Evaluation of osteochondral-like tissues using human freeze-dried cancellous bone and chondrocyte sheets to treat osteochondral defects in rabbits.

Human freeze-dried cancellous bone combined with human chondrocyte sheets have recently been used to construct an osteochondral-like tissue, which resembled a cartilage layer on a subchondral bone layer. Nevertheless, the efficacy of these human tissues in a xenogeneic model has been rarely reported. Therefore, this study aimed to evaluate the potential of human freeze-dried cancellous bones combined with human chondrocyte sheets for the treatment of osteochondral defects in rabbits. The key roles of the extracellular matrix (ECM) and released cytokines in these tissues in osteochondral repair were also assessed. Triple-layered chondrocyte sheets were constructed using a temperature-responsive culture surface. Then, they were placed onto cancellous bone to form chondrocyte sheet-cancellous bone tissues. The immunostaining of collagen type II (COL2) and the proteomic analysis of the human tissues were carried out before the transplantation. In our in vitro study, the triple-layered chondrocyte sheets adhered well on the cancellous bone, and the COL2 expression was apparent throughout the tissue structures. From the proteomic analysis results, it was found that the major function of the secreted proteins found in these tissues was protein binding. The distinct pathways were focal adhesion and the ECM-receptor interaction pathways. Among the highly expressed proteins, laminin-alpha 5 (LAMA5) and fibronectin (FN) not only played roles in the protein binding and ECM-receptor interaction, but also were involved in the cytokine-mediated signaling pathway. At 12 weeks after xenogeneic transplantation, compared to the control group, the defects treated with the chondrocyte sheets showed more hyaline-like cartilage tissue, as indicated by the abundance of safranin-O and COL2 with a partial collagen type I (COL1) expression. At 4, 8, and 12 weeks, compared to the defects treated with the cancellous bone, the staining of safranin-O and COL2 was more apparent in the defects treated with the chondrocyte sheet-cancellous bone tissues. Therefore, the human chondrocyte sheets and chondrocyte sheet-cancellous bone tissues provide a potential treatment for rabbit femoral condyle defect. LAMA5 and FN found in these human xenografts and their culture media might play key roles in the ECM-receptor interaction and might be involved in the cytokine-mediated signaling pathway during tissue repair.

Construction and Evaluation of Osteochondral-Like Tissue Using Chondrocyte Sheet and Cancellous Bone.

The manipulation of human chondrocyte sheets in target areas frequently results in their tearing because they are thin and fragile. In this study, human cancellous bones were used as a supporting material to create chondrocyte sheet-cancellous bone tissues, and their properties were evaluated. Using cell sheet technology, human chondrocytes were constructed into triple-layered chondrocyte sheets that displayed chondrogenic properties. After transferring the chondrocyte sheets onto cancellous bones, the top area of the chondrocyte sheet-cancellous bone tissues exhibited a smooth surface topography without cell sheet floating within 7 days of culture. The immunofluorescence staining of collagen type II (COL2A1) and fibronectin (FN1) was also performed and examined. Using the shotgun proteomic analysis, the proteins associated with cell adhesion, extracellular matrix (ECM) organization, cell-substrate junction assembly, and cell adhesion mediated by integrin were observed in the chondrocyte sheets, cancellous bones, and chondrocyte sheet-cancellous bone tissues. Three integrin members, including integrin ß4 (ITGB4), ITGB6, and ITGB8, were found in the chondrocyte sheets. Only ITGB8 was found in the chondrocyte sheets and chondrocyte sheet-cancellous bone tissues. During 48 h, the mean velocity of the individual cell migration was low, which did not affect the structure and chondrogenic properties of the chondrocyte sheets. Staining of the filamentous actin (F-actin) cytoskeleton in the migratory cells also provided a better understanding of the dynamic communication between the cell cytoskeleton and adhesion molecules through ITGB8, which may play a key role in the attachment of the chondrocyte sheets and the synthesis of the cartilage ECM. Therefore, we suggest that cancellous bone could be used as a supporting material to construct chondrocyte sheet-cancellous bone tissues for potential treatment of osteochondral lesions. Impact Statement We proposed a method to construct an osteochondral-like tissue by placing human chondrocyte sheets onto cancellous bone. The stationary chondrocyte sheets and the low mean velocity of the individual cell migration on the cancellous bone with the expression of COL2A1 indicated that the cancellous bone served as an appropriate supporting material. Moreover, the cellular mechanism for the adhesion of the chondrocyte sheets on the cancellous bone based on ITGB8-mediated adhesion through the rearrangement of filamentous actin provided a better understanding to improve the construction of osteochondral-like tissues, and to predict the repair mechanism in osteoarthritis therapy.

Maintenance of human chondrogenic phenotype on a dendrimer-immobilized surface for an application of cell sheet engineering.

BACKGROUND: Dedifferentiation of chondrocytes during cell expansion is one of the barriers in tissue construction for cartilage repair. To understand chondrocyte behavior and improve cell expansion in monolayer culture, this study investigated the effects of morphological changes and cellular aggregation on the maintenance of chondrogenic capacity by observing the expression patterns of chondrogenic (collagen type II and aggrecan) and dedifferentiation (collagen type I) markers. Primary human chondrocytes were cultured on either a polystyrene surface (PS) or a polyamidoamine dendrimer surface with a fifth-generation (G5) dendron structure to create a one-step process of cell expansion and the maintenance of chondrogenic activities prior to the construction of cell sheets. RESULTS: During the first two passages (P0 - P2), the relative mRNA level of collagen type II decreased in all cultures, while that of collagen type I increased. Remarkably, the level of collagen type II was higher and aggrecan was retained in the chondrocytes, forming cell aggregates and showing some round-shaped cells with less production of stress fibers on the G5 surface compared to fibroblast-like chondrocytes with abundant stress fibers on the PS surface. The numbers of P2 chondrocytes on the G5 and PS surfaces were nearly the same and sufficient for construction of chondrocyte sheets using a temperature-responsive plate. Without a supporting material during cell sheet manipulation, chondrocyte sheets spontaneously detached and exhibited a honeycomb-like structure of stress fibers. Unlike the chondrocyte sheets constructed from cells on the PS surface, the chondrocyte sheets from cells on the G5 surface had higher chondrogenic activities, as evidenced by the high expression of chondrogenic markers and the low expression of dedifferentiation markers. CONCLUSIONS: The one-step process of cell expansion and maintenance of chondrogenic activity could be obtained using the G5 surface. Human chondrocyte sheets were successfully constructed with high chondrogenic activity. These findings may lead to an alternative cultivation technique for human chondrocytes that offers high clinical potential in autologous chondrocyte implantation.

Effect of Cell Sheet Manipulation Techniques on the Expression of Collagen Type II and Stress Fiber Formation in Human Chondrocyte Sheets.

Cell sheet technology is applied to human articular chondrocytes to construct a tissue-like structure as an alternative treatment for cartilage defect. The effect of a gelatin manipulator, as a cell sheet transfer system, on the quality of the chondrocyte sheets was investigated. The changes of important chondrogenic markers and stress fibers, resulting from the cell sheet manipulation, were also studied. The chondrocyte cell sheets were constructed with patient-derived chondrocytes using a temperature-responsive polymer and a gelatin manipulator as a transfer carrier. The properties of the cell sheets, including sizes, expression levels of collagen type II and I, and the localization of the stress fibers, were assessed and compared with those of the cell sheets harvested without the gelatin manipulator. Using the gelatin manipulator, the original size of the chondrocyte cell sheets was retained with abundant stress fibers, but with a decrease in the expression of collagen type II. Without the gelatin manipulator, although the cell shrinkage occurred, the cell sheet with suppressed stress fiber formation showed significantly higher levels of collagen type II. These results support our observations that stress fiber formation in chondrocyte cell sheets affected the production of chondrogenic markers. These densely packed tissue-like structures possessed a good chondrogenic activity, indicating their potential for use in autologous chondrocyte implantation to treat cartilage defects.

Chondrogenesis and hypertrophy in response to aggregate behaviors of human mesenchymal stem cells on a dendrimer-immobilized surface.

OBJECTIVES: To investigate the behaviors of aggregates of human mesenchymal stem cells (hMSCs) on chondrogenesis and chondrocyte hypertrophy using spatiotemporal expression patterns of chondrogenic (type II collagen) and hypertrophic (type X collagen) markers during chondrogenesis. RESULTS: hMSCs were cultured on either a polystyrene surface or polyamidoamine dendrimer surface with a fifth generation (G5) dendron structure in chondrogenic medium and growth medium. At day 7, cell aggregates without stress fibers formed on the G5 surface and triggered differentiation of hMSCs toward the chondrogenic fate, as indicated by type II collagen being observed while type X collagen was undetectable. In contrast, immunostaining of hMSCs cultured on polystyrene, which exhibited abundant stress fibers and did not form aggregates, revealed no evidence of either type II and or type X collagen. At day 21, the morphological changes of the cell aggregates formed on the G5 surface were suppressed as a result of stress fiber formation. Type II collagen was observed throughout the aggregates whereas type X collagen was detected only at the basal side of the aggregates. Change of cell aggregate behaviors derived from G5 surface alone regulated chondrogenesis and hypotrophy, and this was enhanced by chondrogenic medium. CONCLUSIONS: Incubation of hMSCs affects the expression of type II and X collagens via effects on cell aggregate behavior and stress fiber formation.