Experimental Study For Fabrication Of Tissue-engineering Neural Complex And Transplantation Treatment Of Spinal Cord Injury In Rats

Objective: To fabricate tissue-engineering neural complex with bone marrow mesenchymal stem cells(BMSCs) binding different kinds of biological scaffolds; to observe the effect of spinal cord injury on rats treated with transplantation of tissue-engineering neural complex, and to explore its mechanism. Methods: 1, BMSCs were isolated from bone marrow of rats with density gradient centrifugation and adherent culture. Expression of the BMSCs surface marker was detected by flow cytometer. BMSCs of P3 were induced and differentiated into neuron by applying brain-derived neurotrophic factor (BDNF) and alltransretinoic acid(AT-RA) for 7d and 14d. The appearance and function of the BMSCs after differentiation were characterized by Immunohistochemistry and neurophysiology. 2, BMSCs, which were used as seed cells, were inoculated onto the two kinds of biological scaffolds-polymer of lactic and glycolic acids(PLGA) and poly-3-hydroxybutyrate(PHB) coated with the polylysine. And then BDNF and AT-RA were added into the neural complex for 7d. Tissue- tissueing neural complex A and B were fabricated in vitro with PLGA and PHB respectively. The growth and differentiation of BMSCs implanted on biological scaffolds were observed by phase contrast microscope and Immunohistochemistry 3. Twenty eight SD rats were half-transected at T10 cord level and a 4mm segment caudal to the transaction was removed, and then assigned randomly into four groups: experiment group A(tissue-engineering neural complex A transplanted into the spinal cord gap), experiment group B(tissue-engineering neural complex B transplanted into the spinal cord gap), control group C (BMSCs injected into the spinal cord gap), and control group D(nothing positioned into the spinal cord gap). The rats were seeded in a single cage respectively for 12 weeks. The functional recovery and morphological repairing of the injured spinal cords were evaluated by the BBB scale,HRP tracing technique and Immunohistochemistry. Results: 1, The cultured BMSCs in P3 possess typical fibroblast-like morphology and cell surface antigen of BMSCs. The cells induced by BDNF and AT-RA exhibit neuronal phenotype, and show neuron specificenolase (NSE) and microtubule-associated protein-2(MAP-2) positive expression. And the characteristics of neurons induced by BDNF and AT-RA express the electrophysiology function. 2. Inverted phase contrast microscope and scanning electron microscope(SEM) were applying to observe the mophological changing in BMSCs: after neural differentiation of BMSCs induced by BDNF and AT-RA, formation of the three-dimensional network poly was covered with neurite contacting each other. Positive expressing of MAP-2 in BMSCs after differentiation was identified by immunohistochemistry. The tissue-engineering neural complexs were prepared qualificatedly for transplantation treatment of spinal cord injury. 3. The average BBB scales in two experiment groups were better than the control groups for all time points from 14 days(P < 0.05). Further more, BBB scales of the experiment group A was higher than the experiment group B(P <0.05). and the scales of the control group D was the lowest. After 12 weeks, Transplanted distinct in the injured spinal cord was observed by microscope. And the intaction of spinal cord in the experiment group A and the experiment group B was better than the two control groups, and the morphological repairing of the injured spinal cord in the experiment group A was better than that in the experiment group B. The spinal cord on the control group D was wholely discontinued. 4, More labeled motoneurons in contral brain of the experiment groups A and B were observed by utilizing HRP tracting technique, fewer in the control group C and no labeled motoneurons in the control group D. 5, More NF-positive neurons,GAP-43-positive axons and MBP-positive myelins were observed in the experiment groups A and B, fewer in control group C, and no positive neurons ,axons and myelins in the control group D. BCL-2 expression in the two experiment groups were higher than the two control groups, fewer in the control group C and no BCL-2 expression in the control group D. Conclusion: 1.The combination of BDNF and AT-RA is an effective method of neural differentiation for BMSCs. The cells induced by BDNF and AT-RA possess the appearance and function of neurons.2.The biological scaffolds can support the growth and differentiation of BMSCs. The tissue engineering neural complex can be fabricated in vitro by the biological scaffolds seeded with BMSCs.3. Transplantation of tissue engineering neural complex can promote neural and myelin regeneration, structural repairing and functional recovery of half-cut lump spinal cord injury, its curative effects are better than BMSCs transplatation alone. In addition, different scaffold materials have different curative effect on spinal cord injury in rats. The curative effective effect using PLGA for biological scaffold is better than that using PHB.