Repairing 50mm Median Nerve Defects By Tissue Engineered Nerve Seeded With Autologous Bone Marrow Stromal Cells In Rhesus Monkey

Objective:To investigate the biological characteristic of rhesus monkey bone marrow stromal cells(RhBMCSs)and its biocompatibility with poly lactide-co-glycolide acid (PLGA) polymer scaffolds and to explore the possibility of the tissue-engineered nerve graft, which was composed of a chitoan/PLGA conduit and autologous RhBMCSs,to repair long peripheral nerve defect in rhesus monkey.Method:1、RhBMCSs were isolated from bone marrow of adult rhesus monkey by adherent culture. The biological characteristics of RhBMCSs, including surface markers, cell growth,proliferation and differentiation were investigated.2、The second passage of RhBMCSs were co-cultured with PLGA scaffolds. The cells were observed by confocal laser scanning microscope and electric microscope. MTT test was used to detect cell viability. Flow cytometry was employed to detect cell cycle of RhBMCSs. Real-time PCR and western blot were performed to determine the mRNA and protein of neurotrophic factors secreted by RhBMCSs.3、50-mm long median nerve defect in rhesus monkeys were established and the animals were randomly divided into 4 groups(3 monkeys in each group): cell group: the chitosan/PGLA nerve graft seeded with RhBMCSs was utilized to bridge the nerve defect; tube group : chitosan/PGLA nerve graft was used without addition of RhBMCSs; autologous nerve group : autologous nerve graft was used; gap group : no nerve graft was used. The monkeys were monitored for their behavior. Movements of the hand and thumb, and grip strength for obtaining food of both the transplanted and contralateral sides were recorded on photos or videotapes at 3, 6, 9, 12 months post-operatively. 12 months after transplantation, nerve electrophysiological examination and FluoroGold (FG) retrograde tracing were performed to evaluate the effects of nerve regeneration. In addition, histological and immunofluorescence assessment and morphometric analyses were applied to both regenerated nerves and target muscles to assess the nerve regeneration. Routine blood test, blood biochemical tests and coagulogram tests were performed before and 3-, 6-, 9- and 12- months after transplantation. Immunological function, tumor markers and histopathology of important organs were detected at 12-months post- surgery.Results:1、RhBMCSs displayed fibroblastic appearance and expressed surface markers CD73, CD105, CD29and CD90,while do not possess markers CD34, CD45, and CD11b. Examination of the cell cycle profile of RhBMCSs revealed that 12.71% and 3.93% of these cells occurs in phases S and G2 of the cell cycle, while 83.36% remain in the G0/G1 phase. All these characteristics were consistent with stromal stem cells.2、After culture of RhBMCSs on PLGA scaffolds, they were observed to tightly attach to and enwrap the PLGA scaffolds. The results of MTT assay showed that the viability of RhBMCSs cultured in the PLGA extract fluid was not significantly different from that in the plain IMDM. According to the results of cell cycle analysis, no significant difference in cell proliferation, as indicated by proliferation index (G2M + S)/G0G1 + G2M+S) was found between the two groups after 72h of culture. Real-time PCR and western blot analysis revealed RhBMCSs cultured in the PLGA extract fluid and in the IMDM exhibited no significant difference in the mRNA and protein levels of NGF, BDNF, CNTF and bFGF.3、After the nerve graft transplantation, movements of the hand and thumb, and strength for obtaining food of both the transplanted and intact limbs of the monkeys were observed and recorded on a video tape. The functions of the regenerated nerve were gradually recovered in the tube group, cell group and autologous nerve group. The agility of fingers and the thumb to forefinger activity of the monkeys in the cell group and autologous nerve group were better than those in the tube group at 12-months post- surgery. In the gap group, the thumb was contractured, the muscle of thenar was atrophy and no thumb to finger activity was observed. At 12 months following graft placement, the average distal amplitude of the compound muscle action potential (CMAP)in the tube, cell and autologous nerve group was 4.63±0.13mV, 4.45±0.49 mV, 8.89±2.85 mV, respectively, and the autologous nerve group was statistically significant higher than the tube and cell group(P<0.05). The average distal amplitude of CMAP was 3.63±1.14mV, 3.82±0.41 mV, 6.50±2.51 mV, and there were no statistically significant among three groups(P>0.05). The average motor nerve conduction velocity was 12.87±2.14m/s,18.59±4.87m/s,30.21±2.90m/s, and there were no statistically significant among three groups(P>0.05). FluoroGold(FG)-labeled motor neurons, appeared within the anterior horn of spinal cord ipsilateral to the injured side of tube, cell and autologous nerve group were 15292±1005, 15814±600 and 16000±841,and there were no statistically significant among three groups(P>0.05). The percentage of FG-labeled sensory neurons appeared within dorsal root ganglions were 76.14%±7.62%, 79.61%±6.60% and 82.79%±5.11%, there were no statistically significant between the cell and autologous nerve group(P>0.05)while the percentage of labeled cells in the tube group was lower than that in the cell and autologous nerve group(P<0.05).12 months after surgery, the biomaterials of the chitosan and PLGA in cell group and tube group were completely degraded, and regenerated nervous tissue connected the 50mm nerve gap. Meyer’s trichrome staining and double immunofluorescence staining for neurofilament-200 and S-100 showed that regenerated nerve fiber permeated graft from the proximal segment to the distal segment in the 3 grated groups. In the cell group the grafted segment was concentrated by more regenerate nerve fibers than that in tube group. There was no regenerated nervous tissue in non-grafted group. Under the transmission electron microscope, the average myelin sheath’s thickness of myelinated nerve fibres in the distal portion of regenerated nerve were 0.63±0.27μm, 0.82±0.39μm, 1.11±0.58μm and 1.75±1.15μm, respectively; the average diameter of myelinated nerve fibres were 4.09±1.12μm, 4.59±1.39μm, 5.57±2.13μm, and 7.11±3.74μm, respectively, and differences were statistically significant among groups (P < 0.05). Almost no normal nerve structures were observed in the gap group. Masson trichrome staining on muscle of showed that there were comparatively integrated structure and a small amount of collagen fibres among the thenar in the cell and autologous nerve group, while the thenar in the gap group exhibited typical atrophy accompanied by obvious hyperplasia of massive collagen fibres and adipose cells. The average cross-sectional area of thenar in the tube, cell and autologous nerve group was 658.84±420.84μm2, 696.80±357.60μm2, and 1232.54±335.79μm2, respectively, and the autologous nerve group was statistically significant higher than the tube and cell group(P<0.05). The average percentage of collagen fibre area was: 29.62±10.01%、23.04±8.77%、22.65±6.41%,respectively, the tube group was statistically significant higher than the autologous nerve group(P<0.05)while there was no statistically significant between the cell and autologous nerve group( P > 0.05 ). The blood routine test, blood biochemical test and thrombotest test showed that all parameters were within normal range in all groups both before and after surgery, and there were no statistically statistical differences among all groups (P > 0.05 ). At 12 months post- surgery, the immunological and tumor marker detection didn’t show any abnormal in all animals and histopathological examination of heart, liver, lung, kidney, spleen and lymphaden showed no obvious pathological change.Conclusion:1、The biological characteristics of RhBMCSs were consistent with stromal stem cells and may be taken as a promising candidate of seeding cells for nerve-tissue engineering.2、PLGA is a suitable substrate for RhBMCSs growth with little toxicity effect on cell morphology, proliferation and expression of neurotrophic factors, indicating a high degree of biocompatibility and bioaffinity of PLGA material with RhBMCSs.3、Twelve months after implantation of the tissue-engineered nerve graft, which was composed of a chitoan/PLGA conduit and autologous RhBMCSs,the 50-mm media nerve defect of monkeys was repaired by regenerated nerve tissue, and the nerve continuity regained. The regenerated nerve recovered in morphology and physiologic function, and re-inervated its target muscles, facilitating improved recovery in locomotion function.4、Blood test and histopathological examination demonstrated that the tissue-engineered nerve graft reinforced by autologous BMSCs was safe in the body of primate.The findings of this study provided the experimental evidence for clinical application of this tissue-engineered nerve graft to repair extended peripheral nerve defects.