Development Of3D Dynamical Culture System Based On Alginate Gel Beads And Its Application To Cartilage Tissue Engineering

Current clinical treatments for cartilage defects suffer from long-term inefficiency. Tissue engineering offers a promising method to address this problem, although there are many challenges, which include ex vivo expansion of articular chondrocytes and the chondrogenesis of mesenchymal stem cells. Alginate can form hydrogels under cytocompatible conditions, provides a three-dimensional (3D) microenviroment for cells to maintain chondrocytic phenotype. In addition, mechanical stimulation is also important in engineering cartilage tissue. In this work, we aimed at developing a3D dynamic culture system, which combines alginate gel beads with spinner flasks to faciliate the expansion and differentiation of both chondrocytes and mesenchymal stem cells (MSCs).First, rabbit articular chondrocytes (rACs) of passage1(PI) and P4were encapsulated in alginate beads and cultivated in spinner flask for35days, followed by subcutaneous implantation in nude mice for28days. It was shown that3D dynamic culture was not efficient in retaining the phenotype of P1rACs and stimulating the redifferentiation of P4rACs. But, hypertrophic differentiation was not observed for rACs. Then, rabbit mesenchymal stem cells (rMSCs) were induced to chondrogenic differentiation in this system. While significant chondrogenesis of rMSCs was demonstrated, there was hypertrophic differentiation. Notably, cell death was significant for rMSCs. Third, co-culture between MSCs and rACs was carried out in the3D dynamic culture system, which was also designed to be able to separate the two different cells after coculture. To achieve this, magnetic nanoparticles (Fe3O4) was encapsulated in alginate beads along with one type of cells. Suitable concentrations of Fe3O4and alginate were established. In the end, co-culture was carried out in either chondrogenic media with or without2%FBS in this system. In medium without FBS, better chondrogenesis of rMSCs was obtained in co-culture compared to mono-culture of rMSCs. However, cell death was significant. In contrast, in medium with FBS, cells death was attenuated and rMSCs in co-culture demonstrated less efficiency in inducing chondrogenic differentiation of rMSCs. Neverthelss, in this dyanmic co-culture system, the expression of type X collagen was inhabited. Surprisingly, chondrogenic induction of human amniotic mesenchymal stem cells (hAMSCs) through co-culture with rACs was not successful, which was possibly due to the fact that these cells were derived from amniotic membrane with low intrinsic chondrogenic potential.In conclusion,3D dynamic culture is not sufficient to sustain the phenotype of rACs, but to inhibit hypertrophy; it can induce the chondrogenie differentiation of rMSCs, but can not inhibit the hypertrophy. A novel co-culture system based on this3D dynamic culture, which enables easy separation of the two types of cells in co-culture, was further developed. This co-culture system was demonstrated to be able to facilitate better chondrogenesis of rMSCs when in co-culture with rACs. This novel system warrants further optimization for better application in cartilage tissue engineering.