Enhanced chondrogenic differentiation of adipose-derived stem cells by spheroid culture system

Abstract

Damaged articular cartilage has poor intrinsic regenerative capacity, due to its complex and avascular nature. Autologous chondrocyte transplantation (ACT) is a possible treatment, but ACT requires large number of chondrocytes in order to transplant them into the site of defect and the acquisition of choncdocytes relies on in vitro expansion of chondrocytes acquired from patients cartilage. This method involves multiple surgical procedure to harvest and transplant chondocytes, which may cause further cartilage degeneration, and in vitro expansion of chondrocytes can result in dedifferentiation of the cells which could diminish the result of the treatment. Current therapeutic approach is cartilage regeneration with usage of mesenchymal stem cells (MSCs) isolated from various adult tissues such as bone marrow and adipose tissue. Among the sources of MSCs, relatively easy harvest method(eg. liposuction) and repeatable access makes human adipose-derived stem cells (hASCs) an attractive cell source for cartilage regeneration techniques requiring large number of cells. Generally, one large dense cell aggregate called pellet was made and ,by utilizing this system, chondrogenic differentiation of adult stem cells was induced by supplementing transforming growth factor, dexamethasone, and ascorbate-2-phosphate during culture. But diffusional limitation of nutrients and exertion of heavy hypoxia in the pellet core induces cell death which makes it a unfavorable culture system for chondrogenic differentiation. To enhance the efficacy of chondrogenic differentiation of hASCs, spheroid culture system was evaluated. In chapter three, hASCs were aggregated into smaller pellet called spheroid and spinner flask culture system was implemented to enhance cell-cell interaction and overcome unfavorable factors such as diffusional limitation and enhance chondrogenic differentiation of hASCs. We hypothesized that implementation of spheroid culture system could induce mild hypoxia in the core of the spheroid, which is favorable for chondrogenic differentiation, and enhance interaction between the cells. Both of the factor could result in enhanced chondrogenic differentiation of hASCs. Spheroid is generally induced by a method called hanging-drop, where cells of certain concentrations are homogeneously suspended and made into droplets which is hanged upside-down. In this study, we implemented spinner flask to induce spheroid, eliminating the conventional method and thus was able to form spheroids with much less time and labour. Formed spheroids were cultured in the spinner flask for 14 days and chondrogenic differentiation was evaluated. In vitro chondrogenic differentiation of hASCs was enhanced by spheroid culture compared with monolayer culture. The enhanced chondrogenesis was probably attributable to hypoxia-related cascades and enhanced cell-cell interactions in hASC spheroids. Upon hASCs loading in fibrin gel and transplantation into subcutaneous space of athymic mice for four weeks, the in vivo cartilage formation was enhanced by the transplantation of spheroid-cultured hASCs compared with that of monolayer-cultured hASCs. This study shows that spheroid culture may be an effective method for large scale in vitro chondrogenic differentiation of hASCs and subsequent in vivo cartilage formation. In chapter four, we focused on utilizing relatively new material called graphene oxide (GO) to enhance chondrogenic differentiation of hASCs. We hypothesized that presence of the functional groups on GO could interact with extracellular matrix molecules, such as fibronectin (FN) in the culture serum and stably bind growth factors which would help to enhance chondrogenic differentiation of hASCs when interacted with the cell. Using this hypothesis, we interacted GO with the media containing serum and evaluated the proteins absorbed on the GO. GO features both hydrophobic π domains and carboxylic and hydroxyl groups. The π-electron clouds in the GO sheets are capable of interacting with the inner hydrophobic cores of FN and growth factors. It was reported that hydroxyl functional groups stably interact with FN and prevent its deformation. Presence of FN on GO sheets were detected and enhancement of its cellular receptor, integrin expression was detected which could help enhance chondrogenic differentiation. Also, charge-charge interaction of growth factor and GO function groups enabled growth factor absorption onto the GO sheets. FN and growth factor bound GO was than incorporated into spheroids during hanging-drop method for even distribution of GO. As formed spheroids were cultured in the spinner culture and the chondrogenic differentiation of hASC spheroids were evaluated.