The Experimental Research In Construction And Transplantation Of Tissue Engineering Spinal Cord

Objective: To explore construction of tissue engineering spinal cord in vitro, and to study the effects of transplantation of tissue engineering spinal cord on spinal cord half-cut lump injury in rats.Methods: 1. Neural stem cells(NSCs) isolated from the 14d embryonic rat brain were inoculated onto the poly lactic-co-glycolic acid(PLGA), and then NSCs differentiation was induced by brain-derived neurotrophic factor(BDNF). The cells growth and differentiation implanted on PLGA were observed by phase contrast microscope and scanning electron microscope. Immunohistochemistry was applied to identify the cells on PLGA. 2. Two kinds of tissue engineering spinal cord of different pore dimension PLGA(A: 400～500μm; B:200～300μm)were constructed in vitro by inoculating NSCs onto the PLGA for 7 days. Thirty five SD rats were half transected at T₁₀ cord level and a 4mm segment caudal to the transaction was removed, and then divided into five groups randomly: experiment group A transplanted with tissue engineering spinal cord A, experiment group B transplanted with tissue engineering spinal cord B, control group C injected with NSCs alone, control group D positioned with scaffold only, and control group E inserted with nothing. The effects of transplantation of tissue engineering spinal cord on rat's spinal cord injury were evaluated by the BBB scale,HRP tracing technique, immunohistochemistry and Weil myelin staining. Results: Phase contrast microscope and scanning electron microscope showed that PLGA pores were filled with NSCs which shape resembled the irregular geometry of pores. After NSCs differentiation induced by BDNF,formation of the three-dimensional network scaffold was covered with neurite contacted each other. The NSCs were identified by immunohistochemistry. Nestin-positive cells and microtubule associated protein two-positive cells were found in the PLGA before and after differentiation induced by BDNF, indicating that NSCs cultured on PLGA were induced differentiation into neuron cells by BDNF. The average BBB scale in two experiment groups were all higher than that in control groups for all time points from 14 days. In addition, the BBB scale in the experiment group B was higher than that experiment group A, and BBB scale in the experiment group E was the lowest. Statistic showed that these differences had significance(p＜0.05). HRP tracing technique showed that significant amounts of HRP positive neurons in brain were demonstrated in the experiment groups, fewer HRP positive neurons in the control groups C and D were detected, and no labeled neurons were found in control group E. At the same time, more NF-positive neurons, GAP-43-positive axons and regenerate myelinated fibers were observed in experiment groups, fewer in control groups C and D, and no positive neurons and axons in control group E. Nerve regeneration and tissue structure repairing were more obvious in the experiment group B injury region compared to group A and the other control groups. Conclusion: PLGA supports the growth and differentiation of NSCs, The three-dimensional(3D) organization and differentiation direction of NSCs can be regulated by 3D formation of PLGA scaffold and directed by BDNF. Tissue engineering spinal cord can be constructed in vitro by 3D formation of PLGA scaffold and BDNF. Transplantation of tissue engineering spinal cord can promote the structure repairing and functional recovery of half-cut lump spinal cord injury, which effects is better than transplantaion of NSCs and scaffold only. The pore size of the cell scaffold used in tissue engineering spinal cord influences its curative effect. The pore size of 200～300μm utilized in fabricating tissue engi -neering spinal cord is better than that of 400～500μm.