| Spinal cord injury (SCI) is a kind of serious injury in central nervous system diseases, according to statistics:in 2006, the incidence of spinal cord injury around the world is 233-755/million. About 80,000 new cases a year in China, more of which suffered spinal fractures or dislocations caused by the car accident or other accidents, leaving severe disability. Motor and sensory dysfunction caused by spinal cord injury significantly reduced the survival rate and the life quality of the patients.However, the traditional method of treatment is limited, such as fixing spinal fracture and dislocation, removing spinal cord compression and rehabilitating therapy. And the curative effect was still not satisfied. Although in the past two decades, researchers around the world have made great efforts, limited functional recovery was obtained. The treatment of SCI is still a worldwide medical problem.As we know, spinal cord injury often leads to series of pathological changes, such as mechanical forces leading to neuronal cell death directly; and continuity interrupted; distal segment suffered Waller degeneration; the two stumps form a new glia limitans at their ends with a glial scar underneath it and retract apart; and secondary injury increasing damage area and so on. While in human, the gap between the two stumps may be more than 1 cm away. However, nerve regeneration after injury is a complex process, although there will be endogenous reconstruction,such as:axonal sprouting, inflammatory cells, endothelial cells and Schwann cell invade to the damaged area and so on. However, solely relying on these internal reconstruction for improving function is very limited. Over the years, studies have shown that the reasons causing axonal regeneration is limited at least contain the following points:1, Lack of substrate to support axon extension through the damaged area; 2, Lack of the necessary neurotrophic factor; 3, The inhibitor of myelin sheath and cell inhibitory factor were existed at the damaged area; 4, Regenerative capacity of mature neurons is limited; 5, More serious secondary damage caused by inflammatory reaction. And there are more barriers to chronic injury, such as:glial scar formation around the damage zone; the sensitive degree of neurons suffered from chronic injury for regeneration related gene was declined, or even atrophy.For the past 20 years, Broad researchers have made many attempts to overcome a variety of factors influenced nerve regeneration, such as increasing survival of neurons being injured, inhibiting the growth of glial scars, transplanting stem cells or scaffolds, and applying growth factors. All of these have obtained great effect. Subsequently, more and more studies show that the combined treatment strategies are more effective than single treatment strategy for spinal cord injury repairing. For example, the chondroitinase ABC (ChABC) can decompose the sugar chain of chondroitin sulfate proteoglycan (CSPG) molecule combined with scaffolds carrying Schwann cells can facilitate regeneration of axon through the graft-host interface; Schwann cells adhered to scaffolds soaked with neurotrophic factor can help axons regenerate into the internal of scaffold; The application of neurotrophin-3 combined with cAMP promots axon regenerate through the damage zone; Neural stem cells combined with Schwann cells, which over-expressed neurotrophic factor, were transplanted for spinal cord injury, and the function and structure could be ameliorated.Neurotrophic factor have attracted much attention because of its important role in neurite outgrowth and survival of neurons. The members of this family, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), neurotrophin-6 (NT-6) and so on. They are needed for growth, differentiation and maintenance of different subtypes of neuron. Because NT3-mRNA is widely expressed in the central nervous system, and NT-3 can through combining tyrosine kinase receptor C (TrkC) activating Rho GTPases, and acting on the microtubules and actin filaments in cells to promote neurite extension; also, NT-3 is an important media for regulating myelination, can facilitate corticospinal tract (CST) growing, etc., All of these make it especially important in the study of spinal cord injury repairing. However, due to the high price of neurotrophic factors, its application was limited. in order to overcome the shortcomings of exogenous neurotrophin, there has been trying various ways to utilize exogenous neurotrophic factor for spinal cord injury research, including:direct injection at the injury, transplanted micro osmotic pump, transplanted collagen matrix soaked with NT3, the virus carrier transport NT-3 and so on. However, although there are a certain effect for damaged nerve regenerating by these single ways, but the regeneration of neurite are often random and can not reach the original circuit of the spinal cord conduction approximatively,and the function improvement is limited.Currently, due to the tissue engineering development, all kinds of materials were made for spinal cord injury repairing research,such as:Poly lactic glycolic acid copolymer (PLGA), collagen,etc,and the application of scaffold combined with other various strategy has obtain a certain effect. Collagen are widely used in rehabilitation research because of its easy obtaining, good malleability and biocompatibility, etc. Our research group has transformed neurotrophin-3 (NT-3) to a new neurotrophin which has a collagen binding domain,named CBD-NT3, so that it can specifically bind with collagen, and it has already achieved a certain effect. This topic is seeks to explore the role of this collagen-binding NT3 combined with solid and orderly collagen scaffold to promote the growth of cell processes, and to evaluate the feasibility of such combinatorial strategies by cell proliferation and the inflammatory response in vivo.Part One:Ordered Collagen Scaffold Loaded With Collagen-targeting NT-3 Guide The Processes Of Cell Extend DirectionallyObject:To investigate the effect of ordered collagen scaffold loaded with collagen-binding domain neurotrophin-3 (CBD-NT3) on the extension of cellular processes of dorsal root ganglions(DRGs), and explore the significance of this kind of combinatorial strategies in the spinal cord injury repair, and lay the foundation for combining cell transplantation on spinal cord injury.Methods:Rat tail tendon were extracted from SD rat for preparing sufficient collagen scaffold, removing the adhering tissue, and trimming the sample to appropriate size,and then removing cell components and sterilized further. Evaluate the effect of the treatment for scaffolds by HE staining. After be soaked with CBD-NT3, the sterile acellular scaffolds which the cellular components had been removed were co-cultured with dorsal root ganglion cells isolated from SD rat. While setting NT-3,PBS group as control. Cells on the scaffold were stained by Fluorescence dye on the time of 1,3,5d for measuring the length and angle of the processes of each group. After be co-cultured for 3d, dorsal root ganglion cells and morphology of scaffold was observed in scanning electron microcopy. And analyse the impact of topography of scaffold for processes extension.Results:The results shows that the cellular components in scaffold were removed completely after being treated, while there are lots of nuclear staining in untreated scaffold. And the morphology of the scaffolds in two groups are similar; co-cultured for one day, the extension length of processes have no significant difference among the three groups (P>0.05), and then the cells on scaffolds began to stick wall and grew, at the time of the third day,the length of processes elongation of group CBD-NT3 was the longest one than that of group NT3 and group PBS,the difference has Statistical significance (p≤0.05); at the time of the fifth day, the length of processes elongation between group CBD-NT3 and group NT3 were similar, the difference has no Statistical significance (P>0.05),but still longer than group PBS (P<0.05).And the cellular processes on the scaffold have a tendency of extending along the long axis of scaffold.but the cellular processes out of scaffold were random. Angle analysis results indicate that the 95% confidence interval of the angle between the line which the process emerges from the cell soma to the growing tip of the process and the long axis of fiber was (18.8,20.7)degrees, Approximate parallel of the long axis of fiber. And with the extension range increased, extension direction of cellular processes not only have not deviate from the long axis of fiber,but also Have the tendency of parallel; the results of scanning electron microscopy showed that cells can rely on the topography of scaffold for anchoring and growing.Conclusion:By this way, cell components in rat tail tendon was removed adequately; The combinatorial strategies of collagen scaffold with CBD-NT3 can play a double function for oriented guiding and processes extend effectively. This may provide a better therapeutic approach for spinal cord injury.Part Two:Cellular compatibility of collagen scaffold and it's histocompatibility after be transplanted with CBD-NT3Object:To research the influence for cell proliferation of the processed collagen scaffold and it's histocompatibility after be transplanted with CBD-NT3. and evaluate the feasibility of this combinative strategie applied in vivo.Methods:Co-cultured bone marrow stromal cells with collagen scaffold in vitro, Separate bone marrow stromal cells as control group. In 1,2,3d Respectively, measuring the cell proliferation of each group by CCK-8; untreated scaffold and treated scaffold and treated scaffold loaded with CBD-NT3 or NT3 were embedded in SD rats back subcutaneous respectively, Preparation 4 places in each side of every rats,6 rats in total. Specimens were drew After 1 week and 4 weeks respectively, Evaluate the histologic response of buried plants in rat subcision by HE staining.Results:The results shows that the OD value of the cells co-cultured with collagen scaffold has no statistical significance to another group in the 1d,2d, 3d(ld:P=0.339; 2d:P=0.861; 3d:P=0.289). The results of subcutaneous histological response display that:After 1 week and 4 weeks,the reacted cell density out of the processed scaffold was lesser than untreated scaffold, The difference between the two groups has a statistical significance (1 week:P=0.000; 4 weeks: P=0.024);while after 1 week,comparing the two groups, cell density in scaffold has no statistical significance (P=0.067), after 4 weeks, the reacted cell density inside of the processed scaffold was lesser than untreated scaffold, The difference between the two groups has a statistical significance(P=0.000). Comparing the histological response between scaffold loaded with NT3 and scaffold loaded with CBD-NT3, The results shows that:No matter inside scaffold or not,the cell density has no significant difference between the two groups(P>0.05).Conclusion:The processed scaffold doed not affect the cell proliferation of bone marrow stromal cells,and its inflammatory response in vivo is slighter than untreated scaffold; The experiment prove that The combinatorial strategies of collagen scaffold with CBD-NT3 did not aggravate inflammation response in vivo.it can be used for spinal cord injury repair and subsequently combining cells transplantation to treat spinal cord injury in research. |
PDF Full Text Request