue and to enrich for subpopulations with higher CC. These markers might be used as a mean to predict and possibly improve the outcome of cell-based cartilage repair techniques.
Osteoarthritis (OA) is a joint disease involving cartilage degeneration. This study aimed to compare properties of chondrocytes from less-affected (LA-Cartilage) and severely-affected (SA-Cartilage) of human OA articular cartilage. Based on Dougados classification, OA cartilage was classified into two groups; less-affected (Grade 0–1) and severely-affected (Grade 2–3). Chondrocytes from each group were cultured until passage (P) 4. Growth, migration, stem cell properties and chondrogenic properties under normal and inflammatory conditions, and the formation of in vitro 3D cartilage tissues were compared between groups. The growth and migratory properties of LA-chondrocytes and SA-chondrocytes were similar, except that the migration rate of SA-chondrocytes was significantly higher at P0 compared to LA-chondrocytes. Both LA-chondrocytes and SA-chondrocytes expressed mesenchymal stem cell markers and tri-lineage differentiation, but the expression of stem cell markers decre...
Tissue Engineering, 2005
Autologous chondrocyte implantation is currently applied in clinics as an innovative tool for articular cartilage repair. Animal models have been and still are being used to validate and further improve the technique. However, in various species, the outcome varies from hyaline-like cartilage to fibrocartilage. This may be due partly to the spontaneous dedifferentiation of chondrocytes once cultured in vitro. Here we assessed whether the extent of dedifferentiation varies between species and we hypothesized that the level of chondrocyte phenotype stability during expansion may contribute to the maintenance of their chondrogenic commitment and redifferentiation potential. Condyle chondrocytes were harvested from sheep, dog, and human, and expanded for 1, 6, or 12 cell duplications. At each interval, cell phenotype was monitored (morphology and biosynthesis of cartilage markers) and redifferentiation was assessed by an in vitro assay of chondrogenesis in micromass pellet and an in vivo assay of ectopic cartilage formation in immunodeficient mice. Results indicate that, during culture, the sheep chondrocyte phenotype is maintained better than that of human chondrocytes, which in turn dedifferentiate to a lesser extent than dog chondrocytes Accordingly, after expansion, sheep chondrocytes spontaneously reform hyaline-like cartilage; human chondrocytes redifferentiate only under stimulation with chondrogenic inducers whereas, after a few passages, dog chondrocytes lose any capacity to redifferentiate regardless of the presence of inducers. Thus, conditions allowing cartilage formation in one species are not necessarily transposable to other species. Therefore, results with animal models should be cautiously applied to humans. In addition, for tissue-engineering purposes, the number of cell duplications must be, for each species, carefully monitored to remain in the range of amplification allowing redifferentiation and chondrogenesis.
Optimization of Protocol for Isolation of Chondrocytes from Human Articular Cartilage
CARTILAGE, 2019
Objective Cartilage tissue engineering has evolved as one of the therapeutic strategies for cartilage defect, which relies on a large number of viable chondrocytes. Because of limited availability of cartilage and low chondrocytes yield from cartilage, the need for an improve isolation protocol for maximum yield of viable cells is a key to achieving successful clinical constructs. This study optimizes and compares different protocols for isolation of chondrocytes from cartilage. Design We employed enzymatic digestion of cartilage using collagenase II and trypsin. The chondrocytes yield, growth kinetics, aggrecan, and collagen type 2 (COL2) expression were evaluated. Collagen type 1 (COL1) mRNA expression was assessed to monitor the possibility of chondrocytes dedifferentiation. Results Chondrocyte yield per gram of cartilage was significantly higher ( P < 0.05) using collagenase II in Hank’s balanced salt solution (HBSS) compared with 0.25% trypsin. The number of chondrocyte yiel...