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.

Allograft pretreatment for the repair of sciatic nerve defects: green tea polyphenols versus radiation

Pretreatment of nerve allografts by exposure to irradiation or green tea polyphenols can elimi- nate neuroimmunogenicity, inhibit early immunological rejection, encourage nerve regeneration and functional recovery, improve tissue preservation, and minimize postoperative infection. In the present study, we investigate which intervention achieves better results. We produced a 1.0 cm sciatic nerve defect in rats, and divided the rats into four treatment groups： autograft, fresh nerve allograft, green tea polyphenol-pretreated （1 mg/mL, 4~C） nerve allograft, and irradiation-pre- treated nerve allograft （26.39 Gy/min for 12 hours; total 19 kGy）. The animals were observed, and sciatic nerve electrophysiology, histology, and transmission electron microscopy were carried out at 6 and 12 weeks after grafting. The circumference and structure of the transplanted nerve in rats that received autografts or green tea polyphenol-pretreated nerve allografts were similar to those of the host sciatic nerve. Compared with the groups that received fresh or irradiation-pre- treated nerve allografts, motor nerve conduction velocity in the autograft and fresh nerve allograft groups was greater, more neurites grew into the aUografts, Schwann cell proliferation was evident, and a large number of new blood vessels was observed; in addition, massive myelinated nerve fibers formed, and abundant microfilaments and microtubules were present in the axoplasm. Our findings indicate that nerve allografts pretreated by green tea polyphenols are equivalent to trans- planting autologous nerves in the repair of sciatic nerve defects, and promote nerve regeneration. Pretreatment using green tea polyphenols is better than pretreatment with irradiation.

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

Chitosan conduits combined with nerve growth factor microspheres repair facial nerve defects

Microspheres containing nerve growth factor for sustained release were prepared by a compound method, and implanted into chitosan conduits to repair 10-mm defects on the right buccal branches of the facial nerve in rabbits. In addition, chitosan conduits combined with nerve growth factor or normal saline, as well as autologous nerve, were used as controls. At 90 days post-surgery, the muscular atrophy on the right upper lip was more evident in the nerve growth factor and normal sa- line groups than in the nerve growth factor-microspheres and autologous nerve groups. Electro- physiological analysis revealed that the nerve conduction velocity and amplitude were significantly higher in the nerve growth factor-microspheres and autologous nerve groups than in the nerve growth factor and normal saline groups. Moreover, histological observation illustrated that the di- ameter, number, alignment and myelin sheath thickness of myelinated nerves derived from rabbits were higher in the nerve growth factor-microspheres and autologous nerve groups than in the nerve growth factor and normal saline groups. These findings indicate that chitosan nerve conduits com- bined with microspheres for sustained release of nerve growth factor can significantly improve facial nerve defect repair in rabbits.

Autologous transplantation with fewer fibers repairs large peripheral nerve defects

Peripheral nerve injury is a serious disease and its repair is challenging. A cable-style autologous graft is the gold standard for repairing long peripheral nerve defects; however, ensuring that the minimum number of transplanted nerve attains maximum therapeutic effect remains poorly understood. In this study, a rat model of common peroneal nerve defect was established by resecting a 10-mm long right common peroneal nerve. Rats receiving transplantation of the common peroneal nerve in situ were designated as the in situ graft group. Ipsilateral sural nerves（10–30 mm long） were resected to establish the one sural nerve graft group, two sural nerves cable-style nerve graft group and three sural nerves cable-style nerve graft group. Each bundle of the peroneal nerve was 10 mm long. To reduce the barrier effect due to invasion by surrounding tissue and connective-tissue overgrowth between neural stumps, small gap sleeve suture was used in both proximal and distal terminals to allow repair of the injured common peroneal nerve. At three months postoperatively, recovery of nerve function and morphology was observed using osmium tetroxide staining and functional detection. The results showed that the number of regenerated nerve fibers, common peroneal nerve function index, motor nerve conduction velocity, recovery of myodynamia, and wet weight ratios of tibialis anterior muscle were not significantly different among the one sural nerve graft group, two sural nerves cable-style nerve graft group, and three sural nerves cable-style nerve graft group. These data suggest that the repair effect achieved using one sural nerve graft with a lower number of nerve fibers is the same as that achieved using the two sural nerves cable-style nerve graft and three sural nerves cable-style nerve graft. This indicates that according to the ‘multiple amplification＇ phenomenon, one small nerve graft can provide a good therapeutic effect for a large peripheral nerve defect.

A novel artificial nerve graft for repairing longdistance sciatic nerve defects:a self-assembling peptide nanofiber scaffold-containing poly (lactic-co-glycolic acid) conduit

In this study, we developed a novel artificial nerve graft termed self-assembling peptide nanofiber scaffold （SAPNS）-containing poly（lactic-co-glycolic acid） （PLGA） conduit （SPC） and used it to bridge a 10-mm-long sciatic nerve defect in the rat. Retrograde tracing, behavioral testing and histomorphometric analyses showed that compared with the empty PLGA conduit implantation group, the SPC implantation group had a larger number of growing and extending axons, a markedly increased diameter of regenerated axons and a greater thickness of the myelin sheath in the conduit. Furthermore, there was an increase in the size of the neuromuscular junction and myofiber diameter in the target muscle. These findings suggest that the novel artificial SPC nerve graft can promote axonal regeneration and remyelination in the transected peripheral nerve and can be used for repairing peripheral nerve injury.

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

Chitin scaffold combined with autologous small nerve repairs sciatic nerve defects

Although autologous nerve transplantation is the gold standard for treating peripheral nerve defects,it has many clinical limitations.As an alternative,various tissue-engineered nerve grafts have been developed to substitute for autologous nerves.In this study,a novel nerve graft composed of chitin scaffolds and a small autologous nerve was used to repair sciatic nerve defects in rats.The novel nerve graft greatly facilitated regeneration of the sciatic nerve and myelin sheath,reduced atrophy of the target muscle,and effectively restored neurological function.When the epineurium of the small autogenous nerve was removed,the degree of nerve regeneration was similar to that which occurs after autogenous nerve transplantation.These findings suggest that our novel nerve graft might eventually be a new option for the construction of tissue-engineered nerve scaffolds.The study was approved by the Research Ethics Committee of Peking University People's Hospital(approval No.2019 PHE27)on October 18,2019.

Repairing whole facial nerve defects with xenogeneic acellular nerve grafts in rhesus monkeys

Acellular nerve allografts conducted via chemical extraction have achieved satisfactory results in bridging whole facial nerve defects clinically,both in terms of branching a single trunk and in connecting multiple branches of an extratemporal segment.However,in the clinical treatment of facial nerve defects,allogeneic donors are limited.In this experiment,we exposed the left trunk and multiple branches of the extratemporal segment in six rhesus monkeys and dissected a gap of 25 mm to construct a monkey model of a whole left nerve defect.Six monkeys were randomly assigned to an autograft group or a xenogeneic acellular nerve graft group.In the autograft group,the 25-mm whole facial nerve defect was immediately bridged using an autogenous ipsilateral great auricular nerve,and in the xenogeneic acellular nerve graft group,this was done using a xenogeneic acellular nerve graft with trunk-branches.Examinations of facial symmetry,nerve-muscle electrophysiology,retrograde transport of labeled neuronal tracers,and morphology of the regenerated nerve and target muscle at 8 months postoperatively showed that the faces of the monkey appeared to be symmetrical in the static state and slightly asymmetrical during facial movement,and that they could actively close their eyelids completely.The degree of recovery from facial paralysis reached House-Brackmann grade II in both groups.Compound muscle action potentials were recorded and orbicularis oris muscles responded to electro-stimuli on the surgical side in each monkey.Fluoro Gold-labeled neurons could be detected in the facial nuclei on the injured side.Immunohistochemical staining showed abundant neurofilament-200-positive axons and soluble protein-100-positive Schwann cells in the regenerated nerves.A large number of mid-graft myelinated axons were observed via methylene blue staining and a transmission electron microscope.Taken together,our data indicate that xenogeneic acellular nerve grafts from minipigs are safe and effective for repairing whole facial nerve defects in rhesus monkeys,with an effect similar to that of autologous nerve transplantation.Thus,a xenogeneic acellular nerve graft may be a suitable choice for bridging a whole facial nerve defect if no other method is available.The study was approved by the Laboratory Animal Management Committee and the Ethics Review Committee of the Affiliated Wuxi No.2 People's Hospital of Nanjing Medical University,China(approval No.2018-D-1)on March 15,2018.

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.

One-stage human acellular nerve allograft reconstruction for digital nerve defects

Human acellular nerve allografts have a wide range of donor origin and can effectively avoid nerve injury in the donor area. Very little is known about one-stage reconstruction of digital nerve defects. The present study observed the feasibility and effectiveness of human acellular nerve allograft in the reconstruction of 〈 5-cm digital nerve defects within 6 hours after injury. A total of 15 cases of nerve injury, combined with nerve defects in 18 digits from the Department of Emergency were enrolled in this study. After dehridement, digital nerves were reconstructed using human acellular nerve allografts. The patients were followed up for 6-24 months after reconstruction. Mackinnon-Dellon static two-point discrimination results showed excellent and good rates of 89%. Semmes-Weinstein monofilament test demonstrated that light touch was normal, with an obvious improvement rate of 78%. These findings confirmed that human acellular nerve allograft for one-stage reconstruction of digital nerve defect after hand injury is feasible, which provides a novel trend for peripheral nerve reconstruction.

Chemoattractive capacity of different lengths of nerve fragments bridging regeneration chambers for the repair of sciatic nerve defects

A preliminary study by our research group showed that 6-mm-long regeneration chamber bridging is equivalent to autologous nerve transplantation for the repair of 12-mm nerve defects. In this study, we compared the efficacy of different lengths （6, 8, 10 mm） of nerve fragments bridging 6-mm regeneration chambers for the repair of 12-mm-long nerve defects. At 16 weeks after the regeneration chamber was implanted, the number, diameter and myelin sheath thickness of the regenerated nerve fibers, as well as the conduction velocity of the sciatic nerve and gastrocnemius muscle wet weight ratio, were similar to that observed with autologous nerve transplantation. Our results demonstrate that 6-, 8-and 10-mm-long nerve fragments bridging 6-mm regeneration chambers effectively repair 12-mm-long nerve defects. Because the chemoattractive capacity is not affected by the length of the nerve fragment, we suggest adopting 6-mm-long nerve fragments for the repair of peripheral nerve defects.

Acellular nerve xenografts based on supercritical extraction technology for repairing long-distance sciatic nerve defects in rats

Compared to conventional artificial nerve guide conduits (NGCs) prepared using natural polymers or synthetic polymers, acellular nerve grafts (ACNGs) derived from natural nerves with eliminated immune components have natural bionic advantages in composition and structure that polymer materials do not have. To further optimize the repair effect of ACNGs, in this study, we used a composite technology based on supercritical carbon dioxide (scCO_(2)) extraction to process the peripheral nerve of a large mammal, the Yorkshire pig, and obtained an innovative Acellular nerve xenografts (ANXs, namely, CD + scCO_(2) NG). After scCO_(2) extraction, the fat and DNA content in CD + scCO_(2) NG has been removed to the greatest extent, which can better supported cell adhesion and proliferation, inducing an extremely weak inflammatory response. Interestingly, the protein in the CD + scCO_(2) NG was primarily involved in signaling pathways related to axon guidance. Moreover, compared with the pure chemical decellularized nerve graft (CD NG), the DRG axons grew naturally on the CD + scCO_(2) NG membrane and extended long distances. In vivo studies further revealed that the regenerated nerve axons had basically crossed the CD + scCO_(2) NG 3 weeks after surgery. 12 weeks after surgery, CD + scCO_(2) NG was similar to autologous nerves in improving the quality of nerve regeneration, target muscle morphology and motor function recovery and was significantly better than hollow NGCs and CD NG. Therefore, we believe that the fully decellularized and fat-free porcine ACNGs may be the most promising “bridge” for repairing human nerve defects at this stage and for some time to come.

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.

Hypoxic pre-conditioned adipose-derived stem/progenitor cells embedded in fibrin conduits promote peripheral nerve regeneration in a sciatic nerve graft model

Recent results emphasize the supportive effects of adipose-derived multipotent stem/progenitor cells(ADSPCs)in peripheral nerve recovery.Cultivation under hypoxia is considered to enhance the release of the regenerative potential of ADSPCs.This study aimed to examine whether peripheral nerve regeneration in a rat model of autologous sciatic nerve graft benefits from an additional custom-made fibrin conduit seeded with hypoxic pre-conditioned(2%oxygen for 72 hours)autologous ADSPCs(n=9).This treatment mode was compared with three others:fibrin conduit seeded with ADSPCs cultivated under normoxic conditions(n=9);non-cell-carrying conduit(n=9);and nerve autograft only(n=9).A 16-week follow-up included functional testing(sciatic functional index and static sciatic index)as well as postmortem muscle mass analyses and morphometric nerve evaluations(histology,g-ratio,axon density,and diameter).At 8 weeks,the hypoxic pre-conditioned group achieved significantly higher sciatic functional index/static sciatic index scores than the other three groups,indicating faster functional regeneration.Furthermore,histologic evaluation showed significantly increased axon outgrowth/branching,axon density,remyelination,and a reduced relative connective tissue area.Hypoxic pre-conditioned ADSPCs seeded in fibrin conduits are a promising adjunct to current nerve autografts.Further studies are needed to understand the underlying cellular mechanism and to investigate a potential application in clinical practice.

Analysis of risk and protective factors associated with retinal nerve fiber layer defect in a Chinese adult population

AIM:To investigate the risk and protective factors associated with the retinal nerve fiber layer defect(RNFLD)in a Chinese adult population.METHODS:This study was a cross-sectional populationbased investigation including employees and retirees of a coal mining company in Kailuan City,Hebei Province.All the study participants underwent a comprehensive systemic and ophthalmic examination.RNFLD was diagnosed on fundus photographs.Binary logistic regression was used to investigate the risk and protective factors associated with the RNFLD.RESULTS:The community-based study included 14440 participants.There were 10473 participants in our study,including 7120 males(68.0%)and 3353 females(32.0%).The age range was 45-108y,averaging 59.56±8.66y.Totally 568 participants had RNFLD and the prevalence rate was 5.42%.A higher prevalence of RNFLD was associated with older age[P<0.001,odds ratio(OR):1.032;95%confidence interval(CI):1.018-1.046],longer axial length(P=0.010,OR:1.190;95%CI:1.042-1.359),hypertension(P=0.007,OR:0.639;95%CI:0.460-0.887),and diabetes mellitus(P=0.019,OR:0.684;95%CI:0.499-0.939).The protective factors of RNFLD were visual acuity(P=0.038,OR:0.617;95%CI:0.391-0.975),and central anterior chamber depth(P=0.046,OR:0.595;95%CI:0.358-0.990).CONCLUSION:In our cross-sectional community-based study,with an age range of 45-108y,RNFLD is associated with older age,longer axial length,hypertension,and diabetes mellitus.The protective factors of RNFLD are visual acuity and central anterior chamber depth.These can help to predict and evaluate RNFLD related diseases and identify high-risk populations early.

Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold

The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells fol-lowing induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined speciifc neu-ronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuro-nal-speciifc proteins, includingβIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differen-tiation medium differentiated into a multilayered neural network-like structure with long nerve ifbers that was composed of several parallel microifbers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sec-tioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve.

Validation of a novel animal model for sciatic nerve repair with an adipose-derived stem cell loaded fibrin conduit

Despite the regenerative capabilities of peripheral nerves, severe injuries or neuronal trauma of critical size impose immense hurdles for proper restoration of neuro-muscular circuitry. Autologous nerve grafts improve re-establishment of connectivity, but also comprise substantial donor site morbidity. We developed a rat model which allows the testing of different cell applications, i.e., mesenchymal stem cells, to improve nerve regeneration in vivo. To mimic inaccurate alignment of autologous nerve grafts with the injured nerve, a 20 mm portion of the sciatic nerve was excised, and sutured back in place in reversed direction. To validate the feasibility of our novel model, a fibrin gel conduit containing autologous undifferentiated adipose-derived stem cells was applied around the coaptation sites and compared to autologous nerve grafts. After evaluating sciatic nerve function for 16 weeks postoperatively, animals were sacrificed, and gastrocnemius muscle weight was determined along with morphological parameters（g-ratio, axon density ＆ diameter） of regenerating axons. Interestingly, the addition of undifferentiated adipose-derived stem cells resulted in a significantly improved re-myelination, axon ingrowth and functional outcome, when compared to animals without a cell seeded conduit. The presented model thus displays several intriguing features： it imitates a certain mismatch in size, distribution and orientation of axons within the nerve coaptation site. The fibrin conduit itself allows for an easy application of cells and, as a true critical-size defect model, any observed improvement relates directly to the performed intervention. Since fibrin and adipose-derived stem cells have been approved for human applications, the technique can theoretically be performed on humans. Thus, we suggest that the model is a powerful tool to investigate cell mediated assistance of peripheral nerve regeneration.