富血小板血浆诱导骨髓间充质干细胞结合化学萃取去细胞神经修复坐骨神经缺损

背景：周围神经损伤早期许旺细胞尚未大量分裂增殖,此时由于解剖连续性的中断,通过轴浆逆向运输提供的营养因子骤减,缺乏神经营养因子支持的神经元有可能死亡,从而使周围神经不能再生或再生乏力。 目的：观察植入经富血小板血浆诱导的骨髓间充质干细胞结合去细胞神经修复坐骨神经缺损的效果。 方法：取新西兰大耳白兔制备坐骨神经缺损模型,随机抽签法分成4组：去细胞神经组,移植同种异体去细胞神经;骨髓间充质干细胞组,移植同种异体骨髓间充质干细胞结合化学萃取的同种异体去细胞神经：经诱导骨髓间充质干细胞组,移植经富血小板血浆诱导的同种异体骨髓间充质干细胞结合化学萃取的同种异体去细胞神经;自体神经组,移植自体神经。术后进行形态学观察与靶肌肉肌湿质量恢复率、运动神经传导速度、轴突直径和髓鞘厚度的检测。 结果与结论：经富血小板血浆诱导的骨髓间充质干细胞结合化学萃取的去细胞神经移植修复神经的靶肌肉肌湿质量恢复率、运动神经传导速度、轴突直径和髓鞘厚度及形态学观察明显优于移植单纯化学萃取的去细胞神经与骨髓间充质干细胞结合化学萃取的去细胞神经的效果,而与移植自体神经修复结果相似。说明经诱导后的骨髓间充质干细胞在体内具有许旺细胞的部分功能,可作为组织工程化外周神经的种子细胞,用于周围神经缺损的修复。     

Angiogenesis in tissue-engineered nerves evaluated objectively using MICROFIL perfusion and micro-CT scanning

Angiogenesis is a key process in regenerative medicine generally, as well as in the specific field of nerve regeneration. However, no convenient and objective method for evaluating the angiogenesis of tissue-engineered nerves has been reported. In this study, tissue-engineered nerves were constructed in vitro using Schwann cells differentiated from rat skin-derived precursors as supporting cells and chitosan nerve conduits combined with silk fibroin fibers as scaffolds to bridge 10-mm sciatic nerve defects in rats. Four weeks after surgery, three-dimensional blood vessel reconstructions were made through MICROFIL perfusion and micro-CT scanning, and parameter analysis of the tissue-engineered nerves was performed. New blood vessels grew into the tissue-engineered nerves from three main directions: the proximal end, the distal end, and the middle. The parameter analysis of the three-dimensional blood vessel images yielded several parameters, including the number, diameter, connection, and spatial distribution of blood vessels. The new blood vessels were mainly capillaries and microvessels, with diameters ranging from 9 to 301 μm. The blood vessels with diameters from 27 to 155 μm accounted for 82.84% of the new vessels. The microvessels in the tissue-engineered nerves implanted in vivo were relatively well-identified using the MICROFIL perfusion and micro-CT scanning method, which allows the evaluation and comparison of differences and changes of angiogenesis in tissue-engineered nerves implanted in vivo.     

Tissue-engineered rhesus monkey nerve gratfs for the repair of long ulnar nerve defects：similar outcomes to autologous nerve gratfs

Acellular nerve allogratfs can help preserve normal nerve structure and extracellular matrix composition. These allogratfs have low immu-nogenicity and are more readily available than autologous nerves for the repair of long-segment peripheral nerve defects. In this study, we repaired a 40-mm ulnar nerve defect in rhesus monkeys with tissue-engineered peripheral nerve, and compared the outcome with that of autogratf. The gratf was prepared using a chemical extract from adult rhesus monkeys and seeded with allogeneic Schwann cells. Pathomo-rphology, electromyogram and immunohistochemistry ifndings revealed the absence of palmar erosion or ulcers, and that the morphology and elasticity of the hypothenar eminence were normal 5 months postoperatively. There were no signiifcant differences in the mean peak compound muscle action potential, the mean nerve conduction velocity, or the number of neuroiflaments between the experimental and control groups. However, outcome was signiifcantly better in the experimental group than in the blank group. These ifndings suggest that chemically extracted allogeneic nerve seeded with autologous Schwann cells can repair 40-mm ulnar nerve defects in the rhesus monkey. The outcomes are similar to those obtained with autologous nerve gratf.     

Skeletal muscle-derived cells repair peripheral nerve defects in mice

Skeletal muscle-derived cells have strong secretory function,while skeletal muscle-derived stem cells,which are included in muscle-derived cells,can differentiate into Schwann cell-like cells and other cell types.However,the effect of muscle-derived cells on peripheral nerve defects has not been reported.In this study,5-mm-long nerve defects were created in the right sciatic nerves of mice to construct a peripheral nerve defect model.Adult female C57BL/6 mice were randomly divided into four groups.For the muscle-derived cell group,muscle-derived cells were injected into the catheter after the cut nerve ends were bridged with a polyurethane catheter.For external oblique muscle-fabricated nerve conduit and polyurethane groups,an external oblique muscle-fabricated nerve conduit or polyurethane catheter was used to bridge the cut nerve ends,respectively.For the sham group,the sciatic nerves on the right side were separated but not excised.At 8 and 12 weeks post-surgery,distributions of axons and myelin sheaths were observed,and the nerve diameter was calculated using immunofluorescence staining.The number,diameter,and thickness of myelinated nerve fibers were detected by toluidine blue staining and transmission electron microscopy.Muscle fiber area ratios were calculated by Masson’s trichrome staining of gastrocnemius muscle sections.Sciatic functional index was recorded using walking footprint analysis at 4,8,and 12 weeks after operation.The results showed that,at 8 and 12 weeks after surgery,myelin sheaths and axons of regenerating nerves were evenly distributed in the muscle-derived cell group.The number,diameter,and myelin sheath thickness of myelinated nerve fibers,as well as gastrocnemius muscle wet weight and muscle area ratio,were significantly higher in the muscle-derived cell group compared with the polyurethane group.At 4,8,and 12 weeks post-surgery,sciatic functional index was notably increased in the muscle-derived cell group compared with the polyurethane group.These criteria of the muscle-derived cell group were not significantly different from the external oblique muscle-fabricated nerve conduit group.Collectively,these data suggest that muscle-derived cells effectively accelerated peripheral nerve regeneration.This study was approved by the Animal Ethics Committee of Plastic Surgery Hospital,Chinese Academy of Medical Sciences(approval No.040)on September 28,2016.     

富血小板血浆诱导骨髓间充质干细胞修复坐骨神经缺损：并与自体神经修复的效果相比较

目的：通过植入经PRP诱导的BMSCs结合化学萃取的去细胞神经修复坐骨神经缺损,观察其对周围神经的修复作用。 方法：32只新西兰大耳白兔,随机分成4组,即单纯的化学萃取的去细胞神经、BMSCs结合化学萃取的去细胞神经、经PRP诱导的BMSCs结合化学萃取的去细胞神和自体神经修复坐骨神经缺损,检测指标包括形态学观察、靶肌肉肌湿重恢复率、运动神经传导速度（MNCV）及轴突直径和髓鞘厚度等。 结果：结果显示,靶肌肉肌湿重恢复率、MNCV、轴突直径和髓鞘厚度和形态学观察在经PRP诱导的BMSCs结合化学萃取的去细胞神经组明显优于单纯的化学萃取的去细胞神经组和BMSCs结合化学萃取的去细胞神经组,而与自体神经修复组结果相似。 结论：经诱导后的BMSCs在体内具有SC的部分功能,可作为组织工程化外周神经的种子细胞,用于周围神经缺损的修复。     

Bridging sciatic nerve gap using tissue-engineered nerves constructed with neural tissue-committed stem cells derived from bone marrow

BACKGROUND: Schwann cells are the most commonly used cells for tissue-engineered nerves. However, autologous Schwann cells are of limited use in a clinical context, and allogeneic Schwann cells induce immunological rejections. Cells that do not induce immunological rejections and that are relatively easy to acquire are urgently needed for transplantation.OBJECTIVE: To bridge sciatic nerve defects using tissue engineered nerves constructed with neural tissue-committed stem cells (NTCSCs) derived from bone marrow; to observe morphology and function of rat nerves following bridging; to determine the effect of autologous nerve transplantation, which serves as the gold standard for evaluating efficacy of tissue-engineered nerves.DESIGN, TIME AND SETTING: This randomized, controlled, animal experiment was performed in the Anatomical laboratory and Biomedical Institute of the Second Military Medical University of Chinese PLA between September 2004 and April 2006.MATERIALS: Five Sprague Dawley rats, aged 1 month and weighing 100-150 g, were used for cell culture. Sixty Sprague Dawiey rats aged 3 months and weighing 220-250 g, were used to establish neurological defect models. Nestin, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), and S-100 antibodies were provided by Santa Cruz Biotechnology, Inc., USA. Acellular nerve grafts were derived from dogs.METHODS: All rats, each with 1-cm gap created in the right sciatic nerve, were randomly assigned to three groups. Each group comprised 20 rats. Autograft nerve transplantation group: the severed 1-cm length nerve segment was reverted, but with the two ends exchanged; the proximal segment was sutured to the distal sciatic nerve stump and the distal segment to the proximal stump. Blank nerve scaffold transplantation group: a 1-cm length acellular nerve graft was used to bridge the sciatic nerve gap. NTCSC engineered nerve transplantation group: a 1-cm length acellular nerve graft, in which NTCSCs were inoculated, was used to bridge the sciatic nerve gap.MAIN OUTCOME MEASURES: Following surgery, sciatic nerve functional index and electrophysiology functions were evaluated for nerve conduction function, including conduction latency, conduction velocity, and action potential peak. Horseradish peroxidase (HRP, 20%) was injected into the gastrocnemius muscle to retrogradely label the L4 and L5 nerve ganglions, as well as neurons in the anterior horn of the spinal cord, in the three groups. Positive expression of nestin, NSE, GFAP, and S-100 were determined using an immunofluorescence double-labeling method.RESULTS: NTCSCs differentiated into neuronal-like cells and glial-like cells within 12 weeks after NTCSC engineered nerve transplantation. HRP retrograde tracing displayed a large amount of HRP-labeted neurons in L4-5 nerve ganglions, as well as the anterior horn of the spinal cord, in both the autograft nerve transplantation and the NTCSC engineered nerve transplantation groups. However, few HRP-labeled neurons were detected in the blank nerve scaffold transplantation group. Nerve bridges in the autograft nerve transplantation and NTCSC engineered nerve transplantation groups exhibited similar morphology to normal nerves. Neither fractures or broken nerve bridges nor neuromas were found after bridging the sciatic nerve gap with NTCSCs-inoculated acellular nerve graft, indicating repair. Conduction latency, action potential, and conduction velocity in the NTCSC engineered nerve transplantation group were identical to the autograft nerve transplantation group (P>0.05), but significantly different from the blank nerve scaffold transplantation group (P<0.05). CONCLUSION: NTCSC tissue-engineered nerves were able to repair injured nerves and facilitated restoration of nerve conduction function, similar to autograff nerve transplantation.     

Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration.

At present, clinical strategies to repair injured peripheral nerve concentrate on efforts to attain primary suture of the cut nerve ends. If this is not possible, autografts are used to unite the separated nerve segments. Both strategies are based on the recognition that the Schwann cells resident in the peripheral nerve trunk play a crucial role in the regenerative process. Neither strategy may be feasible, however, in extensive or multiple injuries because the amount of autograft material is limited, and allografts are subject to immune rejection. Artificially produced nerve bridges constructed of autologous Schwann cells seeded in guidance channels could be used to overcome these limitations. In the present experiments, the potential of Schwann cells derived from adult nerves and seeded in permselective guidance channels to promote neurite regeneration across an 8 mm nerve gap was evaluated in transected rat sciatic nerves. Immunological sequalae were evaluated by comparing Schwann cells from syngeneic and heterologous rat strains. Schwann cells from either adult outbred (Sprague-Dawley, CD) rats or inbred (Fisher, F) rats were suspended in a Matrigel solution at a density of 80 x 10(6) cells/ml (CD) or 40, 80, or 120 x 10(6) cells/ml (F-40, F-80, and F-120 channels, respectively). Channels containing Schwann cells were compared to sciatic nerve autografts, empty channels, or channels filled with Matrigel alone. One day after seeding permselective synthetic guidance channels with a Schwann cell suspension, a central cable of Schwann cells oriented along the axis of the tube was formed due to syneresis of the hydrogel. By 3 weeks postimplantation, regenerating axons had grown into all channels and autografts. Sciatic nerve autografts supported extensive regeneration, containing 4-5 x 10(4) myelinated axons at the graft midpoint. The ability of channels containing syngeneic Schwann cells to foster regeneration was dependent on the Schwann cell seeding density. At the channel's midpoint, the myelinated axon population in F-120 tubes was intermediate between that in sciatic nerve autografts and F-80 channels, and was significantly higher than in F-40 or control channels. The nerve cable in Schwann cell-containing tubes consisted of larger, more organotypic fascicles than acellular control channels. In contrast, heterologous (CD) Schwann cells elicited a strong immune reaction that impeded nerve regeneration. The present study shows that cultured adult syngeneic Schwann cells seeded in permselective synthetic guidance channels support extensive peripheral nerve regeneration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tissue-engineered rhesus monkey nerve grafts for the repair of long ulnar nerve defects:similar outcomes to autologous nerve grafts

Acellular nerve allografts can help preserve normal nerve structure and extracellular matrix composition.These allografts have low immunogenicity and are more readily available than autologous nerves for the repair of long-segment peripheral nerve defects.In this study,we repaired a 40-mm ulnar nerve defect in rhesus monkeys with tissue-engineered peripheral nerve,and compared the outcome with that of autograft.The graft was prepared using a chemical extract from adult rhesus monkeys and seeded with allogeneic Schwann cells.Pathomorphology,electromyogram and immunohistochemistry findings revealed the absence of palmar erosion or ulcers,and that the morphology and elasticity of the hypothenar eminence were normal 5 months postoperatively.There were no significant differences in the mean peak compound muscle action potential,the mean nerve conduction velocity,or the number of neurofilaments between the experimental and control groups.However,outcome was significantly better in the experimental group than in the blank group.These findings suggest that chemically extracted allogeneic nerve seeded with autologous Schwann cells can repair 40-mm ulnar nerve defects in the rhesus monkey.The outcomes are similar to those obtained with autologous nerve graft.     

Comparison of SB-SDS and other decellularization methods for the acellular nerve graft: Biological evaluation and nerve repair in vitro and in vivo

We aimed to compare the performance of acellular nerves prepared by different decellularization methods, screening out the optimal decellularization protocol, repairing the sciatic nerve defects in rats by the allogeneic transplantation, and evaluating the effect of regenerative nerve on the function reconstruction. The Sondell, SB-SDS, TnBP, and the high/low permeation methods were used to decellularize donor nerves. Nerves without any treatment were as the control group. The histological results were evaluated by HE staining and toluidine blue (TB) staining. The proliferation activity of L929 cells was detected by CCK-8 assay. The adhesion of Schwann cells was observed and quantified by SEM. Balb/c mice were used to evaluate the cellular and humoral immunogenicity of the nerve scaffolds. The rat sciatic nerve defect model was applied to observe the repair effect of acellular nerve scaffold in vivo. To SB-SDS group, it remained the original state of the nerves, with no observed nucleus and axons, the neurotoxicity grade detected by CCK-8 being almost 0, and it kept the largest number of Schwann cells adhered to the acellular nerve and the better morphology. Further, it showed that the selected SB-SDS rats acellular nerve scaffold could promote the nerve repair of the rats by HE staining and TB staining. We could conclude that the acellular nerve matrix prepared by the SB-SDS method effectively removes the cellular components in the nerve tissue and retains the main components of the extracellular matrix of the nerve tissue, whose rats decellularized nerve scaffold could promote the sciatic nerve repair better.     

The role of extracellular matrix in peripheral nerve regeneration: a wound chamber study

Summary A wound chamber model was used for the study of the interaction between axon, Schwann cell and extracellular matrix during peripheral nerve regeneration. Impermeable silicone tubes, 8 mm long and 1.4 mm in internal diameter were sutured to transected rat sciatic nerve and the contents of the tubes were removed at intervals for chemical, histological, immunocytochemical and electron microscopic studies. There was an initial phase of fluid accumulation and the formation of a fibrin/fibronectin clot or cable which connected the cut ends of the nerve. The chamber fluid was shown to have a protein profile similar to that of rat serum. Schwann cells, endothelial cells and fibroblasts migrated first into the cable, apparently mediated by cell-fibrin interaction. Axons buried within the Schwann cell cytoplasm were led into the cable but an axon-fibrin interaction was not observed. After 1 week, the fibrin matrix underwent dissolution, with replacement by collagen. This marked the onset of myelination and the organization of nerve fibers into fascicles. The findings from the present study suggest that the interactions between axon and Schwann cell and between Schwann cell and a changing extracellular matrix are the essential driving force in nerve growth and differentiation during peripheral nerve regeneration.Supported by a grant from the National Science Council of R. O. C. (NSC 80-0412-B075-67)     

无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经修复坐骨神经缺损

背景：作者前期已经成功将无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经,并证明可以促进周围神经再生。 目的：构建组织工程人工神经,观察和验证桥接大鼠坐骨神经缺损后的神经功能恢复情况。 方法：成年雄性SD大鼠60只构建大鼠坐骨神经15 mm缺损模型。随机分成3组,每组20只。桥接大鼠坐骨神经缺损,实验组采用组织工程人工神经,空白对照组采用无细胞组织工程神经支架,自体神经对照组采用自体神经移植。桥接后12周通过大体观察、胫骨前肌湿质量、组织学等方法分析坐骨神经组织学及功能恢复情况。 结果与结论：桥接术后12周：实验组大鼠肢体可以支撑着地,钳夹大鼠手术侧足底皮肤出现逃避反射,足底皮肤s-100蛋白染色呈阳性反应。实验组与自体神经移植组胫骨前肌湿质量比差异无显著性意义(P > 0.05)。实验组辣根过氧化物酶逆行示踪实验显示脊髓、后根神经节均可见数量不等的辣根过氧化物酶标记阳性细胞。实验组移植物与自体神经移植组有髓神经纤维数、髓鞘厚度、神经组织面积比较差异无显著性意义。实验结果验证了无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经修复大鼠坐骨神经缺损,可以促进神经组织学的修复重建和功能的恢复。     

Influence of insulin-like growth factor-I (IGF-I) on nerve autografts and tissue-engineered nerve grafts

To overcome the problems of limited donor nerves for nerve reconstruction, we established nerve grafts made from cultured Schwann cells and basal lamina from acellular muscle and used them to bridge a 2-cm defect of the rat sciatic nerve. Due to their basal lamina and to viable Schwann cells, these grafts allow regeneration that is comparable to autologous nerve grafts. In order to enhance regeneration, insulin-like growth factor (IGF-I) was locally applied via osmotic pumps. Autologous nerve grafts with and without IGF-I served as controls. Muscle weight ratio was significantly increased in the autograft group treated with IGF-I compared to the group with no treatment; no effect was evident in the tissue-engineered grafts. Autografts with IGF-I application revealed a significantly increased axon count and an improved g-ratio as indicator for "maturity" of axons compared to autografts without IGF-I. IGF-I application to the engineered grafts resulted in a decreased axon count compared to grafts without IGF-I. The g-ratio, however, revealed no significant difference between the groups. Local administration of IGF-I improves axonal regeneration in regular nerve grafts, but not in tissue-engineered grafts. Seemingly, in these grafts the interactive feedback mechanisms of neuron, glial cell, and extracellular matrix are not established, and IGF-I cannot exert its action as a pleiotrophic signal.     

A quantitative morphometric analysis of rat spinal cord remyelination following transplantation of allogenic Schwann cells

Quantitative morphometric techniques were used to assess the extent and pattern of remyelination produced by transplanting allogenic Schwann cells into demyelinated lesions in adult rat spinal cords. The effects of donor age, prior culturing of donor cells, prior lesioning of donor nerves, and host immunosuppression were evaluated by transplanting suspensions of 30,000 acutely dissociated or cultured Schwann cells from neonatal, young adult, or aged adult rat sciatic nerves into X-irradiation and ethidium bromide-induced demyelinated dorsal column lesions, with or without co-transplantation of neonatal optic nerve astrocytes. Three weeks after transplantation, spinal cords were processed for histological analysis. Under all Schwann cell transplant protocols, large areas containing many Schwann cell-like myelinated axon profiles could be readily observed throughout most of the lesion length. Within these "myelin-rich" regions, the vast majority of detectable axons showed a peripheral-like pattern of myelination. However, interaxonal spacing also increased, resulting in densities of myelinated axons that were more similar to peripheral nerve than intact dorsal columns. Freshly isolated Schwann cells remyelinated more axonal length than cultured Schwann cells, and cells from younger donors remyelinated slightly more axon length than cells from older donors, but all Schwann cell transplant protocols remyelinated tens of thousands of millimeters of axon length and remyelinated axons at similar densities. These results indicate that Schwann cells prepared under a variety of conditions are capable of eliciting remyelination, but that the density of remyelinated axons is much lower than the myelinated axon density in intact spinal cords.     

Growth of axons into developing muscles of the chick forelimb is preceded by cells that stain with Schwann cell antibodies

A study has been made of the development of limb and muscle nerves in relation to the first appearance of Schwann cells in the flexor digitorum profundus (fdp) and flexor carpi ulnaris (fcu) muscles of the avian forelimb. Schwann cells were identified by immunofluorescent techniques with antibodies to the glycoprotein HNK-1. Myotubes and nerves were identified by using antibodies to myosin and to neurofilament, respectively. At stage 24/25 the brachialis longus inferior (Bli n) and superior (Bls n) nerve trunks within proximal regions of the forelimb were surrounded by Schwann cells. These cells extended in a column for a distance of approximately 100 microns beyond the growing ends of nerves. At stage 26 both interosseus nerve (in n) and the medial-ulnar nerve (m-u n) had formed from the Bli n; each of these branches was surrounded by Schwann cells, which again extended approximately 100 microns beyond the growing ends of the nerves. By stage 26/27 the fdp and fcu muscles were clearly delineated by groups of myotubes. No nerves were detected within these groups; however, Schwann cells were observed between the myotubes. At stage 27 axons had left the in n and m-u n and grown into the fdp and fcu muscles, respectively. These axons were surrounded by Schwann cells. The present observations show that Schwann cells are located ahead of the main limb and muscle nerves as they grow into the fdp and fcu muscles of the limb. It is possible that these Schwann cells play a role in guiding nerves to their correct muscles in the developing chick forelimb.     

以Triton X-100和脱氧胆酸钠为萃取剂制备兔脱细胞神经基质：有最佳时间吗？**

背景：与其他脱细胞神经基质制备方法相比,化学萃取的去细胞同种异系(体)神经组织内细胞成分可以较完全地清除,进一步减少了发生免疫排斥反应的可能性,并能较好地保留神经支架的完整性,但制备过程中的相关问题有许多尚需讨论。 目的：应用Triton X-100和脱氧胆酸钠化学萃取剂处理新西兰大白兔的面神经,提出脱细胞神经基质制备的所需试剂及最佳时间。 设计、时间及地点：随机分组,对比观察细胞学实验,于2009-02/06在滨州医学院附属医院口腔科学实验室完成。 材料：3月龄新西兰大白兔15 只,体质量2.5~3.0 kg。Triton X-100、脱氧胆酸钠由美国Sigma 公司提供。 方法：取兔双侧面神经,在手术显微镜下去除神经表面的脂肪组织和神经外膜,将其分为每段10 mm长,共66条。将66条神经随机分成11 组,每组6条。除正常对照组外,其余10 组均放入培养皿中,先用蒸馏水室温下浸浴12 h ,以使细胞和髓鞘在低渗液体中膨胀,使部分细胞被胀破;然后将其中5组置于3% Triton X-100 12,24,36,48,60 h,室温下振荡,另外5组用3%Triton X-100和4%脱氧胆酸钠先后作用12 h(为1个周期),室温下反复振荡1~5个周期。 主要观察指标：常规苏木精-伊红染色,光镜下观察去细胞程度及纤维管道结构的完整性。 结果：单独应用Triton X-100处理神经即使作用60 h,仍不能将神经中的所有细胞成分去除,且许旺细胞基底膜有较大破坏;Triton X-100配合脱氧胆酸钠处理处理2个周期,可有效地去除神经中的细胞成分及神经纤维髓鞘和轴突,保留完整的许旺细胞基底膜,周边可见神经外膜和束膜。 结论：大白兔面神经经 Triton X-100和脱氧胆酸钠室温处理2个周期,并不停地振荡,可完全去除神经组织中的所有细胞成分,保留完整的许旺细胞基底膜,得到脱细胞的神经基质。 关键词：脱细胞神经;制备条件;基质 doi:10.3969/j.issn.1673-8225.2009.47.007     

Spatial-Temporal progress of peripheral nerve regeneration within a silicone chamber: Parameters for a bioassay

The spatial-temporal progress of peripheral nerve regeneration across a 10-mmgap within a silicone chamber was examined with the light and electron microscope at 2-mm intervals. A coaxial, fibrin matrix was observed at 1 week with a proximal-distal narrowing that extended beyond the midpoint of the chamber. At 2 weeks, Schwann cells, fibroblasts, and endothelial cells had migrated into the matrix from both nerve stumps. There was a delay of 7–14 days after nerve transection and chamber implantation before regenerating axons appeared in the chamber. At 2 weeks, nonmyelinated axons were seen only in the proximal 1–5 mm of the chamber in association with Schwann cells. Axons reached the distal stump by 3 weeks and a proximal-distal gradient of myelination was observed. These observations define the parameters of a morphologic assay for regeneration in this chamber model which can be used to investigate cellular and molecular mechanisms underlying the success of peripheral nerve regeneration.     

Cartilage oligomeric matrix protein enhances the vascularization of acellular nerves

Vascularization of acellular nerves has been shown to contribute to nerve bridging. In this study, we used a 10-mm sciatic nerve defect model in rats to determine whether cartilage oligomeric matrix protein enhances the vascularization of injured acellular nerves. The rat nerve defects were treated with acellular nerve grafting (control group) alone or acellular nerve grafting combined with intraperitoneal injection of cartilage oligomeric matrix protein (experimental group). As shown through two-dimensional imaging, the vessels began to invade into the acellular nerve graft from both anastomotic ends at day 7 post-operation, and gradually covered the entire graft at day 21. The vascular density, vascular area, and the velocity of revascularization in the experimental group were all higher than those in the control group. These results indicate that cartilage oligomeric matrix protein enhances the vascularization of acellular nerves.     

Novel technique for peripheral nerve reconstruction in the absence of an artificial conduit

The purpose of this study is to promote nerve regeneration across a peripheral nerve gap, using a biologic, tissue-engineered nerve (TEN), containing a high density of viable Schwann cells (SCs) in the absence of supportive foreign materials and a tubular system. Isolated SCs from adult rat sciatic nerve were seeded onto biodegradable constructs and implanted into the backs of nude mice to create TENs. Six weeks later, the constructs were harvested, implanted into surgically created sciatic nerve gaps in rats without supportive artificial conduits and compared with both an autograft group and a silicone conduit group using SCs. Two months later, functional assessment was evaluated by walking track analysis and the implanted lesions were imaged by transmission electron microscopy. The axonal number and sciatic function index of the TEN were significantly higher than those of the silicone group and achieved a comparable level to the autograft group. The results indicate that the large number of SCs within their own extracellular matrix appeared sufficient to enable neuronal growth across a nerve gap in the absence of an artificial conduit and that these circumstances may have a positive effect on the supplement of growth factors from the surrounding tissues of implanted TEN.     

Schwann cell endocytosis: a role in nerve regeneration?

Schwann cell plasma membrane vesicles have been shown to increase in numerical density after nerve injury but their function is unclear. In this study, ultrastructural tracers were micro-injected in vivo into crushed rat sciatic nerves after various time intervals to ascertain whether plasma membrane vesicles of Schwann cells are involved in the uptake and utilization of molecules from the endoneurium during axonal regeneration and remyelination. Horseradish peroxidase (HRP), a tracer of fluid-phase endocytosis, was taken up by macrophages and fibroblasts but remained external to Schwann cells throughout the study. After 14-16 days of crush injury, HRP was present within vessel lumina and in cytoplasmic vesicles of pericytes and vascular endothelia. Low-density lipoprotein-gold, which is primarily internalized by receptor-mediated endocytosis, and bovine serum albumin-gold, proposed as a tracer for fluid-phase endocytosis, were internalized by macrophages and fibroblasts but were not taken up by Schwann cells. Although Schwann cells formed pits in the plasma membrane and vesicles were evident in the cytoplasm, none of the tracers used were internalized by Schwann cells. It is suggested that Schwann cell plasmalemmal and cytoplasmic vesicles have a cellular role unrelated to endocytosis or alternatively the Schwann cell basal lamina may function as a diffusion barrier to the tracers employed.     

Synergistic effects of G‐CSF and bone marrow stromal cells on nerve regeneration with acellular nerve xenografts

Peripheral nerve defects result in severe denervation presenting sensory and motor functional incapacitation. Currently, a satisfactory therapeutic treatment promoting the repair of injured nerves is not available. As shown in our previous study, acellular nerve xenografts (ANX) implanted with bone marrow stromal cells (BMSCs) replaced allografts and promoted nerve regeneration. Additionally, granulocyte‐colony stimulating factor (G‐CSF) has been proven to mobilize supplemental cells and enhance vascularization in the niche. Thus, the study aimed to explore whether the combination of G‐CSF and BMSC‐laden ANX exhibited a synergistic effect. Adult Sprague‐Dawley (SD) rats were randomly divided into five groups: ANX group, ANX combined with G‐CSF group, BMSCs‐laden ANX group, BMSCs‐laden ANX combined with G‐CSF group and autograft group. Electrophysiological parameters and weight ratios of tibialis anterior muscles were detected at 8 weeks post‐transplantation. The morphology of the regenerated nerves was assayed, and growth‐promoting factors present in the nerve grafts following G‐CSF administration or BMSCs seeding were also investigated. Nerve regeneration and functional rehabilitation induced by the combination therapy were significantly advanced, and the rehabilitation efficacy was comparable with autografting. Moreover, the expression of Schwann cell markers, neurotrophic factors and neovessel markers in the nerve grafts was substantially increased. In conclusion, G‐CSF administration and BMSCs transplantation synergistically promoted the regeneration of ANX‐bridged nerves, which offers a superior strategy to replace autografts in repairing peripheral nerve injuries.