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.     

BD™ PuraMatrix™ peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration

This study investigated the effects of a membrane conduit filled with a synthetic matrix BD™ PuraMatrix™ peptide (BD) hydrogel and cultured Schwann cells on regeneration after peripheral nerve injury in adult rats.After sciatic axotomy, a 10 mm gap between the nerve stumps was bridged using ultrafiltration membrane conduits filled with BD hydrogel or BD hydrogel containing Schwann cells. In control experiments, the nerve defect was bridged using either membrane conduits with alginate/fibronectin hydrogel or autologous nerve graft. Axonal regeneration within the conduit was assessed at 3 weeks and regeneration of spinal motoneurons and recovery of muscle weight evaluated at 16 weeks postoperatively.Schwann cells survived in the BD hydrogel both in culture and after transplantation into the nerve defect. Regenerating axons grew significantly longer distances within the conduits filled with BD hydrogel when compared with the alginate/fibronectin hydrogel and alginate/fibronectin with Schwann cells. Addition of Schwann cells to the BD hydrogel considerably increased regeneration distance with axons crossing the injury gap and entering into the distal nerve stump. The conduits with BD hydrogel showed a linear alignment of nerve fibers and Schwann cells.The number of regenerating motoneurons and recovery of the weight of the gastrocnemius muscle was inferior in BD hydrogel and alginate/fibronectin groups compared with nerve grafting. Addition of Schwann cells did not improve regeneration of motoneurons or muscle recovery.The present results suggest that BD hydrogel with Schwann cells could be used within biosynthetic conduits to increase the rate of axonal regeneration across a nerve defect.     

FK506 enhances regeneration of axons across long peripheral nerve gaps repaired with collagen guides seeded with allogeneic Schwann cells

We assessed the effects of FK506 administration on regeneration after a 6-mm gap repair with a collagen guide seeded with allogeneic Schwann cells (SCs) in the mouse sciatic nerve. SCs were isolated from predegenerated adult sciatic nerves and expanded in culture using a defined medium, before being seeded in the collagen guide embedded in Matrigel. Functional reinnervation was evaluated by noninvasive methods to determine recovery of motor, sensory, and autonomic functions in the hindpaw over 4 months postoperation. Histological analysis of the regenerated nerves was performed at the end of the study. Using simple collagen guides for tubulization repair, treatment with an immunosuppressant dose of FK506 (5 mg/kg/day) resulted in significant improvement of the onset and the degree of reinnervation. While the introduction of allogeneic SCs did not improve regeneration versus a collagen guide filled only with Matrigel, treatment with FK506 allowed for successful regeneration in all the mice and for significant improvement in the levels of functional recovery. Compared with the untreated group, there was greater survival of transplanted pre-labeled SCs in the FK506-treated animals. Morphologically, the best nerve regeneration (in terms of nerve caliber and numbers of myelinated axons) was obtained with SC-seeded guides from FK506-treated animals. Thus, FK506 should be considered as adjunct therapy for various types of tubulization repair.     

Histological observation on acellular nerve grafts co-cultured with Schwann cells for repairing defects of the sciatic nerve

IN T R O D U C T IO N The bridging of nerve gaps is still one of the m ajor problem s in peripheral nerve surgery. The use of an artificial nerve conduit containing viable Schw ann cell is one of the m ost prom ising ap- proaches to repair nerve defects. …     

Chemoattractive capacity of different lengths of nerve fragments bridging regeneration chambers for the repair of sciatic nerve defects

A preliminary study by our research group showed that 6-mm-long regeneration chamber bridging is equivalent to autologous nerve transplantation for the repair of 12-mm nerve defects. In this study, we compared the efficacy of different lengths (6, 8, 10 mm) of nerve fragments bridging 6-mm regeneration chambers for the repair of 12-mm-long nerve defects. At 16 weeks after the regeneration chamber was implanted, the number, diameter and myelin sheath thickness of the regenerated nerve fibers, as well as the conduction velocity of the sciatic nerve and gastrocnemius muscle wet weight ratio, were similar to that observed with autologous nerve transplantation. Our results demonstrate that 6-, 8-and 10-mm-long nerve fragments bridging 6-mm regeneration chambers effec-tively repair 12-mm-long nerve defects. Because the chemoattractive capacity is not affected by the length of the nerve fragment, we suggest adopting 6-mm-long nerve fragments for the repair of peripheral nerve defects.     

Purification of rat Schwann cells from cultures of peripheral nerve: an immunoselective method using surfaces coated with anti-immunoglobulin antibodies

We report a method for deriving purified rat Schwann cells by immunoselective removal of fibroblasts. Contaminating fibroblasts labeled with antibody against specific surface marker Thy 1.1 are bound on plastic surfaces coated with a second antibody. The efficacy of the method is demonstrated by flow cytometry and by specific Schwann cell Ran-1 immunofluorescence.     

不同无血清培养方式提取SHG44人脑胶质瘤干细胞的比较研究

目的筛选较理想的SHG44人脑胶质瘤干细胞的培养方法。方法采用无血清培养基(含EGF、FGF、B27)以培养皿、培养瓶及悬浮细胞培养板为载体培养SHG44人脑胶质瘤干细胞,通过CD133、Nestin联合Bcl-2标记进行免疫荧光鉴定,并对比其倒置显微镜下细胞形态及干细胞球的形态、大小及数目。结果培养皿和培养瓶初次富集的SHG44胶质瘤干细胞球呈较大不规则形,经二、三次纯化后发现第二与三次纯化后的大部分胶质瘤干细胞呈不规则分化状,大部分胶质瘤干细胞球呈分散、较小、不规则形生长,但第三次比第一、二次有更少的胶质瘤干细胞球却呈更规则球形生长;悬浮细胞培养板提取、纯化的干细胞呈类圆形,基本全部形成胶质瘤干细胞球,且第三次比第一、二次纯化的干细胞球呈更大更规则球形生长;经统计分析悬浮培养板培养纯化的胶质瘤干细胞球较培养皿和培养瓶纯化的胶质瘤干细胞球大且数目多(P0.05);对于悬浮培养板,随着纯化次数增加,胶质瘤干细胞球更大、数目更多(P0.05),且呈更规则球形。结论无血清培养基以悬浮细胞培养板为载体纯化胶质瘤干细胞是较理想的方法。