A composite poly-hydroxybutyrate-glial growth factor conduit for long nerve gap repairs

There is considerable evidence that peripheral nerves have the potential to regenerate in an appropriate microenvironment. We have developed a novel artificial nerve guide composed of poly 3-hydroxybutyrate (PHB) filled with glial growth factor (GGF) suspended in alginate hydrogel. Gaps of 2-4 cm in rabbit common peroneal nerve were bridged using a PHB conduit containing either GGF in alginate hydrogel (GGF) or alginate alone (Alginate), or with an empty PHB conduit (Empty). Tissues were harvested 21, 42 and 63 days post-operatively. Schwann cell and axonal regeneration were assessed using quantitative immunohistochemistry. At 21 days, addition of GGF increased significantly the distance of axonal and Schwann cells regeneration in comparison with that observed in Alginate and Empty conduits for both gap lengths. The axons bridged the 2-cm GGF conduits gap by 63 days, with a comparable rate of regeneration seen in 4-cm conduits. Schwann cells and axonal regeneration quantity was similar for both gap lengths in each group. However, at all time points the quantity of axonal and Schwann cells regeneration in GGF grafts was significantly greater than in both Alginate and Empty conduits, the latter showing better regeneration than Alginate conduits. The results indicate an inhibitory effect of alginate on regeneration, which is partially reversed by the addition of GGF to the conduits. In conclusion, GGF stimulates a progressive and sustainable regeneration increase in long nerve gap conduits.     

Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration

Bone marrow mesenchymal stem cells can trans-differentiate into neuronal phenotypes. We examined the differentiation of marrow stromal cells (MSCs) in culture and during nerve regeneration. MSCs from adult rats were exposed to glial growth factor (GGF) to stimulate glial differentiation. Subsequently differentiated MSCs were retrovirally labelled with green fluorescent protein and transplanted into 1 cm nerve conduits in the rat sciatic nerve. Fifteen days post-operatively the conduits were examined for axonal and Schwann cell regeneration and MSC integration. In vitro, MSCs exposed to GGF expressed S100 and glial fibrillary acidic protein. Following transplantation, MSCs maintained S100 expression and enhanced nerve regeneration, with significant Schwann cell regeneration compared to control (2.7 +/- 0.21 vs. 2.05 +/- .21 mm; P < 0.05). MSCs not exposed to GGF prior to transplantation expressed S100 in vivo indicating glial differentiation in response to local cytokines and growth factors.     

Effects of unidirectional permeability in asymmetric poly(DL-lactic acid-co-glycolic acid) conduits on peripheral nerve regeneration: an in vitro and in vivo study

The high outflow permeability of the nerve conduit used to emit the drained waste generated from the traumatized host nerve stump is critical in peripheral nerve regeneration. Our earlier studies have established that asymmetric conduits fulfill the basic requirements for use as nerve guide conduits. In this study, the inflow characteristics of optimal nerve conduits were further examined using in vivo and in vitro trials. Various asymmetric poly(DL-lactic acid-co-glycolic acid) (PLGA) conduits were controlled by modifying precipitation baths using 0, 20, and 95% isopropyl alcohol, with high-porosity (permeability), medium-porosity (high outflow and low inflow), and low-porosity (permeability), respectively. In the in vitro trial, the Schwann cells and fibroblasts were seeded on either side of the asymmetric PLGA films in a newly designed coculture system that simulated the repaired nerve conduit environment. The results of the directional permeable films indicated the statistically significant proliferation of Schwann cells and the inhibition of the division of fibroblasts in lactate dehydrogenase release and inhibition of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) reduction, compared with the other films. In the in vivo trial, the PLGA conduits seeded with Schwann cells were implanted into 10 mm right sciatic nerve defects in rats. After 6 weeks, implanted conduits were harvested. Histological examination verified that directional permeable conduits had markedly more A-type and B-type myelin fibers in the midconduit and distal nerve. In this work, the directional transport characteristics were established as an extremely important factor to the design and development of optimal nerve guide conduits in peripheral nerve regeneration.     

In vitro and in vivo effects of Ginkgo biloba extract EGb 761 on seeded Schwann cells within poly(DL-lactic acid-co-glycolic acid) conduits for peripheral nerve regeneration

This study investigated the effects of Ginkgo biloba (EGb 761) extract on seeded Schwann cells within poly(DL-lactic acid-co-glycolic acid) (PLGA) conduits by in vitro and in vivo trials for peripheral nerve regeneration. The seeding efficiency of Schwann cells in serum-deprived culture medium, which simulated the environment of mechanical trauma on an injured nerve site, was improved by adding different dosages of EGb 761 (0, 1, 10, 20, 50, 100, 200 microg/mL). The analytical results showed enhanced cell attachment and survival, reduced LDH release and increased MTT values, particularly in the range 10-100 microg/mL. The PLGA nerve conduits seeded with Schwann cells (6 x 10(3) cells) and filled with gelatin containing EGb 761 (0, 10, 50, 100 microg/mL) were implanted to 10-mm right sciatic nerve defects in rats. Autograft was performed as another control. Electromyography was assessed based on the motor unit action potential (MUAP) and fibrillation potential (Fib) at 2, 4, and 6 weeks during all periods. The specimens of the experimental and control groups were harvested for histological analysis at 6 weeks after surgery. The Fib was found to gradually decay, and the MUAP was found not to be present until 4 weeks after surgery. Meanwhile, the experimental groups were all statically better than the control group (without EGb 761) and autografts were observed at 6 weeks, especially at the concentration of 10 microg/mL, where there was higher amplitude of MUAP and a significantly larger number of myelinated axons. This study concluded that a proper concentration of EGb 761 (10-50 microg/mL) promoted seeding efficiency of Schwann cells in a tissue-engineered PLGA conduit. Addition of EGb 761 in Schwann cells-seeded conduit could increase the total number of myelinated axons in nerve regeneration and improve peripheral nerve functional recovery.     

Neuregulin‐1/glial growth factor stimulates Schwann cell migration by inducing α5 β1 integrin–ErbB2–focal adhesion kinase complex formation

After peripheral nerve injury, Schwann cells gain a migratory phenotype and remodel their extracellular matrix to provide a supportive environment for axonal regeneration. The soluble neuregulin‐1 isoform, that is, glial growth factor (GGF), is expressed in regenerating axons of injured peripheral nerves and regulates Schwann cell motility by activating the ErbB family of tyrosine kinase receptors, but how GGF/ErbB signaling contributes to Schwann cell motility remains unclear. Here, we show that GGF stimulates Schwann cell migration by inducing the formation of a protein complex containing the fibronectin receptor α5β1 integrin, ErbB2, and focal adhesion kinase (FAK). ErbB2 co‐localizes and co‐immunoprecipitates with the focal complex members including α5β1 integrin and FAK after GGF treatment. These effects of GGF appear to involve FAK activation, which occurs downstream of ErbB2 stimulation. RNAi‐mediated down‐regulation of α5 integrin expression in primary cultured Schwann cells resulted in significantly decreased interaction between FAK and ErbB2, as well as decreased GGF‐induced migration. An increase in the α5β1 integrin–ErbB2–FAK complex formation was observed in injured nerve Schwann cells, but not uninjured control. Taken together, these data suggest that GGF plays an important modulatory role in Schwann cell migration after nerve crush by inducing α5β1 integrin–ErbB2–FAK complex formation.     

Engineering an artificial nerve graft for the repair of severe nerve injuries

Nerve repair with tubes has a limit to regeneration depending upon the length of the gap. The characteristics of the guide, in terms of permeability, durability and adhesiveness, also influence regeneration. Considering the importance of the cellular component in regeneration, the development of artificial grafts, composed of a biocompatible nerve guide filled with a neurotropic matrix and seeded with Schwann cells (SCs), is an interesting option to enhance nerve regeneration and provide an alternative to the classical autologous nerve graft. We evaluated the ability of SCs transplanted into a nerve guide to improve regeneration after sciatic nerve resection, leaving a 6-mm gap, in the mouse. Syngeneic, isogeneic and autologous SCs were suspended in Matrigel and seeded in resorbable guides, and compared to acellular guides and to nerve autografts. The immunogenicity of the transplanted SCs clearly influenced the outcome. Transplants of autologous SCs resulted in only slightly lower levels of reinnervation than autografts, but higher recovery and number of regenerated axons than transplants of isologous and syngeneic SCs, and than acellular guides. Thus, by combined developments on nerve guides, extracellular matrix components and cell transplantation, an artificial graft has been designed that allows axonal regeneration across long gaps to levels comparable with an autograft.     

Synergistic improvements in cell and axonal migration across sciatic nerve lesion gaps using bioresorbable filaments and heregulin-beta1

The success of entubulation for peripheral nerve regeneration is still limited, especially with long lesion gaps. In this study, we examined if regeneration could be enhanced by constructing implants to both align axonal growth and promote Schwann cell proliferation and migration. Silicone implants were used to bridge a 1.4-cm gap in the rat sciatic nerve. Adult female Sprague-Dawley rats were divided into four groups of tubes containing either 1) Matrigel; 2) Matrigel and heregulin; 3) Matrigel and poly(L-lactic acid) (PLLA) microfilaments; or 4) Matrigel, PLLA microfilaments, and heregulin. Ten weeks postimplantation, the number of axons and Schwann cells were measured at the distal end of implants. Implants with microfilaments displayed better tissue cable formation, increased Schwann cell migration, and regeneration of anti-calcitonin gene-related peptide-positive axons, but not RMDO95-positive axons compared with nonfilament-containing groups. Heregulin treatment caused an increase in Schwann cell number, but it demonstrated no significant improvement in either tissue cable formation or axon number. Extensive regeneration was observed through implants containing Matrigel, microfilaments, and heregulin, which induced significant improvements in the number and longitudinal organization of both Schwann cells and axons. These results indicate that physical guidance of microfilaments and the Schwann cell growth factor, heregulin, act synergistically to improve nerve regeneration across long lesion gaps.     

Low-intensity-ultrasound-accelerated nerve regeneration using cell-seeded poly(D,L-lactic acid-co-glycolic acid) conduits: an in vivo and in vitro study

This study investigated the effects of low intensity ultrasound on seeded Schwann cells within poly(DL-lactic acid-co-glycolic acid) (PLGA) conduits by in vitro and in vivo trials for peripheral nerve regeneration. The possible differences in the ultrasonic effects when using biodegradable and non-biodegradable materials as the conduits were also studied, using silicone rubber tubes as comparisons. In the in vitro study, seeded Schwann cells were cultured in serum deprivation culture medium that simulated the environment of mechanical trauma on injury nerve site. After 12, 24, and 48 h, only the PLGA conduit groups exposed to 0.05 W/cm(2), 3 min/treatment of ultrasound exhibited decreased LDH release and increased MTT values compared to the sham groups. Based on the results of the in vitro experiment in LDH and MTT testing, the silicone conduits with seeded Schwann cells group was ignored in the in vivo study. The PLGA nerve conduits seeded with Schwann cells (9 x 10(3) cells) were implanted to 15-mm right sciatic nerve defects in rats. Each conduit received 12 ultrasonic treatment sessions over 2 weeks after 1 day of rest. Ultrasound was applied as follows: frequency, 1MHz; intensity, 0.3 W/cm(2) (SATP); treatment, 5 min/day. Implanted graft specimens were harvested for histological analysis at 8 weeks following surgery. PLGA groups (with and without Schwann cells) treated with pulsed ultrasonic stimulation were found to have significantly greater number and area of regenerated axons at the mid-conduit of implanted grafts, as compared to the sham groups. Ultrasonic stimulation on silicone groups was found to induce a mass of fibrous tissues that covered the nerve conduits and retarded axon regeneration.     

Use of PLGA 90:10 scaffolds enriched with in vitro-differentiated neural cells for repairing rat sciatic nerve defects

Poly(lactic-co-glycolic acid) (PLGA) nerve tube guides, made of a novel proportion (90:10) of the two polymers, poly(L-lactide): poly(glycolide) and covered with a neural cell line differentiated in vitro, were tested in vivo for promoting nerve regeneration across a 10-mm gap of the rat sciatic nerve. Before in vivo testing, the PLGA 90:10 tubes were tested in vitro for water uptake and mass loss and compared with collagen sheets. The water uptake of the PLGA tubes was lower, and the mass loss was more rapid and higher than those of the collagen sheets when immersed in phosphate-buffered saline (PBS) solution. The pH values of immersing PBS did not change after soaking the collagen sheets and showed to be around 7.4. On the other hand, the pH values of PBS after soaking PLGA tubes decreased gradually during 10 days reaching values around 3.5. For the in vivo testing, 22 Sasco Sprague adult rats were divided into four groups--group 1: gap not reconstructed; group 2: gap reconstructed using an autologous nerve graft; group 3: gap reconstructed with PLGA 90:10 tube guides; group 4: gap reconstructed with PLGA 90:10 tube guides covered with neural cells differentiated in vitro. Motor and sensory functional recovery was evaluated throughout a healing period of 20 weeks using sciatic functional index, static sciatic index, extensor postural thrust, withdrawal reflex latency, and ankle kinematics. Stereological analysis was carried out on regenerated nerve fibers. Both motor and sensory functions improved significantly in the three experimental nerve repair groups, although the rate and extent of recovery was significantly higher in the group where the gap was reconstructed using the autologous graft. The presence of neural cells covering the inside of the PLGA tube guides did not make any difference in the functional recovery. By contrast, morphometric analysis showed that the introduction of N1E-115 cells inside PLGA 90:10 tube guides led to a significant lower number and size of regenerated nerve fibers, suggesting thus that this approach is not adequate for promoting peripheral nerve repair. Further studies are warranted to assess the role of other cellular systems as a foreseeable therapeutic strategy in peripheral nerve regeneration.     

Skin-derived Precursors as a Source of Progenitors for Cutaneous Nerve Regeneration

Peripheral nerves have the potential to regenerate axons and reinnervate end organs. Chronic denervation and disturbed nerve regeneration are thought to contribute to peripheral neuropathy, pain, and pruritus in the skin. The capacity of denervated distal nerves to support axonal regeneration requires proliferation by Schwann cells, which guide regenerating axons to their denervated targets. However, adult peripheral nerve Schwann cells do not retain a growth-permissive phenotype, as is required to produce new glia. Therefore, it is believed that following injury, mature Schwann cells dedifferentiate to a progenitor/stem cell phenotype to promote axonal regrowth. In this study, we show that skin-derived precursors (SKPs), a recently identified neural crest-related stem cell population in the dermis of skin, are an alternative source of progenitors for cutaneous nerve regeneration. Using in vivo and in vitro three-dimensional cutaneous nerve regeneration models, we show that the SKPs are neurotropic toward injured nerves and that they have a full capacity to differentiate into Schwann cells and promote axon regeneration. The identification of SKPs as a physiologic source of progenitors for cutaneous nerve regeneration in the skin, where SKPs physiologically reside, has important implications for understanding early cellular events in peripheral nerve regeneration. It also provides fertile ground for the elucidation of intrinsic and extrinsic factors within the nerve microenvironment that likely play essential roles in cutaneous nerve homeostasis. STEM Cells2012;30:2261-2270.     

The role of microstructured and interconnected pore channels in a collagen-based nerve guide on axonal regeneration in peripheral nerves

The use of bioengineered nerve guides as alternatives for autologous nerve transplantation (ANT) is a promising strategy for the repair of peripheral nerve defects. In the present investigation, we present a collagen-based micro-structured nerve guide (Perimaix) for the repair of 2 cm rat sciatic nerve defects. Perimaix is an open-porous biodegradable nerve guide containing continuous, longitudinally orientated channels for orientated nerve growth. The effects of these nerve guides on axon regeneration by six weeks after implantation have been compared with those of ANT. Investigation of the regenerated sciatic nerve indicated that Perimaix strongly supported directed axon regeneration. When seeded with cultivated rat Schwann cells (SC), the Perimaix nerve guide was found to be almost as supportive of axon regeneration as ANT. The use of SC from transgenic green-fluorescent-protein (GFP) rats allowed us to detect the viability of donor SC at 1 week and 6 weeks after transplantation. The GFP-positive SC were aligned in a columnar fashion within the longitudinally orientated micro-channels. This cellular arrangement was not only observed prior to implantation, but also at one week and 6 weeks after implantation. It may be concluded that Perimaix nerve guides hold great promise for the repair of peripheral nerve defects.     

Collagen filaments as a scaffold for nerve regeneration

This article describes repair of peripheral nerve defect using collagen filaments instead of tubes. Many tube-shaped nerve guides induce regeneration of severed peripheral nerve axons within a limited distance. Substantial regeneration of nerve axons has not been reported without a tubular conduit. Here we show the regeneration of peripheral nerve axons along filaments of collagen without a tube. Cables of collagen filaments were grafted to repair 20-mm defects of rat sciatic nerves. Nerve autografts and collagen tubes were grafted as controls. The mean number and the mean fiber diameter of regenerated myelinated axons were approximately 4800 and 3.3 microm in the distal end of the nerve autograft at 8 weeks postoperatively while in the distal end of the collagen-filaments nerve guide, they were approximately 5500 and 2.3 microm. Collagen tubes failed to bridge the nerve defect. Histologic studies suggest that nerve axons regenerated substantially along the collagen filaments.     

Rational Design of Contact Guiding,Neurotrophic Matrices for Peripheral Nerve Regeneration

Nerve guides filled with magnetically aligned hydrated gels of type I collagen have been shown to impart strong contact guidance cues to elongating neurites in vitro ¹⁵ and to increase the number of regenerating axons in vivo ⁸ relative to an isotropic collagen gel. We have formulated and analyzed a model to determine the conditions under which the target concentration of nerve growth factor (NGF) to support axonal growth can be sustained by entrapping either NGF-secreting cells or NGF-releasing polymer microspheres in the aligned gel. The equation describing NGF concentration with a distributed source term was solved after experimental determination of (1) the rate of NGF release from PLGA 85/15 microspheres, (2) the NGF diffusion coefficient in the gel and nerve guide membrane containing the gel, and (3) the maximum microsphere loading that does not compromise the magnetic alignment of collagen fibrils. We find that for a rat sciatic nerve, when using a 1 mm diameter nerve guide within a commercially available collagen membrane, the microsphere loading limit will prevent the construct's capacity to sustain the target NGF concentration of 1 ng/ml at two months when either wild type Schwann cells or PLGA 85/15 microspheres are used as the NGF source. This target concentration, however, will be maintained when transfected cells described in the literature to hypersecrete NGF are used, or when the microspheres are used if the permeability of the nerve guide membrane can be moderately decreased. For a human median nerve, when using a 5 mm diameter nerve guide within a commercially available membrane, the microspheres are capable of sustaining NGF concentrations above 1 ng/ml to at least 75 days without the need to decrease membrane permeability. © 2003 Biomedical Engineering Society. PAC2003: 8780Xa, 8718Sn, 8715Vv     

坐骨神经瓦勒变性大鼠许旺细胞生物学特性及分泌功能变化

背景：研究表明外周神经损伤后,许旺细胞在基底膜管内形成Bunger带,引导再生轴突延伸,但具体作用机制目前尚不清楚。 目的：观察大鼠坐骨神经损伤后瓦勒变性对许旺细胞生物学特性及分泌功能的影响。 方法：建立大鼠坐骨神经横切模型,分为坐骨神经瓦勒变性组(坐骨神经横断组)和手术对照组。采用神经段单酶消化法分离培养许旺细胞,光镜下观察细胞形态变化,S-100免疫荧光鉴定。取第1代许旺细胞,利用计数法绘制14 d内许旺细胞的生长曲线,MTT法检测14 d内许旺细胞增殖活性,酸性磷酸酶法检测许旺细胞黏附能力,ELISA法检测神经生长因子浓度。 结果与结论：坐骨神经段培养第14天,坐骨神经横断组神经段边缘可见大量许旺细胞,呈线形排列;手术对照组许旺细胞数量少,呈散在分布,两组许旺细胞S-100均呈阳性表达。许旺细胞传代培养第3天,两组许旺细胞均进入对数增长期,随时间延长,细胞数及细胞增殖吸光度值均呈上升趋势,坐骨神经横断组细胞数及增殖吸光度值明显高于手术对照组(P < 0.05);坐骨神经横断组许旺细胞黏附能力明显高于手术对照组(P < 0.05);ELISA法检测示,坐骨神经横断组神经生长因子浓度在培养第4,6,8,10,12,14天时均高于手术对照组(P < 0.05)。结果表明大鼠坐骨神经损伤后两三周,瓦勒变性对许旺细胞生物学功能具有显著影响,可诱导许旺细胞幼稚化,促使许旺细胞在短期内迅速分裂增殖,并分泌大量神经营养因子及细胞外黏附成分,为再生轴突的延伸提供适宜的神经微环境;并增加细胞黏附能力,为外周神经损伤修复提供适宜的神经微环境。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

VEGF enhances intraneural angiogenesis and improves nerve regeneration after axotomy

Whilst there is an increased understanding of the cell biology of nerve regeneration, it remains unclear whether there is a direct interrelationship between vascularisation and efficacy of nerve regeneration within a nerve conduit. To establish this is important as in clinical surgery peripheral nerve conduit grafting has been widely investigated as a possible alternative to the use of nerve autografts. The aim of this study was to assess whether vascular endothelial growth factor (VEGF), a highly specific endothelial cell mitogen, can enhance vascularisation and, indirectly, axonal regeneration within a silicone nerve regeneration chamber. Chambers containing VEGF (500–700 ng/ml) in a laminin‐based gel (Matrigel) were inserted into 1 cm rat sciatic nerve defects and nerve regeneration examined in relation to angiogenesis between 5 and 180 d. Longitudinal sections were stained with antibodies against endothelial cells (RECA‐1), axons (neurofilament) and Schwann cells (S‐100) to follow the progression of vascular and neural elements. Computerised image analysis demonstrated that the addition of VEGF significantly increased blood vessel penetration within the chamber from d 5, and by d 10 this correlated with an increase of axonal regeneration and Schwann cell migration. The pattern of increased nerve regeneration due to VEGF administration was maintained up to 180 d, when myelinated axon counts were increased by 78% compared with plain Matrigel control. Furthermore the dose‐response of blood vessel regeneration to VEGF was clearly reflected in the increase of axonal regrowth and Schwann cell proliferation, indicating the close relationship between regenerating nerves and blood vessels within the chamber. Target organ reinnervation was enhanced by VEGF at 180 d as measured through the recovery of gastrocnemius muscle weights and footpad axonal terminal density, the latter showing a significant increase over controls (P < 0.05). The results demonstrate an overall relationship between increased vascularisation and enhanced nerve regeneration within an acellular conduit, and highlight the interdependence of the 2 processes.     

Addition of fibronectin to alginate matrix improves peripheral nerve regeneration in tissue-engineered conduits

Schwann cell (SC) transplantation has been proposed to encourage peripheral nerve regeneration, but an optimal SC-carrying matrix would be needed. The aim of this study was to characterize how the addition of fibronectin to alginate would affect the outcome of nerve regeneration promoted by Schwann cells embedded in this matrix. Genetically labeled rat SCs were obtained by lacZ gene transduction. SCs were suspended in alginate hydrogel matrix with/without addition of liquid fibronectin, and their viability and growth in the different types of matrices were assessed in vitro by AlamarBlue assay. In vivo assessment of SC transplantation in the matrix was carried out with poly-3-hydroxybutyrate (PHB) conduits to bridge a sciatic nerve gap. The grafted conduits were harvested at 2, 3, and 6 weeks and assessed for the presence of labeled SCs in relation to regrowing axons. The amount and rate of axonal regeneration were assessed by quantitative immunohistochemistry. Addition of fibronectin to alginate hydrogel improved SC viability and growth profile in vitro. X-Gal staining confirmed that SCs transplanted in PHB conduits were viable throughout the time course, and that the labeled SCs were clearly associated with regenerating axons. The regeneration rate was enhanced when liquid fibronectin was added to the alginate matrix. Furthermore, the presence of SCs also enhanced regeneration and there was an additive effect when both SCs and fibronectin were combined with alginate. In conclusion, the addition of fibronectin to alginate hydrogel matrix contributed to improve nerve regeneration, supporting SC viability and augmenting their effect on axonal growth when transplanted in a bioengineered nerve conduit.     

Laminin和Fibronectin对周围神经中的再生轴突及非神经元细胞的作用和影响

本文用近交克隆系(Close cloned)大鼠研究了内源性Laminin和Fibronectin对周围神经的再生轴突及非神经元的雪旺氏细胞、成纤维细胞等的作用和影响。将从供体鼠获得的坐骨神中段经冷冻、加热处理后再用Laminin或Fibronectin抗血清处理;对照组则用正常兔血清处理。将处理后的神经段(10mm)分别植入三组受体鼠体内,术后不同时期取材,电镜观察。术后15天,抗Laminin组和抗Fibronectin组的轴突总数,只有对照组的50%左右。对照组和抗Fibronectin组约90%的轴突走在基底膜管内,而抗Laminin组,再生轴突似不能识别基底膜而生长在基底膜管外。轴突生长总是先于雪旺氏细胞的迁移,而后雪旺氏细胞才生长粘附并包绕轴突。成纤维细胞能够识别伴随有雪旺氏细胞的轴突,并形成神经周膜包绕这些轴突,但它们不能识别空陷的基底膜管,只有当组织中缺乏Fibronectin时,增生的成纤维细胞方在基底膜管外形成神经周膜。在缺乏Laminin的神经段内,巨噬细胞不仅大量增生,还有包随走在基底膜管外单个轴突的趋向。这些结果提示在神经再生的早期,内源性Laminin和Fibronectin不但调节再生神经纤维的生长,对在神经损伤和再生中起重要作用的巨噬细胞和成纤维细胞也有积极的影响。     

Next generation nerve guides: materials, fabrication, growth factors, and cell delivery

Nerve guides are increasingly being used surgically to repair acute peripheral nerve injuries. This is not only due to an increase in the number of commercially available devices, but also clinical acceptance. However, regeneration distance is typically limited to 20-25 mm, in part due to the basic tubular design. A number of experimental studies have shown improvements in nerve regeneration distance when conduits incorporate coatings, internal scaffolds, topographical cues, or the delivery of support cells. Current studies on designing nerve guides for maximizing nerve regeneration focus both on cell-containing and cell-free devices, the latter being clinically attractive as "off the shelf" products. Arguably better results are obtained when conduits are used in conjunction with support cells (e.g., Schwann cells or stem cells) that can improve regeneration distance and speed of repair, and provide informative experimental data on how Schwann and neuronal cells respond in regenerating injured nerves. In this review we discuss the range of current nerve guides commercially available and appraise experimental studies in the context of the future design of nerve guides for clinical use.     

Sensory neurons and Schwann cells during pharmacologic stimulation of the nerve regeneration

Using the model of rat sciatic nerve transection and crush injury we studied influence of pyrimidine derivative xymedon on efficacy of regeneration of myelinated axons, number and phenotype of surviving sensory neurons (expressing GAP-43 and Bcl-2) and Schwann cells (S100, GAP-43, PCNA) on the 7th, 15th, 30th, 60th and 90th day after nerve injury. We found out that xymedon counteracts posttraumatic death of sensory neurons, stimulates regeneration of myelinated fibres and proliferation of Schwann cells.     

Effects of different types of injury to the inferior alveolar nerve on the behavior of Schwann cells during the regeneration of periodontal nerve fibers of rat incisor

The present study reports on different regeneration patterns of axons and Schwann cells in the periodontal ligament of the rat incisor using immunohistochemistry of protein gene product 9.5 (PGP 9.5) and S-100 protein. Three kinds of injury (transection, crush and segmental resection) were applied to the inferior alveolar nerve. In normal animals, PGP 9.5- and S-100-immunoreactivities were detected in the axons and Schwann cell elements of periodontal Ruffini endings, respectively. They were restricted to the alveolus-related part, occurring only rarely in the tooth-related part and in the shear zone (the border between the alveolus-related and tooth-related parts). Both transection and segmental resection caused the complete disappearance of PGP 9.5-immunoreactive nerve fibers in the periodontal ligament, while a small number of them could be found following the crush injury. Regenerating PGP 9.5-reactive nerve fibers appeared at 5 days and 21 days following the transection and segmental resection, respectively. The regeneration of periodontal nerve fibers completed in a period of 21-28 days and 14-21 days following the transection and crush, respectively, but was not completed even at 56 days following the segmental resection. The behavior of Schwann cells during regeneration was similar after the different nerve injuries; spindle-shaped S-100-immunoreactive cells, presumably Schwann cells, appeared in the shear zone and the tooth-related part. These cells disappeared 5-7 days prior to the completion of the regeneration of axonal elements of the periodontal ligament following the transection and crush. Following the segmental resection, in contrast, spindle-shaped S-100-positive cells disappeared from the tooth-related part at 42 days, although the axonal regeneration of periodontal Ruffini endings proceeded even until 56 days. We thus conclude that the duration of the migration of Schwann cells depends on the state of the regeneration of axons.