Repair of sciatic nerve defects using tissue engineered nerves

In this study, we constructed tissue-engineered nerves with acellular nerve allografts in Sprague-Dawley rats, which were prepared using chemical detergents-enzymatic digestion and mechanical methods, in combination with bone marrow mesenchymal stem cells of Wistar rats cultured in vitro, to repair 15 mm sciatic bone defects in Wistar rats. At postoperative 12 weeks, electrophysiological detection results showed that the conduction velocity of regenerated nerve after repair with tissue-engineered nerves was similar to that after autologous nerve grafting, and was higher than that after repair with acellular nerve allografts. Immunohistochemical staining revealed that motor endplates with acetylcholinesterase-positive nerve fibers were orderly arranged in the middle and superior parts of the gastrocnemius muscle; regenerated nerve tracts and sprouted branches were connected with motor endplates, as shown by acetylcholinesterase histochemistry combined with silver staining. The wet weight ratio of the tibialis anterior muscle at the affected contralateral hind limb was similar to the sciatic nerve after repair with autologous nerve grafts, and higher than that after repair with acellular nerve allografts. The hind limb motor function at the affected side was significantly improved, indicating that acellular nerve allografts combined with bone marrow mesenchymal stem cell bridging could promote functional recovery of rats with sciatic nerve defects.

Acellular allogeneic nerve grafting combined with bone marrow mesenchymal stem cell transplantation for the repair of long-segment sciatic nerve defects:biomechanics and validation of mathematical models

We hypothesized that a chemically extracted acellular allogeneic nerve graft used in combination with bone marrow mesenchymal stem cell transplantation would be an effective treatment for long-segment sciatic nerve defects.To test this,we established rabbit models of 30 mm sciatic nerve defects,and treated them using either an autograft or a chemically decellularized allogeneic nerve graft with or without simultaneous transplantation of bone marrow mesenchymal stem cells.We compared the tensile properties,electrophysiological function and morphology of the damaged nerve in each group.Sciatic nerves repaired by the allogeneic nerve graft combined with stem cell transplantation showed better recovery than those repaired by the acellular allogeneic nerve graft alone,and produced similar results to those observed with the autograft.These findings confirm that a chemically extracted acellular allogeneic nerve graft combined with transplantation of bone marrow mesenchymal stem cells is an effective method of repairing long-segment sciatic nerve defects.

Dorsal root ganglion-derived Schwann cells combined with poly(lactic-co-glycolic acid)/chitosan conduits for the repair of sciatic nerve defects in rats

Schwann cells, nerve regeneration promoters in peripheral nerve tissue engineering, can be used to repair both the peripheral and central nervous systems. However, isolation and puriifcation of Schwann cells are complicated by contamination with ifbroblasts. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. In this study, we collected dorsal root ganglia from neonatal rats from which we obtained highly puriifed Schwann cells using serum-free melanocyte culture medium. The purity of Schwann cells （〉95%） using our method was higher than that using standard medium containing fetal bovine serum. The obtained Schwann cells were implanted into poly（lactic-co-glycolic acid）/chi-tosan conduits to repair 10-mm sciatic nerve defects in rats. Results showed that axonal diameter and area were signiifcantly increased and motor functions were obviously improved in the rat sciatic nerve tissue. Experimental ifndings suggest that serum-free melanocyte culture medium is conducive to purify Schwann cells and poly（lactic-co-glycolic acid）/chitosan nerve conduits combined with Schwann cells contribute to restore sciatic nerve defects.

Construction of a three-dimensional bionic nerve conduit containing two neurotrophic factors with separate delivery systems for the repair of sciatic nerve defects

Previous studies of nerve conduits have investigated numerous properties, such as conduit luminal structure and neurotrophic factor incorporation, for the regeneration of nerve defects. The present study used a poly（lactic-co-glycolic acid） （PLGA） copolymer to construct a three-dimensional （3D） bionic nerve conduit, with two channels and multiple microtubule lumens, and incorporating two neurotrophic factors, each with their own delivery system, as a novel environment for peripheral nerve regeneration. The efficacy of this conduit in repairing a 1.5 cm sciatic nerve defect was compared with PLGA-alone and PLGA-microfilament conduits, and autologous nerve transplantation. Results showed that compared with the other groups, the 3D bionic nerve conduit had the fastest nerve conduction velocity, largest electromyogram amplitude, and shortest electromyogram latency. In addition, the nerve fiber density, myelin sheath thickness and axon diameter were significantly increased, and the recovery rate of the triceps surae muscle wet weight was lowest. These findings suggest that 3D bionic nerve conduits can provide a suitable microenvironment for peripheral nerve regeneration to efficiently repair sciatic nerve defects. p

Autologous nerve segment-bridging regeneration chambers for the repair of sciatic nerve defects

Proximal and distal nerve defects exhibit chemotactic growth over certain distances. Our previous studies demonstrated that Schwann cells survive in autologous nerve segments that are bridged by regeneration chambers and secrete various bioactive substances. However, more data are required to determine the required length of regeneration chambers for chemotaxis and nutrition of neural regeneration, as well as the length of repaired nerve defects to replace the effect of autologous nerve grafting. In the present study, sciatic nerve defects of 12, 16, 20 mm were repaired using a regeneration chamber of 6, 8, and 10 mm in length respectively. These were bridged with autologous nerve segments. Results showed that the bridging of two 6-mm long regeneration chambers to repair a 12-mm nerve defect exhibited similar effects to autologous nerve grafting.

Ultrasound imaging of chitosan nerve conduits that bridge sciatic nerve defects in rats

The repair of peripheral nerve injuries with autologous nerve remains the gold standard （Wang et al., 2005; Yao et al., 2010; Deal et al., 2012; Kriebel et al., 2014; Liu et al., 2014; Tamaki et al., 2014; Yu et al., 2014; Zhu and Lou, 2014）. With advances in tissue engineering and biomaterials, tissue-engineered nerve conduits with various biomaterials and structures, such as collagen and chitosan nerve conduits, have already been used in the clinic as alternatives to autologous nerve in the repair of peripheral nerve injury （Wang et al., 2012; Svizenska et al., 2013; Eppenberger et al., 2014; Gu et al., 2014; Koudehi et al., 2014; MoyaDiaz et al., 2014; Novajra et al., 2014; Okamoto et al., 2014; Shea et al., 2014; Singh et al., 2014; Tamaki et al., 2014; Yu et al., 2014）. Therefore, new simple and effective methods

Histological observation on acellular nerve grafts co-cultured with Schwann cells for repairing defects of the sciatic nerve

BACKGROUND： Animal experiments and clinical studies about tissue engineering method applied to repair nerve injury mainly focus on seeking ideal artificial nerve grafts, nerve conduit and seed cells. Autologous nerve, allogeneic nerve and xenogeneic nerve are used to bridge nerve defects, it is one of the methods to promote the repair of nerve injury by culturing and growing Schwann cells, which can secrete various neurotrophic factor activities, in the grafts. OBJECTIVE ： To observe the effect of acellular nerve grafts co-cultured with Schwann cells in repairing defects of sciatic nerve. DESIGN： An observational comparative study.SETTING： Tissue Engineering Laboratory of China Medical University.MATERIALS： The experiment was carried out in the Tissue Engineering Laboratory of China Medical University between April 2004 and April 2005. Forty neonatal Sprague-Dawley rats of 5-8 days （either males or females） and 24 male Wistar rats of 180-220 g were provided by the experimental animal center of China Medical University. METHODS： ① Culture of Schwann cells： The bilateral sciatic nerves and branchial plexus were isolated from the 40 neonatal SD rats. The sciatic nerves were enzymatically digested with collagenase and dispase, isolatd, purified and cultured with the method of speed-difference adhersion, and identified with the SABC immunohistochemical method. ② Model establishment： In vitro Schwann cells were microinjected into 10-mm long acellular nerve grafts repairing a surgically created gap in the rat sciatic nerve. According to the different grafted methods, the animals were randomly divided into three groups： autografts （n=8）, acellular nerve grafts （n=8）, or acellular nerve grafts with Schwann cells （n=8）. ③ The regenerated nerve fiber number and average diameter of myeline sheath after culture were statistically anlayzed. MAIN OUTCOME MEASURES： ① The regenerated nerve ultrastructure, total number and density of myelinated nerve fibers, and the thickness of myeline sheath were observed under electron microscope. ② The images were processed with the Mias-1000 imaging analytical system to calculate the number of myelinated nerve fibers, and the thickness of myeline sheath. RESULTS： All the 24 Wistar rats were involved in the analysis of results. ① Results observed under transmission electron microscope： The regenerated myelinated nerve fibers in the group of acellular nerve grafts with Schwann cells were more even than those in the group of acellular nerve grafts, the number of myelinated nerve fibers and thickness of myelin sheath were close to those in the allografts group （P 〉 0.05）, but significantly different from those in the group of acellular nerve grafts （P 〈 0.05）. ② Results observed under scanning electron microscope： A great amount of Schwann cells with two polars were observed in the group of grafts with Schwann cells, the feature of cultured Schwann cells showed shoulder by shoulder, head to head. ③ The number of myelinated nerve fibers and thickness of myelin sheath analyzed by Mias-1000 imaging system in the group of acellular nerve grafts with Schwann cells were close to those in the autografts group （P 〉 0.05）, but significantly different from those in the group of acellular nerve grafts （P 〈 0.05）.CONCLUSION： Host axonal regeneration is significantly increased after implant of acellular nerve grafts. Acellular nerve grafts with Schwann cells offers a novel approach for repairing the gap of nerve defect.

Effects of subcutaneous implant of peripheral nerve allograft on the regeneration of defected sciatic nerve

BAEKGROUND： Some experimental studies demonstrate that subcutaneous implant of allograft can significantly decrease lymphocyte infiltration and reduce immunological reaction. However, compared with autologous nerve grafting, what is the effect of nerve regeneration after repair？OB]EETIVE： To observe the local nervous status of the detected part of sciatic nerve repaired through subcutaneously implanting peripheral nerve allograft, and compare the effect with fresh autologous nerve grafting.DESTGN ： Contrast observation.SETTING ： Departments of Orthopaedics of Zhengzhou Fifth People＇s Hospital and First Hospital Affiliated to Chongqing Medical University.MATEREALS ： Totally 30 healthy adult Wistar male rats, with body mass of （200±20）g, were enrolled. Optical microscope （Olympus biological microscope BHS, Japan）, Electron microscope （H-600, Japan）,CM-2000 biomedical image analysis system （CM-2000,Beihang） and myoelectricity scanner （KEYPOINT, Denmark） were used in this experiment.METHODS ： This experiment was carried out in the Orthopaedic Laboratory of Chongqing Medical University between October 2000 and April 2002. ① Six rats were chosen as the donors for allogenic nerve grafting, and 15 mm sciatic nerve segment was chosen as graft. The other rats were randomly divided into two groups： allogenic nerve grafting group and autologous nerve grafting group, with 12 rats in each group. In the allogenic nerve grafting group, a skin incision was made on the posterior side of right thigh, and subcutaneous blunt dissection was performed prorsally a little, then allograft was implanted. Two weeks later, sciatic nerve was exposed at the posterior side of left thigh and cut respectively at 5 mm and another 10 mm away from pelvis. The donor nerve （with connective tissue veil） implanted subcutaneously on the right thigh was taken out. Sectioned connective tissue at the proximal end was trimmed and that at the distal end as done but reserved 10 mm in length, and inosculated antegradely at the nerve defect on the left side with 11/0 nylon line. Twelve rats in autologous nerve grafting group underwent a 10 mm sciatic nerve defect inci- sion on the right thigh and implant of fresh sciatic nerve from left thigh. The incision on the left thigh was repaired in situ. ②2,4,8 and 14 weeks after grafting, the nerve specimen of anastomosis segment was observed under optical microscope. Fourteen weeks after grafting, the ultrathin section of distal sciatic nerve was observed under transmission electron microscope. The number and size of regenerated axons at the cross section of anastomosis of proximal and distal sciatic nerve were analyzed with biomedical image analysis system. Neuroelectrophysiological change of in vivo sciatic nerve was detected with myoelectricity scanner.③ t test was used in the comparison of difference of measurement data.MAZN OUTCOME MEASURES ： ① Observation of anastomosis part of sciatic nerve under optical and electron microscopes in the two groups. ② Comparison of motor nerve conduction velocity, latent period and action potential peak as well as the number and size of cross-section of anastomosis part of proximal and distal sciatic nerve between two groups.RESULTS： ①Observation under optical microscope：Two weeks after grafting, neve axon of repaired region broke and medullary sheath denatured in the allogenic nerve grafting group and autologous nerve grafting group. At the same time, vascular engorgement and a little lymphocytes infiltration were found in the autologous nerve grafting group, but those were found worsened in the allogenic nerve grafting group. Four weeks after grafting, the intensity of the inflammatory reaction was similar between two groups, some collagen fibers at the proximal end proliferated; 8 weeks after grafting, the inflammatory reaction ended basically and the collagen fibers proliferated obviously in the two groups. ② Observation under electron microscope： Fourteen weeks after grafting, the structure of epineurium was in integrity and there were no obvious differences in perineurium and endonurium between two groups. A large number of myelinated nerve fibers and a few unmyelinated nerve fibers regenerated. The structure of myelin sheath was in integrity. ③The number and size of regenerated axons of anastomosis of proximal and distal sciatic nerve had no significant difference 14 weeks after grafting [（575.500±7.495） vs（585.700±11.172） axons/visual field ; （389.300±49.073） vs （407.600±0.283） axons/visual field;（6 423.830±119.911 ） vs （6 695.36± 84.287） μm^2/visual field = （ 5 980.110±74.572） vs（5 980.110±74.572） μm^2/visual field] （P 〉 0.05）. ④ Neuroelectrophysiological examination showed that there were no significant differences in motor nerve evoked potential latent pedod[（1.420±0.346）vs （1.237±0.250） ms] , motor nerve conduction velocity [（ 12.120±0.906 ） vs（13.020±0.599） m/s]and latent period of sciatic nerve [（0.500±0.380）vs （1.250±1.067） mV] of rats between two groups （P 〉 0.05）.CONCLUSTON： Although subcutaneous implant of peripheral nerve allograft has some inflammatory reactions, no obvious rejection is found. Repair results of two groups show that subcutaneous implant of allograft can promote nerve regeneration, which is similar to autologous nerve grafting.