Use of chemically extracted muscle grafts to repair extended nerve defects in rats

Nerve regeneration, measured as axonal outgrowth, Schwann cell migration, macrophage invasion, and neovascularisation, was compared after repair of a 15 mm gap in rats' sciatic nerves using autologous muscle grafts made acellular either by freezing and thawing or by chemical extraction. Both extracted and freeze-thawed acellular muscle grafts could be used to bridge the defect. However, axons and Schwann cells, as shown by immunohistochemical staining for neurofilaments and S-100 protein, respectively, grew faster into the extracted muscle grafts than into the freeze-thawed acellular muscle grafts and somewhat more axons were observed in the former graft. There were no significant differences between the two graft types with respect to neovascularisation as showed by staining for endothelial alkaline phosphatase, and limited differences concerning invasion of macrophages (ED1 and ED2) as detected by immunocytochemistry. The results showed that chemically extracted muscle grafts could be used to bridge an extended nerve defect and that such grafts in some aspects were superior to freeze-thawed muscle grafts for extended gaps.     

Acellular muscle with Schwann-cell implantation: an alternative biologic nerve conduit

Denatured or acellular muscle grafts are known to support axonal regeneration. With increasing gap length, failure of regeneration is evident, due to the lack of viable Schwann cells in the graft. The authors created a biologic nerve conduit, in a rat sciatic nerve model, by implanting cultured Schwann cells into an acellular gracilis muscle. Autologous nerve grafts and acellular muscle grafts without Schwann cells served as controls. After 6 weeks, regeneration was assessed clinically, histologically, and morphometrically. Polymerase chain reaction (PCR) analysis showed that the implanted Schwann cells remained viable within the graft. Good regeneration was noted in the muscle-Schwann cell group, while regeneration in the muscle grafts without Schwann cells was significantly impaired. The muscle-Schwann cell graft demonstrated systematic and organized regeneration, including the proper orientation of regenerated fibers. The number of axons regenerating through the muscle-Schwann cell grafts was significantly increased, compared with the acellular muscle without Schwann cells. Implantation of Schwann cells into acellular muscle thus provided a biologic conduit with large basal lamina tubes, as a pathway for regenerating axons. The positive effects of Schwann cells, producing neurotrophic and neurotropic factors, supported axonal regeneration.     

化学去细胞异种神经移植后宿主的许旺细胞在移植物内的增殖

目的 观察化学去细胞异种神经移植后宿主许旺细胞向移植物内增殖的情况。方法 移植化学去细胞兔神经修复大鼠1cm坐骨神经缺损，4个月后处死动物，取移植物行苏木精-伊红染色、S-100免疫组织化学染色及透射电镜观察。结果 再生神经纤维顺利长入移植物，围绕再生纤维有大量胞体S-100免疫组化反应阳性的细胞呈条索状排列，半薄切片显示移植物内再生轴突排列整齐，透射电镜显示有细胞包绕再生轴突并形成髓鞘。结论 化学去细胞异种神经移植后宿主许旺细胞可随再生纤维长入移植物并形成髓鞘。     

Bridging critical nerve defects through an acellular homograft seeded with autologous schwann cells obtained from a regeneration neuroma of the proximal stump

Over the last decade, several models have investigated the usefulness of different biologic and/or synthetic matrices as alternatives to conventional nerve grafts. Still, axonal regeneration did not occur over longer (> 3 cm) distances. One problem may be that a growth-promoting environment not only includes physical cues but also a rich spectrum of different growth factors only provided by reactive Schwann cells. In the current study, we investigated whether a hybrid graft consisting of first-generation autologous Schwann cells seeded onto an acellular auto- or homograft can aid regeneration across a critical nerve defect in a rat model. In this paradigm, Schwann cells were not expanded in vitro but harvested from the proximal stump neuroma at the time of reconstruction and seeded into either an acellular homo- or autograft. Regeneration was then quantitated with functional muscle testing, regular histology, histomorphometry, and retrograde tracing techniques 12 weeks after reconstruction. Results showed successful regeneration over the entire distance regardless of whether Schwann cells were transplanted onto auto- or homologous acellular matrix. Schwann cells did populate both grafts; however, only sensory axons persisted through the entire distance. The functional outcome was dismal with no motor and poor sensory recovery. Control group C with homologous matrix only without Schwann cells showed no signs of directed axonal regeneration. Control group D with autologous reverse graft showed excellent recovery, as was expected. The present experiment sought to create a hybrid graft where the proximal stump neuroma is used as a biological resource for autologous Schwann cells that are seeded unto an acellular matrix, thus providing both physical and chemical support to regenerating axons. The results are encouraging in that successful regeneration was observed over the entire distance; however, only sensory axons had enough regenerative potential to also make end-organ contact. For motor axons, further refinements in conduit preparation have to be done.     

Functional recovery in a tendon autograft used to bridge a peripheral nerve defect

The functional recovery was examined after two different methods had been used to bridge an extended nerve gap. A 15 mm defect in the rat sciatic nerve was repaired using a tendon autograft--a new graft material--on one side and a freeze-thawed muscle graft--a well established experimental material--on the other side. Evaluation after 12 weeks included measurements of tetanic force in the gastrocnemius muscle and computerised morphometry of the tibial nerve. The muscular tetanic force recovered to 26% and 21% of control muscles, respectively, but there were no significant differences between the two types of grafts in any of the measurements. The number of regenerated axons of the tibial nerve correlated with functional recovery as judged by muscular tetanic force in the gastrocnemius muscle. We conclude that the tendon autograft supports functional recovery, as judged by return of muscular tetanic force, to an extent comparable to that of the freeze-thawed muscle graft.     

Functional recovery in a tendon autograft used to bridge a peripheral nerve defect.

The functional recovery was examined after two different methods had been used to bridge an extended nerve gap. A 15 mm defect in the rat sciatic nerve was repaired using a tendon autograft--a new graft material--on one side and a freeze-thawed muscle graft--a well established experimental material--on the other side. Evaluation after 12 weeks included measurements of tetanic force in the gastrocnemius muscle and computerised morphometry of the tibial nerve. The muscular tetanic force recovered to 26% and 21% of control muscles, respectively, but there were no significant differences between the two types of grafts in any of the measurements. The number of regenerated axons of the tibial nerve correlated with functional recovery as judged by muscular tetanic force in the gastrocnemius muscle. We conclude that the tendon autograft supports functional recovery, as judged by return of muscular tetanic force, to an extent comparable to that of the freeze-thawed muscle graft.     

Host responses after acellular muscle basal lamina allografting used as a matrix for tissue engineered nerve grafts1

BACKGROUND: A nerve gap must be bridged by autologous nerve grafts that serve as scaffold and consist of viable Schwann cells that promote regeneration. Owing to the necessary immunosuppression, nerve allografts remain limited to special cases. Alternatively, tissue engineering of peripheral nerves focuses on the implantation of cultured Schwann cells into suitable scaffolds. We established grafts from Schwann cells and basal lamina from acellular muscles. These grafts offer a regeneration that is comparable to autologous nerve grafts. METHODS: Using a rat model (DALEW.1W strain), the present study evaluates the host response to acellular muscle allografts by assessing cellular reaction major histocompatability (MHC) class I and II, lymphocytes, macrophages. The results were compared to untreated muscle allografts. RESULTS: Macroscopically, the untreated muscles showed a strong inflammatory reaction as a sign of rejection, whereas the acellular muscle offered only minor reactions in the periphery of the graft. Expression of MHC I and II and invasion of CD4/CD8 positive cells and macrophages was pronounced after grafting the untreated muscles. Only a moderate reaction was noted for these parameters after acellular grafting. CONCLUSIONS: The acellular muscle graft is not completely free of cellular response; however the reaction is considered to be moderate and is located only in the periphery. To date, synthetic scaffolds that represent endoneurial tube-like structures and allow sufficient adhesion of Schwann cells and axonal regeneration are not available. The decreased response to acellular muscle allografts offers at least a basis for further experiments.     

Successful implantation of Schwann cells in acellular muscles

Acellular muscle grafts can support axonal regeneration over short gaps. Due to the lack of viable Schwann cells in the grafts, failure of regeneration is evident with increasing gap lengths. To create a biological nerve conduit, Schwann cells were implanted into acellular muscle. The grafts were then incubated in vitro and assessed histologically and morphometrically. For cultivation of the Schwann cells, rat sciatic nerves were allowed to predegenerate to obtain a high cell yield. Rat gracilis muscles were harvested and made acellular by a liquid nitrogen treatment. After Schwann cell implantation, the muscles were incubated in vitro for 2, 5, and 7 days. S100-immunostaining, NGF, and N-cadherin, characterized the Schwann cells within the muscle. Viability was assessed by fluoresceine-fluorescence staining. Proliferation was determined by BrdU-DNA incorporation. Cell implantation did not to affect Schwann cell viability. Cells were seen throughout the entire length of the muscle basal lamina. They aligned and formed a cell column. Immunostained for S-100, implanted cells showed 100 percent staining. N-cadherin and NGF were expressed by all of the S-100 positive cells. Predegeneration is considered to be a highly efficacious method, if a high yield of activated Schwann cells is required. The successful implantation of the cells into an acellular muscle provides the possibility of a biologic conduit, offering the advantage of large basal lamina tubes serving as a pathway for regenerating axons. It also provides the beneficial effects of viable Schwann cells that produce neurotrophic and neurotropic factors to support axonal regeneration. Functional outcomes require evaluation in further in vivo studies.     

雪旺细胞在大鼠脱细胞异体神经基膜管中迁移的研究

目的研究大鼠自体雪旺细胞在脱细胞异体神经基膜管中的迁移情况。方法取SD大鼠坐骨神经20mm,用化学萃取法制备脱细胞神经,行大体观察、HE、抗层黏蛋白（Anti-laminin）染色。另取32只雌性SD大鼠,体重250～300g,切取自体坐骨神经2mm,置于分别长10mm的两段大鼠异体脱细胞坐骨神经基膜管之间,形成22mm长的基膜管-自体神经嵌合体,与同样长度的单纯脱细胞神经基膜管埋入肌间隙中,于5、10、15和20d取材,行HE、S-100免疫组织化学染色。结果制备的脱细胞神经基膜管外观呈半透明;HE染色示基膜管内未见细胞核存在,基膜管部分不连续;Anti-laminin染色示基膜管深褐色。基膜管-自体神经嵌合体于术后各时间点HE染色均可见细胞存在,第15天开始可见S-100阳性雪旺细胞;单纯神经基膜管在各时间点HE染色中均可见细胞存在,未见S-100阳性雪旺细胞。结论大鼠坐骨神经的自体雪旺细胞可迁移至两侧异体脱细胞神经基膜管远端,为嵌合自体神经的异体脱细胞神经基膜管修复长段周围神经缺损提供了理论依据。     

A comparison of peripheral nerve regeneration in acellular muscle and nerve autografts.

Regeneration of the rat sciatic nerve through acellular muscle and nerve autografts was evaluated 6-28 days postoperatively by the sensory pinch test, immunocytochemical staining for neurofilaments, and light and electron microscopy. Data points generated by the pinch test were plotted against postoperative time periods and by the use of regression analysis the initial delay period for muscle grafts was determined to 10.3 days. This value was similar to that previously published for acellular nerve grafts (9.5 days), but significantly longer than that for fresh nerve grafts (3.6 days). The calculated regeneration rate (slope of the regression line) for muscle grafts (1.8 mm/day) did not differ significantly (p > 0.05) from that calculated for acellular nerve grafts (2.1 mm/day) or for fresh nerve grafts (1.5 mm/day). The front of regenerating axons shown by axonal neurofilament staining confirmed the pinch test results. Both types of acellular grafts were repopulated with host non-neuronal cells and the muscle graft contained occasional ectopic muscle fibres. Remnants of graft basal laminae were evident at the ultrastructural level. These results indicate the suitability of either acellular muscle or nerve grafts for nerve repair despite their prolonged initial delay periods compared with conventional fresh nerve grafts.     

Reconstruction of peripheral nerves using acellular nerve grafts with implanted cultured Schwann cells

The bridging of nerve gaps is still one of the major problems in peripheral nerve surgery. The present experiment describes our attempt to engineer different biologic nerve grafts in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into 2-cm autologous acellular nerve grafts or autologous predegenerated nerve grafts. Autologous nerve grafts and predegenerated or acellular nerve grafts without implanted Schwann cells served as controls. The regenerated nerves were assessed histologically and morphometrically after 6 weeks. Predegenerated grafts showed results superior in regard to axon count and histologic appearance in comparison to standard grafts and acellular grafts. The acellular nerve grafts showed the worst histologic picture, but axon counts were in the range of standard grafts. The implantation of Schwann cells did not yield significant improvements in any group. In conclusion, the status of activation of Schwann cells and the stadium of Wallerian degeneration in a nerve graft might be key factors for regeneration, rather than total number of Schwann cells. Predegenerated nerve grafts are therefore superior to standard grafts in the rat model. Acellular grafts are able to bridge nerve gaps of up to 2 cm in the rat model, but even the addition of cultivated Schwann cells did not lead to results as good as in the group with autologous nerve grafts.     

猕猴组织工程化周围神经移植物的实验研究

目的 评价利用化学萃取法获得的神经支架制备猕猴组织工程化周围神经移植物修复40mm尺神经缺损的效果。方法 共使用成年猕猴9只，随机取其中的6只，培养自体雪旺细胞，应用已经制备好的去细胞神经为支架材料构建组织工程化周围神经移植体修复猕猴尺神经的40mm缺损。实验分实验组，空白组，正常对照组3组。术后5个月通过形态学，肌电检测和免疫组织化学方法观察。结果 3组均未发现猕猴双手有糜烂或溃疡形成，猕猴小鱼际肌群的饱满度和弹性于术前无明显差别。实验组与正常对照组间小鱼际肌群的运动动作电位潜伏期．最大振幅及再生神经纤维的数目差异均无统计学意义（P〉0．05）。但在实验组与空白组间的差异有统计学意义（P〈0．05）。结论 用自体源雪旺细胞微注入去细胞同种异体神经支架构建的组织工程化神经移植物修复猕猴40mm的尺神经缺损，可取得与自体神经移植相似的效果。     

Regeneration in, and properties of, extracted peripheral nerve allografts and xenografts

When not enough conventional autologous nerve grafts are available, alternatives are needed to bridge nerve defects. Our aim was to study regeneration of nerves in chemically-extracted acellular nerve grafts from frogs, mice, humans (fresh and stored sural nerve), pigs and rats when defects in rat sciatic nerves were bridged. Secondly, we compared two different extraction procedures (techniques described by Sondell et al. and Hudson et al.) with respect to how efficiently they supported axonal outgrowth, and remaining laminin and myelin basic protein (MBP), after extraction. Isografts (rat) and xenografts (mouse) were transplanted into defects in rat sciatic nerves. Acellular nerve allografts from rats, extracted by the Sondell et al's technique, had an appreciably longer axonal outgrowth based on immunohistochemical staining of neurofilaments, than acellular nerve xenografts except those from the pig. Among acellular xenografts there was considerably longer axonal outgrowth in the grafts from pigs compared with those from humans (fresh), but there were no other differences among the xenografts with respect to axonal outgrowth. Axonal outgrowth in acellular nerve xenografts from mice, extracted by the method described by Sondell et al. was longer than in those extracted by Hudson et al's method, while there was no difference in outgrowth between extracted nerve isografts from rats. Electrophoretic analysis of extracted acellular nerve grafts showed remaining laminin, but not MBP, after both extraction procedures. These preserved laminin and removed MBP in acellular nerve grafts. Such grafts can be used to reconstruct short defects in nerves irrespective of their origin. However, selecting and matching a suitable combination of graft and host species may improve axonal outgrowth.     

Neurotrophins and their receptors in early axonal regeneration along muscle-vein-combined grafts

Experimental and clinical studies have shown that a vein segment filled with skeletal muscle used to bridge a peripheral nerve defect (muscle-vein-combined graft) leads to good nerve repair. However, the molecular basis of the nerve fiber regeneration process along this type of graft still remains to be elucidated. The aim of this study was to verify the expression of two neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), as well as their receptors, trkA and p75, in an early stage of axonal regeneration in muscle-vein-combined grafts. Severed rat sciatic nerves were repaired by means of 1-cm-long muscle-vein-combined grafts and withdrawn immediately after surgery (control grafts) and 5 days after surgery. Longitudinal sections of grafts were immunostained by means of the following antibodies: anti-NGF, anti-BDNF, anti-trkA, and anti-p75. An anti-glial fibrillar acid protein (anti-GFAP) antibody was used to recognize Schwann cells. Results showed the presence of a number of GFAP-positive Schwann cells inside the muscle-vein grafts. Many of these cells reacted for NGF, BDNF, and p75, but not trkA. In control grafts, i.e., immediately after surgery, no immunostaining was detected for any of the antibodies used in this study. These observations suggest that, very early after surgery, the muscle-vein-combined graft offers to growing axons an environment particularly favorable for regeneration, providing us with a possible explanation for the efficacy of this grafting technique for peripheral nerve repair.     

组织工程化人工神经修复长段神经缺损实验的初步报告

目的 研究组织化人工神经修复大鼠2．5cm长坐骨神经缺损的效果。方法 90只2个月月龄的Lewis1W雌性大鼠,按手术先后顺序随机分成3个神经移植组,每组30只。A组：用种植同源雪旺细胞并具有内部支架结构的胶原神经管桥接,即组织工程化人工神经组。B组：用无雪旺细胞但具有内部支架结构的胶原神经管桥接,即对照组。C组：自体神经移植组。术后6月,进行神经电生理监测,神经肌肉组织学观察;用S－100和神经微丝蛋白免疫组化染色后,行轴突计数等检测。结果 完成对21只大鼠（每组7只）的实验评估。从A组和C组的胫前肌中均能诱发出波幅明显的神经肌肉复合动作电位（CMAP）,再生轴突已通过移植段神经全长,远端肌肉轻度萎缩。B组中则没有或仅记录到波幅很低的CMAP,移植神经远端结缔纤维组织增生,再生轴突罕见,所支配肌肉明显萎缩。结论 组织工程化人工神经可用来修复大鼠长段神经缺损。     

人类去细胞同种异体神经移植物化学萃取方法的研究

目的：新鲜同种异体神经免疫排斥反应的标靶是细胞、髓鞘。发展新的化学处理方法,清除人类周围神经中的细胞和髓鞘 ,萃取粗大和长段的去细胞神经移植物。方法：切取年轻男性遗体捐献者的长段尺神经,以triton X-100和脱氧胆酸钠溶液按一定浓度和程序进行化学处理。萃取神经及新鲜神经行HE染色、髓鞘染色及纤维素染色,以观察细胞、髓鞘及神经基底膜;免疫组织化学染色以显示许旺细胞基底板层;行半薄切片及超薄切片透射电镜检查,观察超微结构。结果：去细胞神经的延展性及神经外膜的韧弹性良好。细胞和髓鞘被彻底清除,神经基底膜被保留;许旺细胞基底板层保留完好;去细胞神经为一种没有细胞髓鞘及其碎片的空的神经基质管。结论：Triton X-100及脱氧胆酸钠化学处理方法,可有效清除人类周围神经中的细胞及髓鞘,萃取粗大和长段的去细胞神经;该神经移植物保持了网管柱状组织结构,保留了许旺细胞基底板层及板层素。     

Metabosensitive afferent fiber responses after peripheral nerve injury and transplantation of an acellular muscle graft in association with schwann cells

Studies dedicated to the repair of peripheral nerve focused almost exclusively on motor or mechanosensitive fiber regeneration. Poor attention has been paid to the metabosensitive fibers from group III and IV (also called ergoreceptor). Previously, we demonstrated that the metabosensitive response from the tibialis anterior muscle was partially restored when the transected nerve was immediately sutured. In the present study, we assessed motor and metabosensitive responses of the regenerated axons in a rat model in which 1 cm segment of the peroneal nerve was removed and immediately replaced by an autologous nerve graft or an acellular muscle graft. Four groups of animals were included: control animals (C, no graft), transected animals grafted with either an autologous nerve graft (Gold Standard-GS) or an acellular muscle filled with Schwann Cells (MSC) or Culture Medium (MCM). We observed that (1) the tibialis anterior muscle was atrophied in GS, M(SC) and M(CM) groups, with no significant difference between grafted groups; (2) the contractile properties of the reinnervated muscles after nerve stimulation were similar in all groups; (3) the metabosensitive afferent responses to electrically induced fatigue was smaller in M(SC) and MCM groups; and (4) the metabosensitive afferent responses to two chemical agents (KCl and lactic acid) was decreased in GS, M(SC) and M(CM) groups. Altogether, these data indicate a motor axonal regeneration and an immature metabosensitive afferent fiber regrowth through acellular muscle grafts. Similarities between the two groups grafted with acellular muscles suggest that, in our conditions, implanted Schwann cells do not improve nerve regeneration. Future studies could include engineered conduits that mimic as closely as possible the internal organization of uninjured nerve.     

Effects of vascular endothelial growth factor on nerve regeneration in acellular nerve grafts

The purpose of this study was to evaluate the effects of human recombinant vascular endothelial growth factor (VEGF-165) on peripheral nerve axonal sprouting and elongation following peripheral nerve injury and repair. Two-centimeter nerve gaps were created in rat peroneal nerves and repaired with either peripheral nerve autografts, acellular peripheral nerve isografts, or VEGF-165-treated acellular peripheral nerve isografts. Four months postoperatively, the peroneal nerves were harvested and histomorphometric analysis was performed. The reinnervated extensor digitorum longus (EDL) muscles were harvested and weighed. At the proximal nerve gap coaptation site, there was a statistically significant increase in the total number of axons and percent neural tissue in the VEGF-treated acellular nerve graft group, compared with the acellular peripheral nerve isograft and autograft groups. At the distal coaptation site, however, the total number of axons and percent neural tissue was similar in the acellular and VEGF-treated groups, which was significantly less than the autograft group. VEGF-165 treatment of acellular nerve grafts resulted in greater EDL muscle masses than acellular nerve grafts alone. VEGF treatment of acellular peripheral nerve isografts enhances axonal sprouting, resulting in an increased number of axons and percent neural tissue at the proximal nerve graft coaptation site. In the absence of any cellular elements, VEGF-impregnated acellular peripheral nerve grafts do not demonstrate enhanced axonal elongation, as noted by relatively few axons at the distal nerve graft coaptation site.     

构建猕猴组织工程化周围神经的实验研究

目的评价组织工程化周围神经修复猕猴4cm尺神经缺损的实验效果,为临床研究提供资料。方法分别用6种移植物桥接4cm尺神经缺损。A组:自体BMSCs 去细胞同种异体神经支架;B组:自体SCs 去细胞同种异体神经支架;C组:自体BMSCs PLGA支架导管;D组:去细胞同种异体神经支架;E组:PLGA支架导管;F组:自体神经。通过功能学、神经电生理学及组织学研究评价各自的实验效果。结果A、B、C三种组织工程化神经实验组,术后6个月神经电生理和组织学检查,能引起小鱼际肌群产生复合动作电位的潜伏期、复合动作电位的最大振幅、神经传导速度和再生的神经纤维数目与自体神经移植组(F组)相比差异无显著性意义(P>0.05),但分别大于未加细胞的支架组(D、E组),差异有显著意义(P<0.05)。结论用自体源SCs或BMSCs作种子细胞与去细胞同种异体神经支架,或自体源BMSCs与PLGA支架导管构建不同的组织工程化周围神经,修复猕猴4cm尺神经缺损均取得较好的效果。     

组织工程化人工神经实验研究

目的 研究组织工程化人工神经修复大鼠2.5cm长坐骨神经缺损的效果。方法 21只2月龄Lewis lw雌性大鼠随机分成三个神经移植组,每组7只。A组:种植同源雪旺细胞并具有内部支架结构的胶原神经管,即组织工程化人工神经。B组:无雪旺细胞但具有内部支架结构的胶原神经管。C组:自体神经移植组。术后六个月,进行系列神经电生理监测,神经肌肉组织学观察,S-100和神经微丝蛋白(Neurofilament)免疫组化染色,轴突计数等检查。结果 在A组和C组移植神经上均能诱发出波幅明显的神经肌肉复合动作电位(CMAP),再生轴突已通过移植神经全长,远端肌肉轻度萎缩。而B组中没有或仅记录到极小波幅的CMAP,移植神经远端结缔纤维组织增生,再生轴突罕见,所支配肌肉明显萎缩。结论 初步结果显示:组织工程化人工神经可用来修复大鼠长段神经缺损。