Cartilage Tissue Construction In Vitro Based On Artificial Cells

Articular cartilage may be damaged due to illness, trauma or gradually old. But cartilage has limited ability to heal itself. Development of tissue engineering offers a promising way to address clinic therapy of cartilage. In order to improve the distribution and growth of cells in scafford, this study combined the characteristics of microcapsules and hydrogel to construct cartilage tissue, which based on “artificial cells” and filled with low melting point agarose(LMPA). The study explored the possibility cartilage tissue of artificial cell-hydrogel construct cells in vitro(static, shaking and 3D micro-gravity conditions). We expect to testify this construction method is possible and effective through the study, and provide a new way to tissue engineering construction in vitro.Firstly, C5.18 cells were cultured in vitro and growth curve showed it was a kind of rapidly proliferating cells. It went into the logarithmic growth phase after 24 h cultivation, and the population doubling time was 57.8 h with the best sampling time was 48-144 h. C5.18 cells were histological stained in vitro. HE(hematoxylin-eosin) staining showed the typical morphology of chondrocytes. Alcian blue and Safranin staining results indicated C5.18 showing the ability to synthesize glycosaminoglycan(GAG). Masson staining showed C5.18 had the ability to secrete collagen. Under optimized conditions, it could obtain microencapsulated cells with uniform cell distribution, good sphericity, and smooth surface by embedding a different cell density. And the particle size distribution was 150- 280 μm with an average particle diameter was 220 μm. Acridine Orange/Ethidium Bromide(AO/EB) staining showed most were green which meaning high cell viability which did not affect by microencapsulated operating process. Constructs were obtained by mixing artificial cells and LMPA and had regular shape with a certain mechanical strength. It could be found construct cells had high cell viability by AO/EB staining. And construct cells could be further used for in vitro cartilage tissue construction.Secondly, microcapsules and constructs were cultured under static, shaking and 3D micro-gravity conditions with the culture medium with and without b FGF treatment. Then quantified(Cell proliferation, GAG content and Type II collagen(Col-II) secretion) and qualitative(AO/EB, HE, alcian blue, safranin-O and immunohistochemistry staining) data were measured and analysed.For static conditions, cell viability and the corresponding GAG and Col-II synthesis of cells in microcapsules and constructs reached maximum on day 7. Cells in microcapsules and constructs showed positive staining without adding b FGF on day 5. Then stainings were negative accompanied by a decline in amount of cells in the following days. And cells showed a clear advantage on cell proliferation after adding b FGF with the highest activity appeared on day 10. And relevant staining results were positives.For shaking conditions, cells in microcapsules and constructs obtained the maximum viability and corresponding stainings were positive on day 10 without b FGF. The highest viability of cells appeared on day 15 which was accelerated by b FGF. And stainings were positive indicated that the secretion amount of GAG and Col-II chondrocytes reached the maximum, at the same time cells aggregates formation was observed. Although cell aggregates were formed under shaking conditions and b FGF was added, it had not yet reached the desired target of cartilage tissue construction in vitro, due to mass transfer still affected cartilage construction.For 3D micro-gravity conditions, microcapsules and constructs had the highest viability and maximum content of extracellular matrix(ECM)on day 15. And after adding b FGF, cells showed significant advantages. Cell proliferation and secretion rate of ECM were higher with reaching the maximum on day 20. The corresponding staining results appeared positive, and cell aggregates grew to about 100 μm on day 20.In conclusion, C5.18 cells cultured in vitro had high proliferation rate and ability to secrete GAG and collagen. Arificial cells with suitable dimension, enven cell distribution and high viability were prepared through high-voltage electrostatic method. Constructs with a certain mechanical strength and high viability were obtained by mixing artificial cells and LMPA. Large size and high viability of cell aggregates could not be reached under static and shaking conditions. However, under 3D micro-gravity conditions, large cell aggregates which size was about 100 μm, highly active and large ECM formed, which provides possibility for cartilage tissue constructs in vitro.