Study On The Tissue-engineered Nerve To Restore The Injured Rat Sciatic Nerve Defect

1 The culture of bone marrow mesenchymal stromal cells and induce to differentiate into Schwann-like cellsObjective: To master the method for the isolation,culture and amplification of rat bone marrow mesenchymal stromal cells (BMSCs) in vitro and explore their biological characteristics and induce them to differentiate into Schwann-like cells.Methods: BMSCs were collected from degermed femurs, tibias and sternum of 4 to 6-week-old SD rat by flushing the shaft with D-Hank's using a syringe. Cells were disaggregated by gentle pipetting several times and plated in culture flask and re-fed every 2-3 days (L-DMEM with 10% FBS and 1% penicillin-streptomycin).When 95% fusion, cells were digested with 0.125% trypsogen and 0.02% EDTA 2 min and passaged. After successive isolation, purification, subculture and proliferation, the morphology was observed with phase contrast microscope. The growth curve of P2, P4, P6 were drawn with MTT. In order to induce BMSCs differentiate into Schwann-like cells,subconfluent cultures of BMSCs treated with beta-mercaptoethanol (β-ME) followed by all trans retinoic acid and cultured in the presence of forskolin, basic-FGF, PDGF-AA and heregulin, The proliferation and morphology before and after induction of BMSCs was observed with inverted microscope constantly. Immunocytochemistry of S-100 and glail fibrillary acidic protein(GFAP) ,the ratio of positively stained cells was counted. Western blot were used to identify the expressing of S-100 and GFAP.Results: The components of primarily cultured BMSCs were very complex. The marrow cells were round in the beginning and a few cells adhered to flask at 1-2 days, which were in irregular shape such as fusiform, polygon and so on. After changing half of the medium, the cell clone began to proliferate immediately. About 7-9 days later, cells might overgrow the bottom of culture flask and reached over 95% fusion. The cells arranged regularly as a whirlpool. Generated cells stuck on the wall more quickly than primary cells. From the 2nd hour, they began to adhered and completely adhered within 24h. The cell morphology was more uniform and all the cells arranged more regularly. Cells could spread the full flask bottom for 6 or 7 days. After subcultured 10 passages, the cells proliferated rapidly and kept the morphology unchanged. The growth curve of P2, P4, P6 were quite similar and the cells biological character kept stable. The result of growth curve showed that cell growth phase was composed of latency phase, logarithmic phase and platform phase. After the induction, BMSCs displayed morphologies of schwann cells, such as ellipsoidal cell bodies and fences-like array, with two or three sligh cell processes. The positive percentages of S-100 protein expression was 71.34%, GFAP was 68.32%. The expression of GFAP, S-100 protein was detected by western blot, but untreated BMSCs were not.Conclusion: 1 In this part, a simple new method which was used for the isolation, purification and cultivation in vitro of BMSCs from SD rat bone marrow by total marrow culture associating with adhering to flash has been established.The BMSCs could proliferate immediately and keep their biological character stable in vitro. 2 BMSCs could be induced into Schwann-like cells in vitro.2 Preparation and biocompatibility of human acellular amniotic membrane matrixObjective: In this experiment, human acellular amniotic membrane matrix (HAAM) was prepared and its biocompatibility was investigated as a scaffold in tissue engineering. Methods: Fresh human amnion was shaken in 1% tritonX-100 for 24 hours,and then treated with 0.25% trypsin for 4 hours at 37℃. The production was sterilized using ethylene oxide.The HAAM was stained with hematoxylin-eosin (HE) and observed with scanning electron microscope. The HAAM was implanted in the back of SD rats to investigate its histocompatibility.The cytotoxicity of HAAM to Schwann-like cells was measured by methyl thiazolyl tetrazolium method (MTT).The Schwann-like cells were seeded in HAAM,the specimen was observed under inverted microscope and stained with HE.Results: There were no residues of cells in the HAAM, which was a white, thin and semitransparent membrane with good flexibility. The biocompatibility of HAAM in the subcutaneous implant test was good. The cytotoxicity score was graded as one. The Schwann-like cells could be seeded and adhered on the surface of HAAM, then proliferated. Their appearances were the same as those cultured in normal conditions.Conclusion: The HAAM prepared by detergent-enzymatic approach has good biocompatibility. It is an ideal scaffold for tissue engineering.3 Study on using the Schwann-like cells and human acellular amniotic membrane matrix into a tissue -engineered nerve to restore the injured rat sciatic nerve defectObjective:Make of a tissue-engineered peripheral nerve by the Schwann-like cells and human acellular amniotic membrane matrix.Study of the effect on the sciatic nerve defect,so that provide proof for clinical research.Methods: The Schwann-like cells induced from BMSCs as seed cells and HAAM as scaffold material were co-cultured in vitro to construct tissue-engineered artificial nerve for reparing 10mm defect of rat sciatic nerve.Pure HAAM conduit and autografting were as control groups. Twelve weeks after operation,the recovery results and mechanism were analyzed and evaluated by means of gross observation, histology, electrophysiology, image analysis, retrograde tracing, etc.Results: The results showed the ulcer of affected limb healed at 12w after operation in the HAAM compound of schwann-like cells group,the diameter and appearance of graft is similar to the proximal and distal segment of sciatic nerve.Histological examination revealed that a great quantity of myelined nerve fibers with thick myelin regenerated.TEM showed that regenerative nerve fibres demonstrated mature morphology with distinct layers of myelin sheath. Electrophysiological examination,positive neurons labelled with HRP and image analysis showed there was no statistical difference between the HAAM compound of Schwann-like cells group with autografting group.Conclusion: The tissue-engineered peripheral nerve can restore the defect of rat sciatic nerve effectively and there was no marked difference of nerve regeneration when compared with autogenous nerve.4 Effects of brain homogenate on the differentiation of rat bone mesenchymal stormal cells into neuron-like cells following traumatic brain injuryObjective: In this study,the different effects between traumatic brain tissue extracts and normal brain tissue extracts on the differentiation of rat bone mesenchymal stormal cells were observed.Methods: Rat models of moderate brain injury were established by Modified Gruncr Method. The traumatic brain tissue extracts acquired on the point about 24 hours after the injury and normal brain tissue extracts were used to induce 3rd passage of bone mesenchymal stormal cells in vitro. The morphological changes of the cells were observed with inverted phase microscope.The expression of neuron-specific enolase was identified by immunocytochemical technique. The different effects between traumatic brain tissue extracts and normal brain tissue extracts on the differentiation of bone mesenchymal stormal cells were observed.Results: After 24-hours induction with traumatic brain tissue extracts, the cellular bodies changed large,36 hours later, part of the bone mesenchymal stormal cells body contracted into round or spindle shape. 48 hours later, neuron-like cells with two or more prominence could be seen.The immunocytochemical method showed that the ratio of neuron-specific enolase expressing was 54.28±6.03%. The differential ratio of bone mesenchymal stormal cells induced with normal brain tissue extracts was lower, and ratio of neuron-specific enolase expressing was 32.76±3.25%.Conclusion: Bone mesenchymal stormal cells can be induced differentiating into neuron-like cells. The induction proportion induced with traumatic brain tissue extracts is higher than with normal brain tissue extracts.