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The Feasibility Study of Ordered Collagen Scaffold Combining with the Collagen Targeting Neurotrophic Factor-3 on Spinal Cord Injury Repair

Author: TangGuoQiang
Tutor: XuRuXiang；JiangXiaoDan
School: Southern Medical University,
Course: Neurosurgery
Keywords: Collagen Stand Neurotrophin 3 Spinal cord injury Biocompatibility
CLC: R651.2
Type: Master's thesis
Year: 2011
Downloads: 35
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Abstract

Background spinal cord injury (spain cord injury, SCI) is a serious central nervous system trauma, according to statistics: worldwide in 2006, the prevalence rate of spinal cord injury (233-755) / million. In China each year and about about eighty thousand new patients, mostly due to a car accident and fall injuries and other reasons caused spinal fractures, dislocation due often left severely disabled. Motor and sensory dysfunction caused by spinal cord injury, significantly reduced patient survival and quality of life. The traditional treatment is limited to spinal fracture and dislocation of the entire complex, fixed relieve spinal cord compression and rehabilitation treatment, but the treatment of poor efficacy. Over the past two decades, the world researchers do this to make tremendous efforts, but did not achieve significant functional recovery. SCI treatment is still a worldwide medical problem. As we all know, spinal cord injuries are often caused by a series of pathological changes, such as the mechanical force is a direct result of neuronal cell death; interrupt the continuity of the high central; injured segments beyond projections suffer Wallerian degeneration; spinal cord stump glial limiting membrane formation and retraction as well as secondary damage increased primary damage area. In the cases of humans with spinal cord injury, the damage length may reach 1cm, even more extensive. Nerve regeneration after injury, however, is a complex process, although there will be endogenous reconstruction: and axonal sprouting growth, inflammatory cells, endothelial cells, and Schwann cells invade the damaged area. But just rely on these internal repair reconstruction improve the functionality is very limited. Years of research show that in the past caused axonal regeneration is limited because at least contain the following points: 1, the lack to support axons extend through the damaged area of ??the matrix; lack of the necessary neurotrophic support; 3, the damaged area of ??the myelin and cell the presence of inhibitory factor; limited regenerative capacity of mature neurons; inflammatory reaction caused more serious secondary damage; chronic injury there are more obstacles, such as glial scar formation around the damaged area; suffer chronic injury The decline in the degree of sensitivity of the neurons regeneration gene, even atrophy. Nearly 20 years, the majority of researchers for a variety of factors that affect nerve regeneration, do a lot of attempts, such as to improve the survival of neurons after injury, the inhibition of the growth of glial scarring after injury, seed cells or stent grafts as well as to promote the application of renewable factor etc., have made the repair effect. Subsequently, an increasing number of studies have shown that combination therapy strategies used in spinal cord injury repair more effective than a single treatment strategy. Decomposition of chondroitin sulfate proteoglycan (CSPG) molecular sugar chains chondroitinase ABC (ChABC) carrying stent application Schwann cells promote axonal regeneration through the graft - host interface; dip neurotrophic factor Schwann cells stents improve regenerated axons into the bracket internal; the neurotrophic factor cyclic adenosine monophosphate joint application to promote axonal regeneration through the damage zone; Schwann cell co-transplantation of neural stem cells overexpressing neurotrophic factor to promote injury after spinal cord Functional and structural improvements. Neurotrophic factor of concern due to neuronal survival and neurite outgrowth play an important role. Members of his family, including nerve growth factor (Nerve growth factor, NGF), brain-derived neurotrophic factor (Brain-derived neurotrophic factor, BDNF), neurotrophic factor -3 (is neurotrophin-3, NT-3), neurotrophic factor - 4/5 (Neurotrophin-4/5, NT-4/5), neurotrophic factor -6 (Neurotrophin-6, NT-6), and the like. They are growth, differentiation and maintain needed for different subtypes of neurons. NT3-mRNA is widely expressed in the central nervous system, and the NT3 can combine Trk tyrosine kinase receptor C (TrkC) activation downstream of Rho GTPases family, acting on the cell microtubules and actin filaments promote neurite outgrowth extend; NT3 is also important mediators regulating myelin formation, can promote the growth of important motor pathways such as the corticospinal tract (corticospinal tracts, CST), it is particularly important in the study of spinal cord injury repair. However, due to the neurotrophic factor in the high cost of its wide range of application is limited. Has been to try various ways exogenous neurotrophic factor is applied to the study of spinal cord injury in order to overcome the defect of the exogenous administration way, comprising: a damage at the direct injection, transplant osmotic pump, the collagen matrix of the transplant infiltration NT3, viruses carrier transit NT3. However, after this single exogenous administration, although there are some pro-damaged neurite regeneration, but regeneration protrusions often disordered random growth can not be achieved close to the original spinal cord conduction loop function to improve the role of limited. Currently, due to the rapid development of the tissue engineering, various sources of materials such as: polymer materials polylactic acid polyglycolic acid copolymer (PLGA), collagen, etc. is made into conducive stent for study of spinal cord injury, as well as bracket joint made of a variety of strategies and common application to obtain a certain effect. The collagen material due to its easy access, plasticity, and good biocompatibility advantages are widely used to repair the study. Our group through the early neurotrophic factor 3 (NT3) transform, build collagen-binding domain of NT3 (collagen-binding domain neurotrophin-CBD-NT3), so that it can be combined with collagen-specific, and has made more positive effect. This issue through targeted neurotrophic factor ordered collagen scaffold joint collagen -3 guide cell migration processes growth and its biocompatibility research to prove the feasibility of such a joint strategy for spinal cord injury repair. The first part of the orderly collagen scaffold collagen targeting NT3 guide cell processes oriented growth Objective: To study the natural and orderly collagen material combined with collagen targeting domain NT3 extend cell processes affect explore this combination strategy for spinal cord injury repair significance lay the foundation for further joint cell transplantation in the treatment of spinal cord injury. Method: extracted from SD rat tail tendon collagen scaffold was prepared sufficient amount, remove adhesion organizations as much as possible, and trimmed to the appropriate size of the sample cell and sterilized to go further. The effect of the use of the HE staining evaluation stent to cell processing; soaked acellular scaffold after sterilization load CBD-NT3 24h SD rats with neonatal dorsal root ganglion cells (Dorsal root ganglion cells, DRGs) in vitro co-culture while setting NT3 PBS group served as controls. FDA (Fluorescein diacetate) cell line bracket 1,3,5 d staining and measurement analysis the protrusions extending the length and angle of the cells in each group; culture 3d, the use of scanning electron microscopy of training after stent Ultrastructure of dorsal root ganglion cells, analysis of the impact of the stent landscape of cell morphology and neurite extension of. RESULTS: HE staining stent cell components substantially removed after treatment, and the the untreated collagen scaffold visible inside more nuclear staining, and simultaneously visible After treatment, the collagen scaffold untreated collagen material form substantially consistent; Total culture 1d cell protrusions extending the length of the stand was no significant difference (P gt; 0.05), but after stent cells begin adherent growth extends to 3d CBD-NT3 group cell protrusions extending the length of the longest, followed by the three groups the NT3 group, PBS group minimum, the the CBD-of NT3 group of cell processes migrate the length difference between the two groups was statistically significant (P lt; 0.05), but CBD-NT3 group and NT3 cells in culture 5d projection extends The length of the difference was not statistically significant (P gt; 0.05), but still higher than the PBS team leader (P lt; 0.05). Cell growth and extends bracket projections presented along stent long axis oriented growth trend, and cell protrusions outside the stent was disordered random growth; the cell protrusion extending angle further: cell protrusions extending direction of the overall collagen scaffold fiber long axis remained parallel, the angle between lt; 30. . Direction with extended distance and projection extends not only did not deviate from the long axis of the stent fiber, but have a tendency to parallel the trend; scanning electron microscopy showed that the cell is to rely on the stent surface topography anchoring and growth. Conclusion: rat tail tendon after this acellular approach can achieve better go cell effect, but also convenient and easy, while still maintaining the original tendon collagen fiber structure of vertical; NT3 combined with a collagen targeting joint ordered collagen scaffold play a directional guide and to better promote cell protrusions extending the double effect can provide an effective therapeutic approach for spinal cord injury repair research. The second part of the collagen scaffold Cytocompatibility their joint collagen targeting NT3 in vivo transplantation of tissue compatibility purposes: research targeted NT3 in vivo transplantation of tissue cell proliferation as well as its joint collagen treatment collagen scaffold learning This combined strategy of reaction to evaluate the feasibility of in vivo applications. Methods: go the cells were treated with stents and bone marrow stromal cells in vitro co-culture as a control group, to separate bone marrow stromal cells, respectively, in the 1, 2, 3 d use of CCK-8 measurement of cell proliferation; the untreated stents , went cells were treated with stents and load CBD-of NT3 NT3 treated collagen scaffold line Wistar rats back subcutaneous implants, each rat back side in order in accordance with the unprocessed bracket, go cell treatment bracket load there the treated collagen scaffold load CBD-NT3 NT3 the processing order of the collagen scaffold implanted on both sides implants 8, a total of six rats, respectively, drawn in one week and four weeks later, and HE staining Evaluation rats subcutaneously buried plants histological reactions. Results: The treatment by the acellular scaffold with bone marrow stromal cells co-cultured CCK-8 testing found that the co-cultured cells and separate the two groups of cell culture in 1d, 2d, 3d of 0D values ??were not significantly different (respectively 1d: P = 0.520; 2d: P = 0.601; 3d: P = 0.810); the Norplant histological results show: the reaction cell density than the untreated group less deal with the outer periphery of the stent group bracket in one week and four weeks, The difference was statistically significant (one week: P = 0.000; 4 weeks: P = 0.024), in one week, two groups invasive stent inside the cell density no significant difference (P = 0.067), to four weeks unprocessed stent group invasive stent internal cell density than the treatment group, the difference was statistically significant (P = 0.000). Contrast the treated collagen scaffold Joint NT3 treated collagen scaffold joint CBD-NT3 histological reaction: in one week and four weeks, regardless of stent outer periphery reaction cell density or invasive stent inside the cell density were not statistically different (P gt; 0.05). Conclusion: The treatment acellular collagen scaffold does not affect the proliferation of bone marrow stromal cells, and inflammatory response in the body than untreated collagen scaffold slight; CBD-NT3 in vivo implants experiment that does not appear in the first week and after January cause tissue inflammation exacerbated the situation, NT3 treated bracket built jointly with the binding domain of the collagen targeting can be used for joint seed cell transplantation in spinal cord injury repair as well as multi-strategy collaborative study of the treatment of spinal cord injury.

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