Study On Culture Of Bone Marrow Mesenchymal Stem Cells In Cell Scaffolds And Its Induced Differentiation Into Neurons

Objective:To observe the cell growth and adhesion of bone marrow mesenchymal stem cell (BMSC) in several cell scaffolds. Especially, the effect of RADA16-Ⅰ hydrogel on the differentiation of BMSCs induced into neurons were further studied to provide experimental evidence for its application in tissue engineering for repair of nerve injury. Method:1. Bone marrow was flushed out by complete medium from a2-month-old rat’s femur and tibia and the BMSCs were isolated by whole marrow adherence culture. Cells were passaged at1:3when they reach70%-80%confluence and morphological characteristics were observed. Surface antigen CD34/CD44/CD45/CD90of the3rd generation BMSCs were detected by flow cytometry. The4th generation-BMSCs were infected with adenovirus containing empty plasmids (pAdEasy-1-pAdTrack-CMV) to generate BMSC/GFP cell lines.2. We tried to culture BMSCs on three kinds of cell scaffold:polylactic-co-glycolic acid (PLGA), polystyrene (PS) and self-assembled short-peptide hydrogel (RADA16-Ⅰ), and observed cell growth and calculated adhesion rate.3. BMSC/GFP were seeded on cover slip or RADA16-I scaffold and each were divided into two subgroups for induction treatment and relevant control. For induction group, cells were treated with pre-induction medium for24h and then induction medium for another6h. Expression of GFAP, NeuN, Map-2, NF-200of each group were detected by immunofluorescence. Results:1. Primary BMSCs adhered in24h and reached70%-80%confluence that can be passaged in another7days. Flow cytometry assay showed that the BMSCs of the3rd generation were CD34/CD45-negative and CD44/CD90positive and had high homogeneity. BMSCs showed green fluorescence at2days after adenovirus infection.2. The only59%adhesion rate was seen in the PS scaffold group, scanning electron microscopy showed that relatively more cells adhered to the PLGA surface but not much distributed inside; cells grow evenly in the RADA16-Ⅰ hydrogel and the gel strength is similar to that of brain, which may be beneficial for application in transplantation for nerve damage.3. Induced differentiation of BMSCs into neurons showed no significant difference in RADA16-Ⅰ scaffold group and coverslip group (P>0.05). Conclusion:1. BMSCs isolated by whole marrow adherent culture and subsequent passage have high homogeneity and were merely damaged, thus can be used as seed cells for tissue engineering.2. Compared with PS and PLGA scaffold, RADA16-Ⅰ hydrogel scaffold better suits cell growth and adhesion.3. RADA16-Ⅰ hydrogel scaffold has no significant effect on the induced differentiation of BMSCs into neuron and can be used as a suitable cell scaffold for transplantation.