Repair of rat sciatic nerve gap by a silk fibroin-based scaffold added with bone marrow mesenchymal stem cells

Tissue-engineered nerve grafts (TENGs), typically consisting of a neural scaffold included with support cells and/or growth factors, represent a promising alternative to autologous nerve grafts for surgical repair of large peripheral nerve gaps. Here, we developed a new design of TENGs by introducing bone marrow mesenchymal stem cells (MSCs) of rats, as support cells, into a silk fibroin (SF)-based scaffold, which was composed of an SF nerve guidance conduit and oriented SF filaments as the conduit lumen filler. The biomaterial SF had been tested to possess good biocompatibility and noncytoxicity with MSCs before the TENG was implanted to bridge a 10-mm-long gap in rat sciatic nerve. Functional and histological assessments showed that at 12 weeks after nerve grafting, TENGs yielded an improved outcome of nerve regeneration and functional recovery, which was better than that achieved by SF scaffolds and close to that by autologous nerve grafts. During 1-4 weeks after nerve grafting, MSCs contained in the TENG significantly accelerated axonal growth, displaying a positive reaction to S-100 (a Schwann cell marker). During 1-3 weeks after nerve grafting, MSCs contained in the TENG led to gene expression upregulation of S100 and several growth factors (brain-derived neurotrophic factor, ciliary neurotrophic factor, and basic fibroblast growth factor). These results suggest that the cell behaviors and neurotrophic functions of MSCs might be responsible for their promoting effects on peripheral nerve regeneration.     

Peripheral nerve tissue engineering: autologous Schwann cells vs. transdifferentiated mesenchymal stem cells

Mesenchymal stem cells (MSCs) were evaluated as an alternative source for tissue engineering of peripheral nerves. MSCs, transdifferentiated MSCs, or Schwann cells cultured from male rats were grafted into devitalized autologous muscle conduits bridging a 2-cm sciatic nerve gap in female rats. The differentiation potential of MSCs and transformed cultivated MSCs into Schwann cell-like cells was exploited using a cocktail of cytokines. Polymerase chain reaction of the SRY gene confirmed the presence of the implanted cells in the grafts. After 6 weeks, regeneration was monitored clinically, histologically, and morphometrically. Autologous nerves and cell-free muscle grafts were used as control. Revascularization studies suggested that transdifferentiated MSCs, in contrast to undifferentiated MSCs, facilitated neo-angiogenesis and did not influence macrophage recruitment. Autologous nerve grafts demonstrated the best results in all regenerative parameters. An appropriate regeneration was noted in the Schwann cell-groups and, albeit with restrictions, in the transdifferentiated MSC groups, whereas regeneration in the MSC group and in the cell-free group was impaired. The results indicate that transdifferentiated MSCs implanted into devitalized muscle grafts are able to support peripheral nerve regeneration to some extent, and offer a potential for new therapeutic strategies.     

Bone marrow stromal cells differentiated into functional Schwann cells in injured rats sciatic nerve

Bone marrow stromal cells (MSCs) have been shown to differentiate into various lineage cells including neural cells in vitro and in vivo. We therefore examined whether MSCs can differentiate into Schwann cells in injured peripheral nerves, After cultured in vitro, PKH-67-labeled MSCs were injected into the mechanically injured rat sciatic nerves. Three weeks after injection, immunofluorescent examinations were carried out. MSCs had been incorporated around the injured nerves and differentiated into Schwann cells. MSCs had accumulated mainly in the epineurium around the injured nerve. The incorporated cells partially expressed GFAP, S-100, and P75. These results confirmed the possibility that MSCs have the ability to differentiate into Schwann cells, and that injection of MSCs into the injured peripheral nerve would help repair damaged nerve.     

Collagen nerve conduits--assessment of biocompatibility and axonal regeneration

Bridging of nerve gaps is still a major problem in peripheral nerve surgery. Alternatively to autologous nerve grafts tissue engineering of peripheral nerves focuses on biocompatible conduits to reconstruct nerves. Such non-neural conduits fail to support regeneration over larger gaps due to lacking viable Schwann cells that promote regeneration by producing growth factors and cell guiding molecules. This problem may be overcome by implantation of cultivated Schwann cells into suitable scaffolds. In the present experiments we tested a collagen type I/III tube as a potential nerve guiding matrix. Revascularization, tolerance and Schwann cell settlement were evaluated by light, fluorescence and scanning electron microscopy after different implantation times. The conduits were completely revascularized between day 5 and 7 post-operatively and well integrated into the host tissue. Implanted Schwann cells adhered, survived and proliferated on the inner surface of the conduits. Nevertheless, bridging a 2 cm gap of the sciatic nerve of adult Wistar rats with these collagen/Schwann cell conduits led to a disappointing regeneration compared to controls with autologous grafts. From these results, we conclude that a sufficient biocompatibility of bioartificial nerve conduits is a necessary prerequisite, however, it remains only one of several parameters important for peripheral nerve regeneration.     

Peripheral nerve explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve

Summary We have conducted experiments in the adult rat visual system to assess the relative importance of an absence of trophic factors versus the presence of putative growth inhibitory molecules for the failure of regeneration of CNS axons after injury. The experiments comprised three groups of animals in which all optic nerves were crushed intra-orbitally: an optic nerve crush group had a sham implant-operation on the eye; the other two groups had peripheral nerve tissue introduced into the vitreous body; in an acellular peripheral nerve group, a frozen/thawed teased sciatic nerve segment was grafted, and in a cellular peripheral nerve group, a predegenerate teased segment of sciatic nerve was implanted. The rats were left for 20 days and their optic nerves and retinae prepared for immunohistochemical examination of both the reaction to injury of axons and glia in the nerve and also the viability of Schwann cells in the grafts. Anterograde axon tracing with rhodamine-B provided unequivocal qualitative evidence of regeneration in each group, and retrograde HRP tracing gave a measure of the numbers of axons growing across the lesion by counting HRP filled retinal ganglion cells in retinal whole mounts after HRP injection into the optic nerve distal to the lesion. No fibres crossed the lesion in the optic nerve crush group and dense scar tissue was formed in the wound site. GAP-43-positive and rhodamine-B filled axons in the acellular peripheral nerve and cellular peripheral nerve groups traversed the lesion and grew distally. There were greater numbers of regenerating fibres in the cellular peripheral nerve compared to the acellular peripheral nerve group. In the former, 0.6–10% of the retinal ganglion cell population regenerated axons at least 3–4 mm into the distal segment. In both the acellular peripheral nerve and cellular peripheral nerve groups, no basal lamina was deposited in the wound. Thus, although astrocyte processes were stacked around the lesion edge, a glia limitans was not formed. These observations suggest that regenerating fibres may interfere with scarring. Viable Schwann cells were found in the vitreal grafts in the cellular peripheral nerve group only, supporting the proposition that Schwann cell derived trophic molecules secreted into the vitreous stimulated retinal ganglion cell axon growth in the severed optic nerve. The regenerative response of acellular peripheral nerve-transplanted animals was probably promoted by residual amounts of these molecules present in the transplants after freezing and thawing. In the optic nerves of all groups the astrocyte, microglia and macrophage reactions were similar. Moreover, oligodendrocytes and myelin debris were also uniformly distributed throughout all nerves. Our results suggest either that none of the above elements inhibit CNS regeneration after perineuronal neurotrophin delivery, or that the latter, in addition to mobilising and maintaining regeneration, also down regulates the expression of axonal growth cone-located receptors, which normally mediate growth arrest by engaging putative growth inhibitory molecules of the CNS neuropil.     

成年金黄地鼠大脑运动皮质神经元轴突的再生

在用ＨＲＰ逆行追踪法确定金黄地鼠大脑运动皮质的准确位置后，将截取的三组实验动物的自体坐骨神经分别插入之。存活８周后，在坐骨神经移植段的远端，注射２０％ＨＲＰ溶被１μｌ，做全脑及脊髓颈段连续冰冻切片，观察ＨＲＰ逆行标记的运动皮质锥体细胞。结果发现：在移植的正常坐骨神经段的诱导下，运动皮质神经元的受损轴突可以再生并长入到该移植段内（ｎ＝１２，平均＝９．７５），用预先渍变法未能导致运动皮质轴突再生神经元数目的增加（ｎ＝９，平均＝７．５），而经冻化处理后的移植神经则未能诱导出运动皮质神经元的轴奕再生。本文结果证明，周围神经移植法可诱导成年金黄地鼠运动皮质神经元的轴突再生，雪旺氏细胞的存在及其功能活动与锥体神经元轴突再生密切相关。     

脱细胞枪乌贼神经的制备及其生物相容性

背景：脱细胞哺乳动物神经有有髓纤维直径小、细胞成分不易彻底清除、复合细胞不均匀的缺点。 目的：制备脱细胞枪乌贼神经,并探讨其生物相容性。 方法：用Hasse化学萃取法制备脱细胞枪乌贼神经,观察其脱细胞的效果、管道及基底膜的完整性,用MTT法检测脱细胞枪乌贼神经与细胞相容性,用皮下植入实验检测其组织相容性。 结果与结论：制备的脱细胞枪乌贼神经细胞及髓鞘清除彻底、神经纤维膜管及基底膜保留完整,且膜管粗大。脱细胞枪乌贼神经皮下植入植入后1和2周材料周围有炎症细胞浸润。植入后4 周材料周围有薄层纤维结缔组织囊,材料内部及周围只有少量炎症细胞。结果证实,采用Hasse化学萃取法制备的脱细胞枪乌贼神经,细胞清除彻底,神经纤维膜管及基底膜保留完整,与异种细胞及组织相容性良好。     

人发角蛋白丝束修复大鼠坐骨神经损伤的实验研究

目的：观察人发角蛋白(HHK)丝束桥接体诱导坐骨神经再生过程中形态字变化：方法：制备坐骨神经损伤SD大鼠动物模型，分别植入HHK或HHK 胶原屏障膜，术后2d、2、3、6、9、12周行组织学观察。结果：术后2d到2周，断端的施万细胞去分化，沿着HHK束表面纵向分裂增殖。术后3周HHK开始降解，施万细胞大量增生：HHK周围有很多巨噬细胞和多核巨细胞，并出现轴突和大量微血管：术后6周，HHK丝周围可见大量新生的神经纤维。术后9周，HHK降解显著，有明显的神经外膜和束膜。术后12周，HHK完全降解，其部位被新的神经纤维取代。HHK丝束 胶原膜屏障膜组与HHK丝束组没有明显区别。结论：HHK具有良好的桥接作用，坐骨神经沿着HHK再生时在其外周就能由微血管和结缔组织形成一屏障膜，无需外加屏障膜。     

Extracellular matrix of human amnion manufactured into tubes as conduits for peripheral nerve regeneration

The human amnion consists of the epithelial cell layer and underlying connective tissue. After removing the epithelial cells, the resulting acellular connective tissue matrix was manufactured into thin dry sheets called amnion matrix sheets. The sheets were further processed into tubes, amnion matrix tubes (AMTs), of varying diameters, with the walls of varying numbers of amnion matrix sheets with or without a gelatin coating. The AMTs were implanted into rat sciatic nerves. Regenerating nerves extended in bundles through tubes of 1-2 mm in diameter and further elongated into host distal nerves 1-3 weeks after implantation. Morphometrical analysis of the regenerated nerve cable at the middle of each amnion matrix tube 3 weeks after implantation was performed. The average numbers of myelinated axons were almost the same (ca. 80-112/10(4) microm(2)) in AMTs of 1-2 mm in diameter, as in the normal sciatic nerve (ca. 95/10(4) microm(2)). No myelinated fibers were found in AMTs composed of multiple thin tubes of 0.2 mm in diameter. The myelinated axons were thinner in implanted tubes than those in the normal sciatic nerve. The rate of occurrences of myelinated axons less than 4 microm in diameter was significantly higher in the AMTs, whereas axons in the normal sciatic nerve were diverse in distribution, with the highest population at 8-12 microm in diameter. Reinnervation to the gastrocnemius muscle was demonstrated electrophysiologically 9 months after implantation. It was concluded that the extracellular matrix sheet from the human amnion is an effective conduit material for peripheral nerve regeneration.     

Mesenchymal stem cells in a polycaprolactone conduit promote sciatic nerve regeneration and sensory neuron survival after nerve injury

Despite the fact that the peripheral nervous system is able to regenerate after traumatic injury, the functional outcomes following damage are limited and poor. Bone marrow mesenchymal stem cells (MSCs) are multipotent cells that have been used in studies of peripheral nerve regeneration and have yielded promising results. The aim of this study was to evaluate sciatic nerve regeneration and neuronal survival in mice after nerve transection followed by MSC treatment into a polycaprolactone (PCL) nerve guide. The left sciatic nerve of C57BL/6 mice was transected and the nerve stumps were placed into a biodegradable PCL tube leaving a 3-mm gap between them; the tube was filled with MSCs obtained from GFP+ animals (MSC-treated group) or with a culture medium (Dulbecco's modified Eagle's medium group). Motor function was analyzed according to the sciatic functional index (SFI). After 6 weeks, animals were euthanized, and the regenerated sciatic nerve, the dorsal root ganglion (DRG), the spinal cord, and the gastrocnemius muscle were collected and processed for light and electron microscopy. A quantitative analysis of regenerated nerves showed a significant increase in the number of myelinated fibers in the group that received, within the nerve guide, stem cells. The number of neurons in the DRG was significantly higher in the MSC-treated group, while there was no difference in the number of motor neurons in the spinal cord. We also found higher values of trophic factors expression in MSC-treated groups, especially a nerve growth factor. The SFI revealed a significant improvement in the MSC-treated group. The gastrocnemius muscle showed an increase in weight and in the levels of creatine phosphokinase enzyme, suggesting an improvement of reinnervation and activity in animals that received MSCs. Immunohistochemistry documented that some GFP+ -transplanted cells assumed a Schwann-cell-like phenotype, as evidenced by their expression of the S-100 protein, a Schwann cell marker. Our findings suggest that using a PCL tube filled with MSCs is a good strategy to improve nerve regeneration after a nerve transection in mice.     

Cat peripheral nerve regeneration across 50 mm gap repaired with a novel nerve guide composed of freeze-dried alginate gel

We have developed a novel artificial nerve guide composed of biodegradable freeze-dried alginate gel covered by polyglycolic acid mesh, and evaluated its effect on peripheral nerve regeneration, using a 50-mm gap cat sciatic nerve model. Functional reinnervation of motor and sensory nerves occurred 13 weeks after implantation, as demonstrated by recovery of compound muscle action potential (CMAP) and somatosensory evoked potential (SEP). For histologic evaluation, samples of tissue were harvested from the grafted material segment 7 months after operation. Many newly developed nerve fasciculi were found, and the implanted nerve guidance material had completely disappeared with little inflammation. These results indicate that freeze-dried alginate gel allows the nerve to regenerate across longer gaps than described in previous literature.     

Neurotropism in nerve regeneration: an immunohistochemical study

A rectangular pseudomesothelial-lined chamber was used to elucidate the hypothesis that in adult rats neurotrophic factors are formed after nerve injury and may influence regeneration of peripheral nerves. The proximal end of a cut sciatic nerve was inserted into one corner of the chamber. In one group of animals the distal end of the cut sciatic nerve was implanted in the diagonally opposite corner of the chamber. In another group we just introduced the proximal end of the sciatic nerve; no distal implant was used. The organization, length and direction of the nerve fibres, regenerating from the proximal end of the sciatic nerve, was visualized immunohistochemically with the aid of antibodies against neurofilaments at 2, 3 and 4 weeks after surgery. When a distal sciatic nerve segment was used, nerve fibres regenerating from the proximal cut end of the sciatic nerve showed an organized growth across the chamber, formed bundles and grew into the diagonally implanted nerve piece. If there was no distal implant, the growth of the randomly directed nerve fibres ceased after about two weeks, resulting in formation of a neuroma-like structure. Increased immunoreactivity of the trophic peptide insulin-like growth factor I (IGF-I, somatomedin C) was demonstrated in the regenerating nerve, most evidently in reactive Schwann cells. It is concluded that a positive neurotropic effect is exerted on growing nerve fibres by injured, reactive peripheral nerve tissue. There could tentatively be a relation between nerve regeneration and local formation of trophic factors.     

Tubulation with dental pulp cells promotes facial nerve regeneration in rats

Dental pulp is an easily obtainable source of viable cells for potential use in peripheral nerve regeneration. We prepared artificial conditions for nerve regeneration using a silicone tube containing a collagen gel embedded with rat dental pulp cells, and we examined its effectiveness for repairing a gap in the rat facial nerve. Twelve days after transplantation, defective facial nerves connected with silicone tubes containing dental pulp cells were repaired more rapidly than control tubes containing the collagen gel alone. When a tube containing green fluorescent protein (GFP)-positive dental pulp cells was transplanted into a facial nerve gap in a GFP-negative rat, we observed regenerated nerves with GFP-positive cells at 2 weeks posttransplantation. The regenerated nerves included Tuj1-positive axons, RECA1 and GFP double-positive blood vessels, and S100 and GFP double-positive Schwann-like supportive cells. Osmium-toluidine blue staining revealed that the regenerated nerves contained myelinated fibers. Moreover, fluorescent retrograde tracing analysis by application of Fluoro-Gold into the regenerated nerves demonstrated the presence of Fluoro-Gold-positive motor neurons in the facial nucleus of the rat brain. These results suggest that the transplanted dental pulp cells formed blood vessels and myelinating tissue and contributed to the promotion of normal nerve regeneration.     

Human adipose-derived mesenchymal stem cells systemically injected promote peripheral nerve regeneration in the mouse model of sciatic crush

Mesenchymal stem cells (MSCs) represent a promising therapeutic approach in nerve tissue engineering. To date, the local implantation of MSC in injured nerves has been the only route of administration used. In case of multiple sites of injury, the systemic administration of cells capable of reaching damaged nerves would be advisable. In this regard, we found that an intravenous administration of adipose-derived MSC (ASC) 1 week after sciatic nerve crush injury, a murine model of acute axonal damage, significantly accelerated the functional recovery. Sciatic nerves from ASC-treated mice showed the presence of a restricted number of undifferentiated ASC together with a significant improvement in fiber sprouting and the reduction of inflammatory infiltrates for up to 3 weeks. Besides the immune modulatory effect, our results show that ASC may contribute to peripheral nerve regeneration because of their ability to produce in culture neuroprotective factors such as insulin-like growth factor I, brain-derived neurotrophic factor, or basic fibroblast growth factor. In addition to this production in vitro, we interestingly found that the concentration of glial-derived neurotrophic factor (GDNF) was significantly increased in the sciatic nerves in mice treated with ASC. Since no detectable levels of GDNF were observed in ASC cultures, we hypothesize that ASC induced the local production of GDNF by Schwann cells. In conclusion, we show that systemically injected ASC have a clear therapeutic potential in an acute model of axonal damage. Among the possible mechanisms promoting nerve regeneration, our results rule out a process of trans-differentiation and rather suggest the relevance of a bystander effect, including the production of in situ molecules, which, directly or indirectly through a cross-talk with local glial cells, may modulate the local environment with the down-regulation of inflammation and the promotion of axonal regeneration.     

Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration

Surgical repair of severe peripheral nerve injuries represents not only a pressing medical need, but also a great clinical challenge. Autologous nerve grafting remains a golden standard for bridging an extended gap in transected nerves. The formidable limitations related to this approach, however, have evoked the development of tissue engineered nerve grafts as a promising alternative to autologous nerve grafts. A tissue engineered nerve graft is typically constructed through a combination of a neural scaffold and a variety of cellular and molecular components. The initial and basic structure of the neural scaffold that serves to provide mechanical guidance and optimal environment for nerve regeneration was a single hollow nerve guidance conduit. Later there have been several improvements to the basic structure, especially introduction of physical fillers into the lumen of a hollow nerve guidance conduit. Up to now, a diverse array of biomaterials, either of natural or of synthetic origin, together with well-defined fabrication techniques, has been employed to prepare neural scaffolds with different structures and properties. Meanwhile different types of support cells and/or growth factors have been incorporated into the neural scaffold, producing unique biochemical effects on nerve regeneration and function restoration. This review attempts to summarize different nerve grafts used for peripheral nerve repair, to highlight various basic components of tissue engineered nerve grafts in terms of their structures, features, and nerve regeneration-promoting actions, and finally to discuss current clinical applications and future perspectives of tissue engineered nerve grafts.     

Central and peripheral nerve regeneration by transplantation of Schwann cells and transdifferentiated bone marrow stromal cells

In contrast to the peripheral nervous system (PNS), little structural and functional regeneration of the central nervous system (CNS) occurs spontaneously following injury in adult mammals. The inability of the CNS to regenerate is mainly attributed to its own inhibitorial environment such as glial scar formation and the myelin sheath of oligodendrocytes. Therefore, one of the strategies to promote axonal regeneration of the CNS is to experimentally modify the environment to be similar to that of the PNS. Schwann cells are the myelinating glial cells in the PNS, and are known to play a key role in Wallerian degeneration and subsequent regeneration. Central nervous system regeneration can be elicited by Schwann cell transplantation, which provides a suitable environment for regeneration. The underlying cellular mechanism of regeneration is based upon the cooperative interactions between axons and Schwann cells involving the production of neurotrophic factors and other related molecules. Furthermore, tight and gap junctional contact between the axon and Schwann cell also mediates the molecular interaction and linking. In this review, the role of the Schwann cell during the regeneration of the sciatic (representing the PNS) and optic (representing the CNS) nerves is explained. In addition, the possibility of optic nerve reconstruction by an artificial graft of Schwann cells is also described. Finally, the application of cells not of neuronal lineage, such as bone marrow stromal cells (MSCs), in nerve regeneration is proposed. Marrow stromal cells are known as multipotential stem cells that, under specific conditions, differentiate into several kinds of cells. The strategy to transdifferentiate MSCs into the cells with a Schwann cell phenotype and the induction of sciatic and optic nerve regeneration are described.     

Sciatic nerve repair by reinforced nerve conduits made of gelatin-tricalcium phosphate composites

This study proposes a biodegradable GGT composite nerve guide conduit containing genipin-cross-linked gelatin and tricalcium phosphate (TCP) ceramic particles in peripheral nerve regeneration. The proposed genipin-cross-linked gelatin annexed with TCP ceramic particles (GGT) conduit was dark bluish and round with a rough and compact surface. Water uptake and swelling tests indicated that the hydrated GGT conduit exhibited increased stability with not collapsing or stenosis. The GGT conduit had higher mechanical properties than the genipin-cross-linked gelatin without TCP ceramic particles (GG) conduit and served as a better nerve guide conduit. Cytotoxicity tests revealed that the GGT conduit was not toxic and that it promoted the viability and growth of neural stem cells. The experiments in this study confirmed the effectiveness of the GGT conduit as a guidance channel for repairing a 10-mm gap in rat sciatic nerve. Walking track analysis showed a significantly higher sciatic function index score and better toe spreading development in the GGT group than in the silicone group 8 weeks after implantation. Gross examination revealed that the diameter of the intratubular newly formed nerve fibers in GGT conduits exceeded those in silicone tubes after the implantation period. Histological observations revealed that the morphology and distribution patterns of nerve fibers in the GGT conduits at 8 weeks after implantation were similar to those of normal nerves. The quantitative results indicated the superiority of the conduits over the silicone tubes. Motor functional and histomorphometric assessments demonstrate that the proposed GGT conduit is a suitable candidate for peripheral nerve repair.     

Electrospinning of Matrigel to Deposit a Basal Lamina-Like Nanofiber Surface

Schwann cell basal lamina is a nanometer-thin extracellular matrix layer that separates the axon-bound Schwann cells from the endoneurium of the peripheral nerve. It is implicated in the promotion of nerve regeneration after transection injury by allowing Schwann cell colonization and axonal guidance. Hence, it is desired to mimic the native basal lamina for neural tissue engineering applications. In this study, basal lamina proteins from BD Matrigel (growth factor-reduced) were extracted and electrospun to deposit nonwoven nanofiber mats. Adjustment of solute protein concentration, potential difference, air gap distance and flow rate produced a basal lamina-like construct with an average surface roughness of 23 nm and composed of 100-nm-thick irregular and relatively discontinuous fibers. Culture of embryonic chick dorsal root ganglion explants demonstrated that the fabricated nanofiber layer supported explant attachment, elongation of neurites, and migration of satellite Schwann cells in a similar fashion compared to electrospun collagen type-I fibers. Furthermore, the presence of nanorough surface featues significantly increased the neurite spreading and Schwann cell growth. Sciatic nerve segment incubation also showed that the construct is promigratory to nerve Schwann cells. Results, therefore, suggest that the synthetic basal lamina fibers can be utilized as a biomaterial for induction of peripheral nerve repair.     

Recellularized nerve allografts with differentiated mesenchymal stem cells promote peripheral nerve regeneration

Chemical-extracted acellular nerve allografting, containing the natural nerve structure and elementary nerve extracellular matrix (ECM), has been used for peripheral nerve-defect treatment experimentally and clinically. However, functional outcome with acellular nerve allografting decreases with increased size of gap in nerve defects. Cell-based therapy is a good strategy for repairing long nerve defects. Bone-marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ADSCs) can be induced to differentiate into cells with Schwann cell-like properties (BMSC-SCs or ADSC-SCs), which have myelin-forming ability in vitro and secrete trophic nerve growth factors. Here, we aimed to determine whether BMSC-SCs or ADSC-SCs are a promising cell type for enriching acellular grafts in nerve repair. We evaluated axonal regeneration distance by immunofluorescence staining after 2-week implantation. We used functional and histomorphometric analysis to evaluate 3-month regeneration of the novel cell-supplemented tissue-engineered nerve graft used to bridge a 15-mm-long sciatic nerve gap in rats. Introducing BMSC-SCs or ADSC-SCs to the acellular nerve graft promoted sciatic nerve regeneration and functional recovery. Nerve regeneration with BMSC-SCs or ADSC-SCs was comparable to that with autografting and Schwann cells alone and better than that with acellular nerve allografting alone. Differentiated bone-marrow-or adipose-derived MSCs may be a promising cell source for tissue-engineered nerve grafts and promote functional recovery after peripheral nerve injury.     

Generation of Schwann Cell-Derived Multipotent Neurospheres Isolated from Intact Sciatic Nerve

Schwann cells (SCs) are the supporting cells of the peripheral nervous system and originate from the neural crest. They play a unique role in the regeneration of injured peripheral nerves and have themselves a highly unstable phenotype as demonstrated by their unexpectedly broad differentiation potential. Thus, SCs can be considered as dormant, multipotent neural crest-derived progenitors or stem cells. Upon injury they de-differentiate via cellular reprogramming, re-enter the cell cycle and participate in the regeneration of the nerve. Here we describe a protocol for efficient generation of neurospheres from intact adult rat and murine sciatic nerve without the need of experimental in vivo pre-degeneration of the nerve prior to Schwann cell isolation. After isolation and removal of the connective tissue, the nerves are initially plated on poly-D-lysine coated cell culture plates followed by migration of the cells up to 80?% confluence and a subsequent switch to serum-free medium leading to formation of multipotent neurospheres. In this context, migration of SCs from the isolated nerve, followed by serum-free cultivation of isolated SCs as neurospheres mimics the injury and reprograms fully differentiated SCs into a multipotent, neural crest-derived stem cell phenotype. This protocol allows reproducible generation of multipotent Schwann cell-derived neurospheres from sciatic nerve through cellular reprogramming by culture, potentially marking a starting point for future detailed investigations of the de-differentiation process.