Biological conduits combining bone marrow mesenchymal stem cells and extracellular matrix to treat long-segment sciatic nerve defects

The transplantation of polylactic glycolic acid conduits combining bone marrow mesenchymal stem cells and extracellular matrix gel for the repair of sciatic nerve injury is effective in some respects, but few data comparing the biomechanical factors related to the sciatic nerve are available. In the present study, rabbit models of 10-mm sciatic nerve defects were prepared. The rabbit models were repaired with autologous nerve, a polylactic glycolic acid conduit + bone marrow mesenchymal stem cells, or a polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel. After 24 weeks, mechanical testing was performed to determine the stress relaxation and creep parameters. Following sciatic nerve injury, the magnitudes of the stress decrease and strain increase at 7,200 seconds were largest in the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel group, followed by the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells group, and then the autologous nerve group. Hematoxylin-eosin staining demonstrated that compared with the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells group and the autologous nerve group, a more complete sciatic nerve regeneration was found, including good myelination, regularly arranged nerve fibers, and a completely degraded and resorbed conduit, in the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel group. These results indicate that bridging 10-mm sciatic nerve defects with a polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel construct increases the stress relaxation under a constant strain, reducing anastomotic tension. Large elongations under a constant physiological load can limit the anastomotic opening and shift, which is beneficial for the regeneration and functional reconstruction of sciatic nerve. Better regeneration was found with the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel grafts than with the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells grafts and the autologous nerve grafts.     

Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

Human umbilical cord-derived mesenchymal stem cells(h UCMSCs) represent a promising young-state stem cell source for cell-based therapy. h UCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the paracrine effects of h UCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that h UCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with h UCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These findings suggest that paracrine action may be a key mechanism underlying the effects of h UCMSCs in peripheral nerve repair.     

Delayed peripheral nerve repair: methods,including surgical ‘cross-bridging' to promote nerve regeneration

Despite the capacity of Schwann cells to support peripheral nerve regeneration, functional recovery after nerve injuries is frequently poor, especially for proximal injuries that require regenerating axons to grow over long distances to reinnervate distal targets. Nerve transfers, where small fascicles from an adjacent intact nerve are coapted to the nerve stump of a nearby denervated muscle, allow for functional return but at the expense of reduced numbers of innervating nerves. A 1-hour period of 20 Hz electrical nerve stimulation via electrodes proximal to an injury site accelerates axon outgrowth to hasten target reinnervation in rats and humans, even after delayed surgery. A novel strategy of enticing donor axons from an otherwise intact nerve to grow through small nerve grafts(cross-bridges) into a denervated nerve stump, promotes improved axon regeneration after delayed nerve repair. The efficacy of this technique has been demonstrated in a rat model and is now in clinical use in patients undergoing cross-face nerve grafting for facial paralysis. In conclusion, brief electrical stimulation, combined with the surgical technique of promoting the regeneration of some donor axons to ‘protect' chronically denervated Schwa nn cells, improves nerve regeneration and, in turn, functional outcomes in the management of peripheral nerve injuries.     

Skin-derived precursor cells enhance peripheral nerve regeneration following chronic denervation

While peripheral nerves demonstrate the capacity for axonal regeneration, outcome following injury remains relatively poor, especially following prolonged denervation. Since axon-deprived Schwann cells (SCs) in the distal nerve progressively lose their ability to support axonal growth, we took the approach of using skin-derived precursor cells (SKPs) as an accessible source of replacement SCs that could be transplanted into chronically denervated peripheral nerve. In this study, we employed a delayed cross-reinnervation paradigm to assess regeneration of common peroneal nerve axons into the chronically denervated rodent tibial nerve following delivery of SKP-derived SC (SKP-SCs). SKP-SC treated animals exhibited superior axonal regeneration to media controls, with significantly higher counts of regenerated motorneurons and histological recovery similar to that of immediately repaired nerve. Improved axonal regeneration correlated with superior muscle reinnervation, as measured by compound muscle action potentials and wet muscle weights. We therefore conclude that SKPs represent an easily accessible, autologous source of stem cell-derived Schwann cells that show promise in improving regeneration through chronically injured nerves.     

Mesenchymal stem cell treatment for peripheral nerve injury: a narrative review

Peripheral nerve injuries occur as the result of sudden trauma and lead to reduced quality of life.The peripheral nervous system has an inherent capability to regenerate axons.However,peripheral nerve regeneration following injury is generally slow and incomplete that results in poor functional outcomes such as muscle atrophy.Although conventional surgical procedures for peripheral nerve injuries present many benefits,there are still several limitations including scarring,difficult accessibility to donor nerve,neuroma formation and a need to sacrifice the autologous nerve.For many years,other therapeutic approaches for peripheral nerve injuries have been explored,the most notable being the replacement of Schwann cells,the glial cells responsible for clearing out debris from the site of injury.Introducing cultured Schwann cells to the injured sites showed great benefits in promoting axonal regeneration and functional recovery.However,there are limited sources of Schwann cells for extraction and difficulties in culturing Schwann cells in vitro.Therefore,novel therapeutic avenues that offer maximum benefits for the treatment of peripheral nerve injuries should be investigated.This review focused on strategies using mesenchymal stem cells to promote peripheral nerve regeneration including exosomes of mesenchymal stem cells,nerve engineering using the nerve guidance conduits containing mesenchymal stem cells,and genetically engineered mesenchymal stem cells.We present the current progress of mesenchymal stem cell treatment of peripheral nerve injuries.     

Schwann cell‐derived exosomes enhance axonal regeneration in the peripheral nervous system

Axonal regeneration in the peripheral nervous system is greatly supported by Schwann cells (SCs). After nerve injury, SCs dedifferentiate to a progenitor‐like state and efficiently guide axons to their original target tissues. Contact and soluble factors participate in the crosstalk between SCs and axons during axonal regeneration. Here we show that dedifferentiated SCs secrete nano‐vesicles known as exosomes which are specifically internalized by axons. Surprisingly, SC‐derived exosomes markedly increase axonal regeneration in vitro and enhance regeneration after sciatic nerve injury in vivo. Exosomes shift the growth cone morphology to a pro‐regenerating phenotype and decrease the activity of the GTPase RhoA, involved in growth cone collapse and axon retraction. Altogether, our work identifies a novel mechanism by which SCs communicate with neighboring axons during regenerative processes. We propose that SC exosomes represent an important mechanism by which these cells locally support axonal maintenance and regeneration after nerve damage. GLIA 2013;61:1795–1806     

The influence of cultured Schwann cells on regeneration through acellular basal lamina grafts

Acellular basal lamina grafts have been shown to be less immunogenic in comparison to cellular grafts, but possess a limited potential for supporting axonal regeneration through them. The present study describes the effect of cultured Schwann cells on enhancing regeneration through acellular grafts. 2 cm long acellular grafts, and in vitro Schwann cell populated acellular grafts were used to repair a surgically created gap in the host peroneal nerve. The transplants were analyzed at 1, 2, 4 and 8 weeks to determine their ability to support axonal regeneration. Host axonal regeneration through Schwann cell cocultured acellular grafts occurred rapidly and was significantly better as compared to non-cultured acellular grafts. The results demonstrate a beneficial effect of Schwann cell culture pretreatment on regeneration through acellular grafts and an improved recovery of the target muscle. The procedure of first preparing acellular grafts with subsequent coculture with Schwann cells offers a novel approach for the repair of injured nervous tissue.     

Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects

Tissue-engineering as laboratory based alternative to human autografts and allografts provides "custom made organs" cultured from patient's material. To overcome the limited donor nerve availability different biologic nerve grafts were engineered in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into acellular autologous matrices: veins, muscles, nerves, and epineurium tubes. Autologous nerve grafts, and the respective biogenic material without Schwann cells served as control. After 6 weeks regeneration was assessed clinically, histologically and morphometrically. The PCR analysis showed that the implanted Schwann cells remain within all the grafts. A good regeneration was noted in the muscle-Schwann cell-group, while regeneration quality in the other groups (with or without Schwann cells) was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. All venous and epineurium grafts had a more disorganized regeneration. Seemingly, the lack of endoneural tube like structures in vein grafts lead to impaired regeneration. And, apparently, the beneficial effects of implanted Schwann cells into a large luminal structure can only be demonstrated to a limited extent if endoneural like structures are lacking. A tube offers less area for Schwann cell adhesion and it is more likely to collapse. This underlines the role of the basal lamina, or at least an inner structure acting as scaffold in axonal regeneration. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biogenic graft with basal lamina tubes as pathway for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

The role of Schwann cells and basal lamina tubes in the regeneration of axons through long lengths of freeze-killed nerve grafts

The ability of long acellular nerve grafts to support axonal regeneration was examined using inbred rats. Grafts (40 mm long) of tibial/plantar nerves were used either as live grafts or after freeze-drying to render the grafts acellular. The grafts were sutured to the proximal stump of severed tibial nerves in host animals which were then killed 1-12 weeks later. Axons rapidly regenerated through the living grafts but only extended 10-20 mm into the acellular grafts. This distance was achieved by 6 weeks and thereafter no significant further axonal extension occurred in the acellular grafts. A few naked axons lacking Schwann cell contact were identified in all acellular grafts, but became more numerous near the distal extent of axonal penetration into 6-12 week grafts. These axons contained large numbers of neurofilaments. When the distal 20 mm of 6 week acellular grafts (segments into which axons had not penetrated) were sutured to freshly severed tibial nerves, axons grew readily into the grafted tissue to a maximum distance of 9 mm. It is therefore likely that the limits to axonal regeneration through initially acellular grafts were set by factors intrinsic to the severed nerve. It is suggested that the limited migratory powers of Schwann cells may be one such factor. The concept that basal lamina tubes are not essential for axonal regeneration but may act as low resistance pathways for both axonal elongation and Schwann cell migration is discussed.     

Spatiotemporal progress of nerve regeneration in a tendon autograft used for bridging a peripheral nerve defect

We have previously shown that a tendon autograft from the rat tail can support regeneration across a gap in the continuity of the rat sciatic nerve. In this study, we characterized the spatiotemporal progress of regeneration in such a graft bridging a 10-mm defect in the sciatic nerve of the rat. Regeneration was assessed 7, 10, 14, or 18 days postoperatively, by immunocytochemistry for axons, Schwann cells, and macrophages and histochemistry for blood vessels. Axonal regrowth into the grafts showed an initial delay period of 6.8 days, whereafter axons grew at a rate of 1.0 mm/day. Schwann cells grew into the grafts from both the proximal and distal nerve segments, proximally just ahead of the axonal front. Macrophages were initially preferentially located at the periphery of the grafts, but gradually increased inside the grafts. Blood vessels entered the grafts from both the proximal and distal aspects of the severed nerve. The onset of vascularization appeared to coincide with axonal regeneration into the grafts.     

Repair of extended peripheral nerve lesions in rhesus monkeys using acellular allogenic nerve grafts implanted with autologous mesenchymal stem cells

Despite intensive efforts in the field of peripheral nerve injury and regeneration, it remains difficult in humans to achieve full functional recovery following extended peripheral nerve lesions. Optimizing repair of peripheral nerve injuries has been hindered by the lack of viable and reliable biologic or artificial nerve conduits for bridging extended gaps. In this study, we utilized chemically extracted acellular allogenic nerve segments implanted with autologous non-hematopoietic mesenchymal stem cells (MSCs) to repair a 40 mm defect in the rhesus monkey ulnar nerve. We found that severely damaged ulnar nerves were structurally and functionally repaired within 6 months following placement of the MSC seeded allografts in all animals studied (6 of 6, 100%). Furthermore, recovery with the MSC seeded allografts was similar to that observed with Schwann cell seeded allografts and autologous nerve grafts. The findings presented here are the first demonstration of the successful use of autologous MSCs, expanded in culture and implanted in a biological conduit, to repair a peripheral nerve gap in primates. Given the difficulty in isolating and purifying sufficient quantities of Schwann cells for peripheral nerve regeneration, the use of MSCs to seed acellular allogenic nerve grafts may prove to be a novel and promising therapeutic approach for repairing severe peripheral nerve injuries in humans.     

The HMGB1 signaling pathway activates the inflammatory response in Schwann cells

Schwann cells are not only myelinating cells, but also function as immune cells and express numerous innate pattern recognition receptors, including the Toll-like receptors. Injury to peripheral nerves activates an inflammatory response in Schwann cells. However, it is unclear whether specific endogenous damage-associated molecular pattern molecules are involved in the inflammatory response following nerve injury. In the present study, we demonstrate that a key damage-associated molecular pattern molecule, high mobility group box 1(HMGB1), is upregulated following rat sciatic nerve axotomy, and we show colocalization of the protein with Schwann cells. HMGB1 alone could not enhance expression of Toll-like receptors or the receptor for advanced glycation end products(RAGE), but was able to facilitate migration of Schwann cells. When Schwann cells were treated with HMGB1 together with lipopolysaccharide, the expression levels of Toll-like receptors and RAGE, as well as inflammatory cytokines were upregulated. Our novel findings demonstrate that the HMGB1 pathway activates the inflammatory response in Schwann cells following peripheral nerve injury.     

Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects

Abstract

Tissue-engineering as laboratory based alternative to human autografts and allografts provides "custom made organs" cultured from patient's material. To overcome the limited donor nerve availability different biologic nerve grafts were engineered in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into acellular autologous matrices: veins, muscles, nerves, and epineurium tubes. Autologous nerve grafts, and the respective biogenic material without Schwann cells served as control. After 6 weeks regeneration was assessed clinically, histologically and morphometrically. The PCR analysis showed that the implanted Schwann cells remain within all the grafts. A good regeneration was noted in the muscle-Schwann cell-group, while regeneration quality in the other groups (with or without Schwann cells) was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. All venous and epineurium grafts had a more disorganized regeneration. Seemingly, the lack of endoneural tube like structures in vein grafts lead to impaired regeneration. And, apparently,the beneficial effects of implanted Schwann cells into a large luminal structure can only be demonstrated to a limited extent if endoneural like structures are lacking. A tube offers less area for Schwann cell adhesion and it is more likely to collapse. This underlines the role of the basal lamina, or at least an inner structure acting as scaffold in axonal regeneration. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biogenic graft with basal lamina tubes as pathway for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

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

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

Edaravone combined with Schwann cell transplantation may repair spinal cord injury in rats

Edaravone has been shown to delay neuronal apoptosis, thereby improving nerve function and the microenvironment after spinal cord injury. Edaravone can provide a favorable environment for the treatment of spinal cord injury using Schwann cell transplantation. This study used rat models of complete spinal cord transection at T9. Six hours later, Schwann cells were transplanted in the head and tail ends of the injury site. Simultaneously, edaravone was injected through the caudal vein. Eight weeks later, the PKH-26-labeled Schwann cells had survived and migrated to the center of the spinal cord injury region in rats after combined treatment with edaravone and Schwann cells. Moreover, the number of PKH-26-labeled Schwann cells in the rat spinal cord was more than that in rats undergoing Schwann cell transplantation alone or rats without any treatment. Horseradish peroxidase retrograde tracing revealed that the number of horseradish peroxidase-positive nerve fibers was greater in rats treated with edaravone combined with Schwann cells than in rats with Schwann cell transplantation alone. The results demonstrated that lower extremity motor function and neurophysiological function were better in rats treated with edaravone and Schwann cells than in rats with Schwann cell transplantation only. These data confirmed that Schwann cell transplantation combined with edaravone injection promoted the regeneration of nerve fibers of rats with spinal cord injury and improved neurological function.     

Biological conduit small gap sleeve bridging method for peripheral nerve injury: regeneration law of nerve fibers in the conduit

The clinical effects of 2-mm small gap sleeve bridging of the biological conduit to repair peripheral nerve injury are better than in the traditional epineurium suture, so it is possible to replace the epineurium suture in the treatment of peripheral nerve injury. This study sought to identify the regeneration law of nerve fibers in the biological conduit. A nerve regeneration chamber was constructed in models of sciatic nerve injury using 2-mm small gap sleeve bridging of a biodegradable biological conduit. The results showed that the biological conduit had good histocompatibility. Tissue and cell apoptosis in the conduit apparently lessened, and regenerating nerve fibers were common. The degeneration regeneration law of Schwann cells and axons in the conduit was quite different from that in traditional epineurium suture. During the prime period for nerve fiber regeneration(2–8 weeks), the number of Schwann cells and nerve fibers was higher in both proximal and distal ends, and the effects of the small gap sleeve bridging method were better than those of the traditional epineurium suture. The above results provide an objective and reliable theoretical basis for the clinical application of the biological conduit small gap sleeve bridging method to repair peripheral nerve injury.     

Autologous nerve graft repair of different degrees of sciatic nerve defect: stress and displacement at the anastomosis in a three-dimensional fnite element simulation model

In the repair of peripheral nerve injury using autologous or synthetic nerve grafting, the magnitude of tensile forces at the anastomosis affects its response to physiological stress and the ultimate success of the treatment. One-dimensional stretching is commonly used to measure changes in tensile stress and strain; however, the accuracy of this simple method is limited. Therefore, in the present study, we established three-dimensional finite element models of sciatic nerve defects repaired by autologous nerve grafts. Using PRO E 5.0 finite element simulation software, we calculated the maximum stress and displacement of an anastomosis under a 5 N load in 10-, 20-, 30-, 40-mm long autologous nerve grafts. We found that maximum displacement increased with graft length, consistent with specimen force. These findings indicate that three-dimensional finite element simulation is a feasible method for analyzing stress and displacement at the anastomosis after autologous nerve grafting.     

Cell replacement therapy for central nervous system diseases

The brain and spinal cord can not replace neurons or supporting glia that are lost through traumatic injury or disease. In pre-clinical studies, however, neural stem and progenitor cell transplants can promote functional recovery. Thus the central nervous system is repair competent but lacks endogenous stem cell resources. To make transplants clinically feasible, this field needs a source of histocompatible, ethically acceptable and non-tumorgenic cells. One strategy to generate patient-specific replacement cells is to reprogram autologous cells such as fibroblasts into pluripotent stem cells which can then be differentiated into the required cell grafts. However, the utility of pluripotent cell derived grafts is limited since they can retain founder cells with intrinsic neoplastic potential. A recent extension of this technology directly reprograms fibroblasts into the final graftable cells without an induced pluripotent stem cell intermediate, avoiding the pluripotent caveat. For both types of reprogramming the conversion efficiency is very low resulting in the need to amplify the cells in culture which can lead to chromosomal instability and neoplasia. Thus to make reprogramming biology clinically feasible, we must improve the efficiency. The ultimate source of replacement cells may reside in directly reprogramming accessible cells within the brain.     

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.     

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。