Biocompatibility Evaluation Of Electrospun Regenerated Spider Dragline Silk With Bone Marrow Mesenchymal Stem Cells And Study Of Adipose Differentiation Of Bone Marrow Mesenchymal Stem Cells

Bone-marrow mesenchymal stem cells (BMSCs) have generated a great deal of excitement and expectation as a potential source of cells for cell-based therapeutic strategies in regenerative medicine, primarily because of their intrinsic ability to self-renew and differentiate into functional cells of multiple tissues including fat, bone, muscle, neural and cartilage. They are easily isolated and expanded with high efficiency and induced to differentiate under well defined culture conditions.1. Isolation, in vitro expansion of bone marrow mesenchymal stem cellsThe high purification of bone marrow mesenchymal stem cells were obtained by incontinuous density Percoll gradient centrifugation technique. BMSCs expressed CD29, CD90, CD44, CD106, and did not express CD34 and CD45.2. Biocompatibility evaluation of electrospun regenerated spider dragline silk with bone marrow mesenchymal stem cellsTo evaluate the biocompatibility of electrospun regenerated spider dragline silk (SHSH) with bone marrow mesenchymal stem cells (BMSCs), cells were cultured on SHSH or PLL. Using live/dead assay, cell viability was assessed 24, 48, and 72 h after seeding. BrdU incorporation was used to label the dividing cells and the proliferation of BMSCs was assessed at 0-24 h, 24-48 h, and 48-72 h. Adipogenic differentiation of BMSCs was analyzed by oil red O staining for lipid accumulations. Results showed that SHSH, like PLL, which is usually used as a substrate for cell cultures, supports the growth of BMSCs. Dead cells were hardly detected on SHSH and BMSCs underwent normal proliferation, except for the first 48 h after seeding, during which less BrdU positive cells were present than those culture on PLL. Furthermore, BMSCs on SHSH gave rise to adipocytes upon adipogenic differentiation. Together, these data indicate that SHSH has a good biocompatibility with BMSCs, and that, combined with its excellent mechanical property, SHSH would be one of the promising scaffolds in the field of tissue engineering and regenerative medicine.3. Differentiation of bone marrow mesenchymal stem cells into adipocyteTo evaluate the key point of adipogenic differentiation of BMSCs, adipogenic medium was removed after 12 h, 18 h, 24 h, 48 h and 72 h, and then growth medium (L-DMEM+10% FCS) was added to maintain the differentiation of BMSCs. Adipogenic differentiation of BMSCs was analyzed by oil red O staining for lipid accumulations at 6 d and 9 d. Result showed that cells that were differentiated for 12 h, 18 h stained negative, but cells that were differentiated for 24 h, 48 h, or 72 h stained positive, indicating that there is a key point during the adipogenic differentiation of BMSCs, around 24 h of differentiation, after which cells are destined to differentiate to adipocytes regardless of the culture medium being the adipogenic medium or the growth medium.Meanwhile, we analyzed the expression of Rac1, Cdc42, and RhoA by western blotting and the activity of Rac1, Cdc42, and RhoA by pull-down assay in MSCs that were induced by adipogenic differentiation medium for 12 h, 18 h, 24 h, or 48 h. Results showed that during the first 48 h, expressions of Rac1, RhoA, Cdc42 have changed, and during the first 24 h, Rac1-GTP expression increased, and then decresed during the next 24 h. RhoA-GTP, by contrast, displayed an opposite tendency in expression. During the first 24 h, RhoA-GTP expression decresed, while increased in the next 24 h. Cdc42-GTP expression kept stable during the first 24 h, and then decreased after 24 h of differentiation.Together, there is a key point during the adipogenic differentiation of BMSCs around 24 h of differentiation, and expressions of Rac1, Cdc42, and RhoA have a conspicuous change around 24 h.