Peripheral Nerve Regeneration Using Tissue Engineering Technique

Long gap injuries of peripheral nerve are currently treated through autologous peroneal nerve grafts or cable-like sutures of multi-strand grafted nerve fibers. However, these methods are limited due to graft-available, donor-site dysfunction or even morbidity. Meanwhile, fully recovering the functions of reconstructed tissue and organ grafts depend on the control and adjustment of peripheral nerves. On the basis of the model in rabbits on axonal regeneration and nerve functional recovery after 2-month acellular sciatic nerve allografts, this project is studying various parameters, such as the axonal quantity, conduction velocity and neurobehavioral assessment (motor, nociception, proprioception functions) at different time points after acellular sciatic nerve allografts seeded with autologous Schwann cells, to understand the importance of autologous Schwann cells in accelerating functional recovery of regenerated sciatic nerve. As a result, the study can provide powerful and appropriate experimental and theoretical foundations for the feasibility of the fully nerve functional recovery of injured peripheral nerves regeneration using acellular nerve allografts with autologous Schwann cells as well as tissue and organ regenerations.PART I ESTABLISHMENT OF THE MODEL IN RABBITS ON ALLOGENIC ACELLULAR SCIATIC NERVE SCAFFOLD[Method] After 5cm-long sciatic nerve segments were cut from donor rabbits, they were immersed in distilled water, which was replaced several times for 24-48hours at 4℃. Then these segments were treated by0.05%Trypsin at 37℃ for 24 hours and exposed to 1% Triton and 0.1% Ammonium Hydroxide at 4℃ for 72 hours for cell lyses. All these steps should be under 250 rpm shaking. After that, they were washed thoroughly with distilled water several times and frozen at—20℃ for 20-30 minutes and lyophilized for 24 hours. Before sterilized with ethylene oxide, packaged and stored until needed, the acellular nerve grafts should be coated with 2% PLGA.[Result] After a multiple-step treatment of decellularization, all nuclear or cytoplasmic cellular components in the sciatic nerve segments have been chemically removed, which are confirmed through histological staining (H&E, Movet staining), immunohistochemistry and immunoflurescence. The nerve matrices still maintained their structural architecture within the intact nerve sheath. In order to correct the fragility of the mechanical property of decellularized sciatic nerves, 2%PLGA were coated on the surface of acellular nerve grafts to recover matrices' flexibility. There are still a lot of porous structures in decellularized nerves detected by Scanning Eelectron Microscope (SEM) after 2%PLGA coated, which are good for autologous cell attachment and migration.[Conclusion] The allografts acellular sciatic nerve scaffold can be made through 0.05%Trypsin, 1%Triton and 0.1%Ammonium Hydroxide treatment and lyophilization technique. Meanwhile, that 2% PLGA is coated on the surface of decellularized sciatic nerve segments can recover their flexibility, which has been lost after decellularization process, and still maintain the porous structures. The recovery of the flexibility provides the best condition for acellular sciatic nerve allografts.PART II ESTABLISHMENT OF THE METHOD ONAUTOLOGOUS SCHWANN CELLS' HARVEST, CULTURE ANDEXPANSION IN VITRO[Method] Sciatic nerve segment, taken out through a sterilized surgical biopsy, was soaked and shaken in the dissociation medium at a 37°C water bath for 30 minutes. After decant off the dissociation medium, replace with 10%FBS/DMEM and wash 3 times, the nerve segment was triturated by aspirated and expelled liquid through a 10ml-20gauge needle and slowly filtered through a 20um nylon mesh. Then, centrifuge the cell suspension for 10 minutes at 1500 rpm. After that, decant the media from the tube and replace with 1ml 10%FBS/DMEM to dissociate the cell pellet by repeatedly pipetted and add cell suspension in a PDL pre-coated flask for incubation. 24 hours later, remove all media from flask and wash, then re-feed culture flask by adding Fibroblast inhibition medium (Ara-c) for 2-3 days. After that, remove all media and add Schwann cell culture medium (10%FBS/DMEM/rhNGF/Forskolin) for primary cell culture. When the cells have grown to confluence, they should be passaged.[Result] After 10-day culture, light microscope indicates that the cultured primary cells are adherent growth. The cells have characteristic shapes in culture: small, bipolar or multi-polar with a strongly refraction light and a fairly regular mono-layer arrangement (contact inhibition). Immunocytochemistry(anti-S₁₀₀) staining has shown that S₁₀₀ is positive in the cultured primary cells. Meanwhile, the specific target protein S₁₀₀ of Schwann cell is also detected through Western Blotting.[Conclusion] Schwann cells can be successfully harvested, cultured and proliferated in vitro, followed by using Dissociation Medium, Fibroblast inhibition Medium and Schwann cell Culture Medium. However, nerve cells proliferate slower than any other cells, as well as passage. Therefore, it is hard to collect enough quantities of Schwann cells to seed onto the scaffold for sciatic nerve regeneration just under limited passages (15 passages on average).PARTâ…¢ SCIATIC NERVE REGENERATION USING ACELLULAR ALLOGRAFTS SEEDED WITH AUTOLOGOUSSCHWANN CELLS[Method] After volume measured, 5cm gap acellular sciatic nerve grafts (n=30) were treated with 1× PBS for 24 hours at 4℃. Then, two-thirds grafts (n=20) were seeded with Schwann cells 20 minutes before implantation. After recipient rabbit was anesthetized well, the sciatic nerve on the left side was exposed and 5cm segment of the nerve was removed from the midthigh level sterilizedly. The 5cm-long acellular nerve allograft (20 seeded with Schwann cell and 10 without Schwann cell) was interposed across the gap. The graft was anastomosed microsurgically to the epineurium of the proximal and distal nerve stumps with three or four sutures (9-0 absorbable ethicon) at each junction. Loading with rhNGF around the junctions, the wound was closed. The right side sciatic nerves (n=30) served as normal controls, while acellular nerve allografts without Schwann cell (n=10) served as negative controls.[Result] After 6-month implantation, the findings from grafts only, grafts with Schwann cells and normal sciatic nerves indicate: 1. The axon quantities are 8900 ± 1060, 12000 ± 980 and 6800 ± 740, respectively. Statistical evaluation has shown a highly significant difference (P<0.01) between them. Meanwhile, the axon quantities between grafts only and grafts with Schwann cells are considered to be statistically significant (P<0.01). 2. The conduction velocities are 31.7 ± 5.92m/s, 47.22 ± 4.89m/s and 49.6 ± 3.24m/s, respectively. Statistical evaluation has shown a highly significant difference (P<0.01) between grafts only and normal controls and no significant difference (P>0.05) between grafts with Schwann cells and normal controls. 3. The extensor postural thrusts are 175 ± 8.6g, 196 ±5.6g, and 200±6.8g, respectively. Statistical evaluation has shown a highly significant difference (P<0.01) between grafts only and normal controls and no significant difference (P>0.05) between grafts with Schwann cells and normal controls. 4. The temperatures are 67 + 5.1℃ , 66 ± 3.8℃ and 65 ± 2.7℃, respectively. Statistical evaluation hasshown no significant difference (P>0.05) between them. 5. Pain nociception are 97.8 ± 5.4%, 98.4 ± 3.8% and 100 ± 2%, respectively. Statistical evaluation has shown no significant difference (P>0.05) between them.[Conclusion] 1. After 6-month implantation, the number of axons detected from either grafts only or grafts with Schwann cells is much more than that of normal controls, which is benefit for the functional recovery of regenerated sciatic nerve. The study has shown that grafts with Schwann cells have more assets than grafts only for nerve regeneration. 2. After 6-month implantation, the conduction velocity and extensor postural thrust of grafts with Schwann cells have been completely recovered, while those of grafts only have not yet. 3. After 6-month implantation, the sensory functions of either grafts only or grafts with Schwann cells have been fully recovered.