摘要
去细胞肌肉组织工程支架可作为生物工程支架支持神经元轴突再生。研究表明羊膜上皮细胞可分泌多种神经营养因子,促进神经元轴突的生长,是治疗神经系统疾病良好的种子细胞。本实验将化学去细胞肌肉做为支架材料,以大鼠羊膜上皮细胞做为种子细胞,共同移植入大鼠脊髓半横断损伤处,观察它们对脊髓损伤的治疗效果。
本研究通过冻融和化学萃取方法制备了两种去细胞肌肉组织工程支架,并把它们分别植入大鼠半横断脊髓缺损处,4W后观察两种去细胞肌肉中的轴突再生情况。与冻融去细胞肌肉相比,化学去细胞肌肉可以更好的促进损伤脊髓的轴突再生,支架内再生轴突定向分布。化学去细胞肌肉支架移植不引起明显的炎症反应,其内血管再生良好,对胶质瘢痕的形成有一定抑制作用,化学去细胞肌肉与脊髓具有良好的生物相容性。羊膜上皮细胞与化学去细胞肌肉共同移植入大鼠脊髓(联合移植)后,与单纯化学去细胞肌肉支架移植比较,促进了轴突的再生,并在一定程度上促进了5-HT能神经纤维的再生,更重要的是,联合移植明显的促进了髓鞘的生成,通过BBB评分和神经电生理方法检测发现,联合移植更好的促进了大鼠患肢的功能恢复。
总之,本研究证明化学去细胞肌肉组织工程支架可有效促进损伤脊髓的轴突再生,具有良好的生物相容性;并成功的利用羊膜上皮细胞与支架的共移植治疗大鼠的脊髓损伤,为脊髓损伤的组织工程治疗提供了理论和实验依据。
Spinal Cord Injury (SCI) is a serious debilitating illness resulting from transport, mining accidents and sporting accidents. It is reported that China currently has about 400 000 patients with SCI with an annual increase of about 1 million. The people afflicted with SCI are a big burden to both the society and the family. The pathophysiology of spinal cord injury is characterized by the primary injury followed by secondary injury processes that cause spinal cord hemorrhage, edema, axonal and neuronal necrosis, cavity formation, infarction and subsequent prolonged demyelination, and ultimately lead to the loss of motor and sensory function of spinal cord. At present, the treatment of spinal cord injury, on the one hand, is to prevent secondary pathological changes caused by mechanical injury from aggravating spinal cord damage, and to protect the injured spinal cord. On the other hand the treatment is to promote axonal regeneration and neural pathway reconstruction of the damaged area, to obtain functional recovery of damaged spinal cord. Spinal cord protection strategies can not promote axonal regeneration, but only minimize the destruction of axons and neurons caused by secondary damage. So the promotion of axon regeneration is the key to promote the rehabilitation of damaged spinal cord function in the treatment of spinal cord injury.
The reasons for the mammalian central nervous system unable to regenerate include:①poor regenerative capacity of adult mammalian central nervous system②astrocytes stimulated by the immune response and excitotoxity surround the injured area, creating the glial scar which is the obstacle of axonal regeneration and neural pathway reconstruction.③inhibitors such as Nogo, MAG and proteoglycans to axonal regeneration within the injured spinal cord. Recent studies show that the neurotrophic factors, cell transplantation, tissue engineering, gene therapy and other means can promote axonal regeneration after spinal cord injury which improve the injured tissue microenvironment. The studies also shows that tissue engineering scaffolds in spinal cord injury not only can reduce the glial scar formation and guide axonal regeneration by forming a bridge structure, but also become an important aid tool for cell transplantation. For example, treatments for spinal cord injury by neurotrophic factors and cell transplantation are difficult to overcome the glial scar, but the neural tissue engineering approaches have acquired certain effect. Especially in the case when the primary injury results into partial fracture of spinal cord, nerve tissue engineering scaffolds can provide support and guidance for the regeneration of axons through the gap. Currently, nerve tissue engineering has made great development, but there is no one which can be used in clinical applications. Studies have shown that peripheral nerves can promote axonal regeneration of
injured spinal cord. However, the weaknesses of peripheral nerve limit its further application. In the course of neural transplantation, the study found that extracellular matrix (laminin, fibronectin, heparan sulphate) of neural basement membrane plays an important role in axon regeneration. The skeletal muscle basement membrane is very similar to neural basement membrane, resulting the possibility of using muscle tissue to replace nerve as nerve tissue engineering scaffold. However, the transplantation of fresh skeletal muscle may produce a strong immune rejection. This problem can be solved by decellularization technology which can remove the skeletal muscle cells, effectively eliminating the antigenicity of skeletal muscle as a graft, and keep basement membrane components, making the transplant possible. Indeed, tissue engineering studies have proven that acellular muscle can replace nerve to promote axonal regeneration of injured peripheral nerve. Acellular muscles as nerve tissue engineering scaffolds have many advantages. First of all, the extracellular matrix components of acellular muscle have an important role in cell migration, adhesion, growth and metabolism. Second, the arrangement of acellular muscle structure is similar to that of neural tube, which provides sufficient space to support axonal growth, and is very important for the induction of neuronal regeneration. In addition acellular muscle eliminate cellular components and reduce the immunogenicity, possibly allowing allograft or xenograft. So acellular muscle may be the ideal tissue engineering scaffold for the treatment of spinal cord injury.
The pathological process of spinal cord injury is very complicated. So the effect of a single treatment is often limited. At present, one common strategy of the nerve tissue engineering is to combine the tissue engineering scaffold and cell transplantation for treatment of spinal cord injury. The choice of seed cells is an important aspect of tissue engineering. Several factors must be considered, such as the role of neurotrophic factors released by seed cells, access ways, ethical issues and etc. Amniotic epithelial cells can secrete BDNF, NT-3 and NGF and other neurotrophic factors. After spinal cord injury ,the application of NT-3, BDNF and NGF not only reduce the edema and the blood- spinal cord barrier injury, providing a protective effect on neurons, but also can promote the regeneration of injured spinal cord axons and be beneficial to functional recovery. The study of amniotic epithelial cell transplantation in rat spinal cord contusion injury model found that amniotic epithelial cell transplantation promote axonal regeneration of injured spinal cord and recovery of hind limb function of the animals. In addition, the amniotic membrane is a product derived from fetal which is exposure to maternal immune system surveillance. The cell surface has low human leucocyte antigen-DR expression and no expression of HLA-A, B, C antigens. So its immunogenicity is very low. Amniotic epithelial cells also secrete a variety of soluble molecules which can reduce the specific and non-specific immunological activity of immune cells and play an anti-inflammatory role. And the transplantation of amnion cells did not present the risk of tumorigenicity. Amniotic membrane is a part of postpartum waste tissue. So it has plenty source and no ethics problem. Therefore, amniotic epithelial cells may be the ideal seed cells for spinal cord repair.
This study was to fabricate chemically extracted acellular muscle and freeze-thawed acellular muscle scaffolds, using them to treat hemisected adult rat spinal cord, and comparing their roles in promoting axon regeneration. After going through screening, chemically extracted acellular muscle scaffolds and amniotic epithelial cells were co-transplanted into the hemisected adult rat spinal cord, to observe the effects of treatment. Details are as follows.
1. The fabrication of acellular muscle scaffolds, observation of scaffold’s histological structure, and identification of scaffold’s composition
The acellular muscle scaffolds were prepared by SDS and TritonX-100 extraction or freeze-thaw method. Conventional HE staining and Weigert-Van Gieson staining were used to observe the scaffold’s histological structure, and to identify the scaffold’s composition. The dry-wet weight method was used to detect water content of scaffolds. Image-Pro Plus 6 image analysis software was used to measure the diameter of scaffold’s pipeline. HE staining showed that chemically extracted acellular muscle scaffold completely removes the cell components and retain the fiber components of extracellular matrix, with the spatial configuration of fiber components being the pipeline structure parallel with the muscle, suggesting that the chemically extracted acellular muscle scaffold was successfully made. HE staining showed that in freeze-thawed acellular muscle, nucleus and stripes of cytoplasm disappeared, suggesting that cell structures were damaged and freeze-thaw method also removed living cells. It also showed that freeze-thawed scaffold contained a large number of residual muscle cell components. Between them were the parallel pipeline structures, suggesting that freeze-thawed acellular muscle scaffolds were prepared successfully. Weigert-Van Gieson staining showed that the chemically extracted acellular muscle scaffold was mainly composed of collagen and elastic fibers and that the freeze-thawed scaffold, apart from the above components, also included components of residual myocytes. The dry-wet weight measurements showed that the two scaffolds contained large amounts of water, which is helpful for the transport of oxygen and nutrients. Image analysis showed that the diameters of pipeline in two kinds of scaffolds are about 100 micrometers, which will help the migration and adhesion of cells.
2. Screening of the acellular muscle scaffolds
Chemically extracted acellular muscle and freeze-thawed acellular muscle scaffolds were respectively implanted into the hemisected adult rat spinal cord. At 7d, 14d and 28d after transplantation, ED-1 immunohistochemistry showed, both compared with the injured group, chemically extracted acellular muscle and freeze-thawed acellular muscle scaffolds don't cause significant foreign body rejection. At 28d after transplantation, Holmes silver staining revealed that the chemically extracted acellular muscle can better promote axonal regeneration after spinal cord injury, with regenerated axons in a strikingly organized and linear fashion.
3. The biocompatibility of chemically extracted acellular muscle grafts as biomatrices in experimental spinal cord injury in rats
We further verified the biocompatibility of chemically extracted acellular muscle scaffold. At 28d after transplantation, alkaline phosphatase staining showed that angiogenesis is good in chemically extracted acellular muscle scaffold. GFAP immunohistochemistry showed that the distribution of astrocytes around the chemically extracted acellular muscle scaffold changed in some areas, suggesting the formation of the glial scar is inhibited. HRP neural tracing results showed that chemically extracted acellular muscle scaffold can promote the regeneration of nerve fibers in the spinal cord itself. Therefore, the chemically extracted acellular muscle scaffold has good biocompatibility with rat spinal cord.
4. Chemically extracted acellular muscle scaffold seeded with amniotic epithelial cells to promote spinal cord repair
Amniotic epithelial cells and chemically extracted acellular muscle grafts were co-transplanted into the hemisected adult rat spinal cord. At 28d after transplantation, the immunofluorescence staining found, in comparison with pure scaffold transplantation, co-transplantation promoted the regeneration of nerve axons, and to some extent, facilitated the 5-HT nerve fiber regeneration and, more importantly, significantly promoted the myelination of nerve fibers. The BBB tests and nerve electrophysiological methods also detected the cotransplantation promote functional recovery of rat limb better.
In conclusion, this study showed the immune response after chemically extracted acellular muscle scaffold transplantation is weak. The chemically extracted acellular muscle scaffolds possess good biocompatibility and can promote robust regrowth of axons in spinal cord with regenerating axons in a strikingly organized and linear fashion. This study also successfully used co-transplantation of amniotic epithelial cells and chemically extracted acellular muscle scaffolds to treat spinal cord injury, providing a theoretical and experimental basis for the tissue engineering for spinal cord injury.
引文
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