| Introduction:Regeneration and functional reconstruction after spinal cord injury (SCI) has been a difficult problem to solve in the medical community. SCI, the annual incidence of spinal cord injury is 20-40/1,000,000 in most countries, is in a rising trend year by year with the rapid development of economy and improving the efficiency of transportation in our country. Morbidity, often leading to lower quality of life, is very high after SCI. And the large number of paraplegia patients with severe dysfunction brought their families and society huge economic losses and social burden.For a long time, people don't think the central nervous system (CNS) possess regenerative capacity. The regeneration of CNS was not known by people gradually since the Richardson published a paper in Nature. In recent years, the experimental results showed that the CNS regeneration capability related to the type of nerve cells, development degree and its distance from the center of the injury, as well as producing waste by myelin degradation after injury, damaging ion steady state, changing of neurotransmitter release, neurotransmitter and receptor dysfunction and immune inflammatory reaction.As spinal cord complex anatomical structures would be changed by SCI, which damaging the normal physiology function of spinal cord, so the recovery of the corresponding anatomical structure after injury is the necessary premise of functional recovery. Tissue engineering materials, due to their advantages such as good mechanical properties and plasticity, is becoming a possible direction of regeneration after SCI repair. Many studies have shown that biological materials, as a carrier of the neural stem cells, can not only be carrying neurotrophic factors and other cytokines, but be providing mechanical support, reducing the formation of glial scar in traumatic area after SCI. An ideal biological material should have good biocompatibility, not cause other adverse reactions; possess good plasticity and mechanical properties in order to be processed into variety of objective forms; need to have good interface to facilitate adhesion cell growth; also shall be stable or adjustable degradation rate in order to adapt to different cells and nutritional factors play a role of time; and degradation products should be non-toxic side effects. Existing tissue engineering materials can be roughly divided into the following categories:(1) the natural biodegradable polymer; (2) synthetic biodegradable polymers, (3) synthesis non-biodegradable polymers, (4) composite materials. The application forms of these materials contains support, microspheres, electrospinning, casing, et al.Other drugs have not yet been widely endorsed or have been restricted by other factors to practical application while methyl prednisolone has been universally recognized and used in SCI.This study investigated the feasibility of the poly-propylene carbonate (PPC) electrospun fibers combined with chitosan microspheres for the repair of spinal cord injury. We also researched the effect of the locally sustained releasing drugs by this composite biodegradable implant for axonal regeneration and functional restoration after the experimental SCI.Production of biodegradable drug-loading polymer materialsThe sustained delivery materials have been applied in SCI increasingly due to its good biocompatibility, biodegradable and sustained release drugs and other characteristics. Most of biodegradable polymer materials would be degraded to acid in the body, which brought the possibility of secondary injury after SCI, so the poly(propylene carbonate) (PPC), which can be eventually degraded into carbon dioxide and water in the body and has good biological compatibility, has been chose in our experiment. Chitosan, which has good biocompatibility and degradability and can dissolve in water, can load protein drugs and keep their activity.Preparation of PPC electrospinning containing db-cAMP alone:Powder PPC and dbcAMP were weighed exactly and poured into a 30 mm × 50 mm weighing bottle. Acetonitrile was added into the bottle, and then the mixture was vortexed for 6 h until the powder was dissolved in the solution completely. The weight ratio of PPC and dbcAMP was 9:1. The solution was used for electric spinning operation while the grounding flat tin foil was used as a receiver.Preparation of chitosan microspheres containing chondroitinase ABC:Chitosan (CS) was weighed and poured into a 30 × 50 mm bottle that was filled with 1% w/w aqueous acetic acid solution. The mixture was then stirred for 6 h until the powder was completely dissolved in the solution, then got A solution. PVP was dissolved in deionized water to create a 1% PVP solution (B solution). ChABC was then added to the PVP solution (C solution), and isovolumetric CS and PVP solutions were mixed and stirred for 0.5 h (D solution). The D solution was dropped into the tripolyphosphate 5% w/w solution, and the resulting mixture was stirred for 3 h. The final mixture was centrifuged at 2000 g for 30 min; the precipitate was washed twice with deionized water and then freeze-dried. Finally, we obtained CS microspheres either with or without ChABC.The preparation of electrospinning-microspheres contains ChABC and electrospinning-microspheres contains both ChABC and db-cAMP:CS microspheres contains ChABC were weighed and poured into a 30 × 50 mm bottle filled with dichloromethane, followed by PPC db-cAMP powder was added to the bottle, and the mixture was stirred for 6 h until the powder completely dissolved in the solution, then we got milky solution. The electrospinning process was performed while the grounding flat tin foil was used as a receiver. The electrospinning can be collected while the distance and voltage between the nozzle and a grounded collection target was 15 cm and 10 kV. We can got the electrospinning contains ChABC alone while former method without the db-cAMP powder.The formation of spinal cord hemisection model of ratWe would like to learn the possibility of application in SCI for sustained delivery materials, observe the axon regeneration and whether it can penetrate out of the glial scar, so we chose the model of spinal cord hemisection which benefit to observing axon regeneration. The process is described as follows:Female rats were anesthetized and fixed on the operating table.The T7-T9 spinous were exposed after shaving and disinfection. A laminectomy was then performed, and after the posterior median artery was identified, the spinal cord was right hemitransected using a No.11 scalpel blade. According to different groups, respective materials were implanted into the injury site after confirm to stop the bleeding,Postoperatively, animals were given penicillin for the first three days to prevent infection (intraperitoneal injection,200,000 U), and bladder voiding was performed three times a day until automatic micturition recovered.Research on the feasibility of application and effects of the electrospnning containing db-cAMP in spinal cord hemisection model of ratAfter model was built and the materials were implanted into the injury site, we closed the wound and given them postoperative care. The rat motor function recovery, which evaluated by BBB rating, were observed and tissues were picked in 1,2,3,4 weeks. Immunotluorescence and immunohistochemical staining was applied to observe axon regeneration, scarring and cavitation formation and to evaluate the effects of materials for functional recovery axon regeneration after spinal cord hemisection.In research on the application of PPC electrospinning contains db-cAMP alone for the rat spinal cord hemisection injury, the electrospinning has been produced with the mixture of PPC and db-cAMP. Then the release data was received in vitro test which simulated in vivo.The results showed that the release time of db-cAMP could be extended to 8 days with the electrospinning and the release rate was stable. BBB was applied to evaluated the motor function of rats in the time points of 1,2,3,4 weeks after SCI, then immunofluorescence and immunohistochemical technique was used to analysis axon regeneration and scarring in the damaged region. The results of BBB showed that functional recovery of experimental group was better than control group, Immunohistochemistry and immunofluorescence results described that the experimental group rats had more axon growth within glial scar, axon regeneration was more active and thickness of scar was thinner. Our results showed that PPC electrospinning can be applied in the rat SCI and provide a new drug delivery options for the local slow-release drug in SCI.Research on the feasibility of application and effects of the electrospnning containing both db-cAMP and ChABC in spinal cord hemisection model of ratBased on PPC electrospinning, we introduced the CS microspheres into our system, united the protein drugs ChABC and electrospinning, broaden ways of the application of protein drugs in SCI and increased the treatment method for SCI. We proved the possibility of applying biodegradable polymer materials PPC electrospinning and chitosan microspheres in SCI again. Meanwhile, we also proved the ettects of db-cAMP and protein drug ChABC in SCI by sustained delivery. Our results showed that the application of above two kinds of single drug had a certain effect, but the role of promoting axon regeneration and functional recovery was not significant. Considering repair barriers were formed by different factors, so this experiment finally adopts the combination of both drugs application. In our study, we would like to prove the possibility of the combination of two drugs in our system, verify whether it has synergy on spinal cord repair and whether would be influenced each other, and compare it to single drug respectively.Drug release curves were detected respectively. The results showed that there no significant difference among the three groups. BBB was applied to evaluated the motor function of rats in the time points of 1,2,3, 4 weeks after SCI, then immunofluorescence and immunohistochemical technique was used to analysis axon regeneration and scarring in the damaged region. The results showed that functional recovery and axon regeneration of all experimental groups was better than the control group, the glial scar was loose and not continuous in the containing ChABC group. In addition, there was no significant difference between db-cAMP group and ChABC group, and compared to the both single drug groups, there were more axon regeneration and higher BBB scores in the combination group (p<0.05).This study investigated the feasibility of the poly-propylene carbonate (PPC) electrospun fibers combined with chitosan microspheres for the repair of spinal cord injury. We also researched the effect of the locally sustained releasing drugs by this composite biodegradable implant for axonal regeneration and functional restoration after the experimental spinal cord injury.The main results of this study are:1. The biodegradable PPC electrospun fibers and chitosan microspheres have good biocompatibillity and favorable capacity of drug sustained-release. The process of drug sustained-release could be controlled and regulated via changing the parameters of morphology of the fibers or microspheres, strain rate, and elongation at break.2. db-cAMP loading PPC electrospun fibers could promote axonal regeneration, restore the motor function and decrease the glial scar formation.3. Chondroitinase ABC loading chitosan microspheres combined with PPC electrospun fibers could smoothly release the chondroitinase ABC. This could prevent the scar wall formation within the post-injury cavity, which facilitated the axon to pass through the injured region and promoted the motor functional recovery.4. Composite implant consisting of microspheres and fibers could release chondroitinase ABC and dibutyryl cyclic adenosine monophosphate (db-cAMP) simultaneously. Compared with the single drug implants, composite implant with combined drugs could reduce the post-injury cavity, restore the remnant neural fibers, decrease the scar, make the interface of cavity and scar irregular and thereby significantly improve the motor function recovery after the spinal cord injury.Our study demonstrated that PPC electrospun fibers and chitosan microspheres could release drugs steadily in the injured site of the spinal cord and the speeds of implant degradation and drugs release could be controlled and regulated. This modality of composite implant can offer wonderful vehicle for the local sustained release of therapeutic agents. This combined implant can also supply new method for research of repair post injury, anti-tumor, and degenerative disease in nervous system. Meanwhile, our study demonstrated that this composite biodegradable implant had promising perspective for clinical application. |
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