Silk Fibroin With Peripheral Nerve Tissue In Vitro Biocompatibility Study

Part One Biocompatibility Study of Silk Fibroin and Dorsal Root Ganglia (DRGs) in VitroObjective: To explore the biocompatibility of silk fibroin and dorsal root ganglia and the feasibility of its usage as a bridging graft in injured peripheral nerve.Methods: Dorsal root ganglia from 1～2 day old SD rats were co-cultured with silk fibroin for 21 days, and regular observation were conducted under invert microscope every day. On the 4th and 7th day, photos were taken to record the status of the cocultures, meanwhile, fluorescent immunocytochemistry and scanning electron microscopy (SEM) were also performed. Finally, the specimens were observed through transmission electron microscope (TEM) after 21 day coculture.Results: Amount of cells crawled out from the DRGs and migrated far along the silk fibroin after 4 days of co-culture, three more days later, many more cells were found to crawl out and twist even encapsule the fibers. Immunocytochemistry and morphological observation under SEM suggested that most of these cells were schwann cells. After 21 days culture, observation under TEM revealed that the processes of cells around the fibers extend into the axons to wrap up them partly or compltetely without abnormality inside the cells.Conclusions: Schwann cells from DRGs can move along, twist and even encapsule the silk fibers after coculture, which suggests that there's a fine biocompatibility of silk fibroin and DRGs. Part Two The Effect of Silk Fibroin Extraction on Cultured Schwann Cells (SCs)Objective: To investigate the effect of silk fibroin extraction on the growth, proliferation and some effections of cultured schwann cells, determine whether silk fibroin has any toxicity to Scs, and make a further discussion on the possibilities of application of this biomaterial to peripheral nerve repair.Methods: Actively growing Scs were obtained from the sciatic nerve of 1～2 day old SD rats. The morphological changes of Scs cultured in silk fibroin extraction were observed under light microscope and photos were taken at 24h, 48h, 72h after cultivation. MTT assay was conducted to detect the cell vitality of Scs at 12h, 24h, 48h, 72h and the 7th day after cultivation. Flow cytomter was used to determine the changes in cell cycle and the proportion of S100 positive cells after 24h of culture. RT-PCR was carried out to determine the changes in mRNA expression of NGF, BDNF and S100 in Scs after 12h, 24h and 48h of culture. Western blotting and immunocytochemistry were performed to estimate the changes in expression of NGF, BDNF and S100 at 24h, 48h and 72h of culture. Results: Little change was observed in morphological, vitality, the cell proportion at ceil cycles and expression of NGF, BDNF and S100 at protein and mRNA levels for Scs cultured in silk fibroin extraction when compared with Scs cultured in L15 at corresponding time points.Conclusion: Schwann cells cultured in silk fibroin extraction show no change of morphology, vitality, proliferation ability and expression of specific proteins which are quite important to peripheral nerve repair, namely, NGF, BDNF and S100. We thus conclude that silk fibroin practically have no toxicity to schwann cells.