Formation of a Tissue-Engineered Cartilage Structure Based on Decellularized Cartilage Microparticles in a Perfusion Bioreactor
摘要
Objective: The formation of a tissue equivalent based on injectable form of microdispersed scaffold— microparticles of decellularized porcine cartilage (DecCp)—seems to be a promising technology for repairing cartilage tissue defects. The aim of this work was to obtain and comparatively study a tissue-engineering construct (TEC) based on DecCp microparticles and mesenchymal stromal cells (MSCs) under static conditions and in perfusion bioreactor. Materials and methods: The decellularization process included freeze-thaw cycles (–196°C/+37°C), the use of surfactants (Triton X-100 and sodium dodecyl sulfate), as well as DNase treatment. The morphology of the surface and the nearest subsurface layer of the samples was studied using scanning electron microscopy. Each TEC consisted of 5 × 105 MSCs and 5 mg DecCp. Results: It was found that, compared with static conditions, the cultivation of MSCs on DecCp microparticles in a perfusion bioreactor for 14 days allows increasing the proliferative activity of cells with subsequent chondrogenic differentiation, as evidenced by the ability of the cellular component of cartilage to synthesize extracellular matrix (ECM), characteristic of cartilage tissue, histochemical analysis of which revealed the presence of collagen and glycosaminoglycans (GAG). Conclusion: The possibility of forming cartilage TECs based on DecCp and MSCs under 3D cultivation conditions both under static conditions and in a perfusion bioreactor was shown. Cultivation of MSCs on DecCp under flow conditions at a rate of 0.5 mL/min contributed to an increase in cell proliferative activity compared with static conditions, and also supported the ability of cells to synthesize ECM, characteristic of cartilage tissue, histochemical analysis of which revealed the presence of total collagen and GAG, which may be evidence of chondrogenic differentiation of MSCs.