Acellular Spinal Cord Scaffold Seeded With Mesenchymal Stem Cells Promotes Long-Distance Axon Regeneration And Functional Recovery In Spinal Cord Injured Rats

Background:The spinal cord is an important part of the central nervous system, and It is almost have no ability to regenerate by itself after spinal cord injury. The most cases of spinal cord injury always result in loss of limb movements and sensory function, or even death. Those patients caused enormous impact of life and society. Stem cells and tissue engineering made some progress in the treatment of spinal cord injury in recent years, and it is a hot research topic. However, there is still no effective treatment to meet clinical needs. Typically, natural non-intervention in the case of spinal cord injury experienced primary injury and secondary injury pathological processes which include:①The death and apoptosis of spinal cord nerve cells, and lack of ability to repair by itself;②The lack of neurotrophic factors and micro-environment change;③The presence of inhibitory factor in Central nervous system;④Boot system degradation causes axons can’t be growth in the right direction;⑤Proliferation of fibroblasts, astrocytes, microglia cells and epithelial cells to form glial scar in the Injury site, which hinder spinal cord regeneration. Moreover, the primary damaged cells release toxins and can cause secondary damage on both up and down sides of the spinal cord tissue. Due to the complexity of the pathology of spinal cord injury, the treatment strategy of spinal cord injury should include a number of factors and variety of treatment measures. It is not only to protect the remnants of nerve neuron survival, but also can regulate the micro-environment of the injured spinal cord and promote axonal regeneration and reconstruction of nerve reflex loop. Now days, the spinal cord injury treatment principles are as follows:①Supple neurotrophic factors to promote axonal survival and regeneration;②Reduce inhibitory factors in the central nervous system;③Reduce cell degeneration and losses;④Cells transplantation to replacement of the damaged cells;⑤Reduce scarring and cavities in favor of the axonal growth. Unfortunately, so far there are no animal studies of spinal cord injury meet the clinical requirements. Because of many defects in the traditional allograft and synthetic scaffolds, Such as:a limited cell sources, cell function transformation, ethical issues and immune rejection, among them the most important is that the artificial scaffold cannot mimic the three-dimensional structure of the extracellular matrix in the target tissue. Not to mention the regeneration of axons through the bracket, it is easy to get lost disseminated growth direction. How can we overcome these unfavorable factors for nerve regeneration? Using tissue engineering technology as the cells, scaffolds, and growth factors may be able to help spinal cord injury recovery and reconstruction. Basic and clinical research in recent years also confirmed that tissue engineering techniques can help restore tissue function and structure reconstruction. The progress of seed cells in biological scaffold graft repair of spinal cord injury has to be potential research strategy in spinal cord injury repair.Using Stem cell seeded in scaffold as a graft in treatment of SCI is a hot research point. Stem cell has ability of self-replication and differentiation potential, and it’s the origin of cells of various tissues and organs in living organisms. Microenvironments determine the capable of self-renewal and differentiation for different types of cells to replace diseased or aging cells. Typically, Stem cells divide into embryonic stem cells, and adult stem cells. The adult stem cells can be derived from the bone marrow, peripheral blood, cord blood, and they have the nerve cell differentiation potential and the secretions of growth factors, prompted impaired axonal regeneration through the spinal cord cavity damage zone, and to whom provide beneficial environment or replacement of damaged cells. The Umbilical cord blood mesenchymal stem cells have the following obvious advantages compared to other sources:①Rich sources and readily available;②The acquisition convenient of donor with no pain;③Cord blood is in the primitive stage of immune system after transplantation, which caused light acute and chronic graft-versus-host response in the incidence and severity;④Cord blood have primitive hematopoietic stem cells, mesenchymal stem cells and endothelial progenitor cells, have a stronger proliferation, differentiation capacity;⑤Umbilical cord blood in a variety of relatively few opportunities for infection.The ideal scaffold should have many different characteristics:bionic three-dimensional structure and pore network to communicate with each other, have similar biomechanical properties with the target tissue, good biological compatibility and the cytokines transmission function to promote and guide cell and tissue regeneration. Recently It is reported the acellular spinal cord matrix scaffolds have a high degree of bionic characteristics, compared to a variety of artificial scaffold composite spinal cord tissue characteristics. It has Potential as spinal cord repair scaffold which means bridge on both sides of spinal cord, promote the host regeneration of nerve cells and inhibit glial scar formation; It would be able to guide the implanted seed cell proliferation, directed differentiation and migration and guide axons directional extension to rebuild spinal cord loops, and promote the repair of spinal cord function.The acellular spinal cord scaffolds repair of spinal cord injury in vivo has not reported yet. This study aim to use acellular spinal cord scaffold seeded with xenograft cells of human umbilical cord blood-derived mesenchymal stem cells as the graft to repair spinal cord injury and observe the function recovery of rat were, to explore the mechanism for spinal cord injury by histology, apoptosis and local immune status. this study may provided the basis and foundation for the future clinical treatment. Objective:1. Prepared the acellular spinal cord scaffold, observing the characteristics of the skeleton structure of the spinal cord matrix; isolated human umbilical cord blood-derived mesenchymal stem cells in vitro and identification;2. Explore the acellular spinal cord scaffold seeded with human umbilical cord blood-derived mesenchymal stem cells promote axonal growth regeneration and functional recovery effect of long distance of the spinal cord injury, and explore its mechanism.3. Explore the mechanisms of the rat spinal cord injury treat with the acellular spinal cord scofflds seeded with umbilical cord blood mesenchymal stem cells from the point of view of apoptosis and local immune status.Chapter I Regeneration of Spinal cord with Acellular Spinal Cord Scaffolds:An in vivo study in rat model of Hemi-sectioned spinal cord injuryObjective:Acellular spinal cord (ASC) scaffold was prepared through chemical extraction, and in vivo promote spinal cord regeneration on lateral hemisected thoracic spinal cord in adult SD Rats. And to test the effect of ASC scaffold on functional recovery after spinal cord injury in vivo study by supporting adhesion, proliferation of host neural cells and axonal remyelination.Methods:Twenty-four adult Sprague Dawley (SD) rats were conducted lateral hemisection in thoracic spinal cord to establish SCI model. The ASC scaffolds were implanted into the SCI lesion gaps in ASC group (n=6) while the lesion only as injury only group(n=6). The Basso, Beattie, and Bresnahan (BBB) locomotor test were conducted to assess neurologic function weekly for8weeks.14day and56day after SCI, six rats in each group were sacrificed and measured under histological, immunohistochemical and MBP examination.Results:The ASC scaffold general looking is milky white, the lack of toughness and softness; The cross-section of it is a network structure of the extracellular matrix constituent, The most of the cells and myeloid have removed. Moreover, The ASC scaffold arranged in parallel fibrous structure and cross-section has a three dimensional network structure. In animal test the ASC scaffold could integrate well with the host spinal cord at the spinal cord gap and have beneficial effect on functional recovery (p<0.05). HE staining showed the group of spinal cord injury destroy the damaged spinal cord dorsal and ventral right part. The ASC scaffold graft group:The ASC scaffold have very good integration into the rat spinal cord hemisection defect and seen a large number of cell nuclei distributed in the ASC scaffold. Immunohistochemical showed few astrocytes was observed on ASC graft, forming an astrocyte-free area and, also, a few neural stem cells and neurons were observed on grafts. Among them, oligodendrocytes is main cell type, distributed in ASC graft at day56post-SCI. Some remyelination of ingrown axons had taken place inside in ASC scaffold.Conclusion:ASC Scaffold, as a potential scaffold for SCI, could provide a microenvironment favorable to differentiation or proliferation of host oligodendrocyte and promote axon remyelination.Chapter II Acellular Spinal Cord Scaffold Seeded with Mesenchymal Stem Cells Promotes Long-Distance Axon Regeneration and Functional Recovery in Spinal Cord Injured RatsObjective:Stem cells based experimental therapies are partially successful for the recovery of Spinal Cord Injury (SCI). Recently, acellular spinal cord (ASC) scaffolds which mimic native extracellular matrix (ECM) have been successfully prepared. This study aimed to investigate whether the spinal cord lesion gap could be bridged by implantation of bionics designed ASC scaffold, either alone or seeded with human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), and their effects on functional improvement.Methods:A laterally hemisected SCI lesion was performed in adult Sprague-Dawley (SD) rats (n=36) and ASC scaffolds were implanted into the lesion immediately, either with or without seeded hUCB-MSCs. All rats were behaviorally tested using the Basso-Beattie-Bresnahan (BBB) test once a week for8weeks. we detect nerve cells by immunofluorescence at second and eighth week after SCI, and completed BDA tracer at eighth weekResults:The three passgers of umbilical cord blood mesenchymal stem cells identified by flow cytometry:the cells with low expression of CD14(0.31%), CD34(0.82%), CD45(0.23%), HLA-DR (0.26%), and high expression following adhesion molecule CD90(99.99%), CD29(92.95%), CD44(99.98%) CD105(99.92%) and CD73(99.88%), and it is consistent with identification of mesenchymal stem cell phenotype reported before; human umbilical cord blood mesenchymal stem cells seeded in ASC scaffold were cultured in vitro for two days, and gross looking is white, soft and distribution of5-bromodeoxyuridine (Brdu)-positive human umbilical cord blood-derived mesenchymal stem cells in the scaffold of extracellular matrix composed of a three-dimensional network. Behavioral analysis showed that there was significant locomotor recovery improvement in combined treatment group (ASC scaffold and hUCB-MSCs) as compared with the control SCI only group (no scaffold)(p<0.01). Brdu-labeled hUCB-MSCs could also be observed in the implanted ASC two weeks after implantation. Moreover, host neural cells (mainly oligodendrocytes) were able to migrate into the graft. Biotin-dextran-amine (BDA) tracing test demonstrated that myelinated axons successfully grew into the graft and subsequently promoted axonal regeneration at lesion sites.Conclusion:Our study provides the first experimental evidence that ASC scaffold seeded with hUCB-MSCs is able to bridge a spinal cord cavity and promote long-distance axon regeneration and functional recovery in SCI ratsChapter Ⅲ The local immune regulation and apoptosis research on Acellular Spinal Cord Scaffold Seeded with Mesenchymal Stem Cells to repair spinal cord hemisection defectsObjective:explore the apoptosis and immune regulation mechanisms of rat spinal cord hemisection model of spinal cord injury repaired by Acellular Spinal Cord Scaffold Seeded with Mesenchymal Stem Cells Method:Spinal cord acellular scaffold seed with human umbilical cord blood-derived mesenchymal stem cells as graft.36adult SD rats were randomized to three groups:group A, the SCI only group (n=12); group B, SCI+ASC scaffold (n=12); group C, SCI+ASC+hUCB-MSCs (n=12); The rat underwent spinal cord hemisection respectively implanted corresponding group. Two weeks after operation, Three animals of each group detect markers of immune cells and Tunel-positive cells by immunofluorescence and count positive cells; another three animals in each group under caspase-3activation degree detection, counting the OD value. Using analysis of variance to test the statistical differences in each group the number of positive cells and caspase-3activation OD values, differences between groups of homogeneity of variance with LSD test, P<0.05indicates statistical difference.Results:two weeks after operation the group of SCI+ASC+hUCB-MSCs significantly inhibited Tunel positive cells (P<0.05) and caspase-3activation (P <0.05), it means this group inhibit apoptosis of local neural cells; On the contrary, the ASC scaffold group did not inhibit apoptosis. Moreover, the group of SCI+ASC+hUCB-MSCs significantly inhibited neutrophil (P<0.05), macrophages (microglia)(P<0.05), T lymphocytes (P<0.05) proliferation in spinal cord injury area. While, The ASC scaffold group doesn’t significant inhibit immune response, It compared with SCI only group that there is no obvious differences on inflammatory positive cells (P>0.05). Furthermore the three groups was no significant difference on1gM positive expression (P>0.05).Conclusion:The Acellular Spinal Cord Scaffold Seeded with Mesenchymal Stem Cells to repair spinal cord hemisection defects promote founction recovery through suppression of local immune cells, and inhibit apoptosis.