| BackgroundsSpinal cord injury(SCI) is a kind of disabling condition of the central nervous system.In the world,the incidence of the SCI is about 10.4-83 in one million people every year.Less than 1%of the patients who sustain significant injury to their spinal cord recover complete neurological function and many of these injuries result in partial or complete paralysis,82%of them are the young men aged from sixteen to thirty-two years old.The people who are afflicted with SCI is a big burden to both of the society and the families.SCI initiates a chain of events that lead to cell death,scarring and the loss of function.The initial trauma injures cells and induces swelling.The damaged cells release toxins that cause necrosis of the cells above and below the injuried spinal cord level.The lesion site that results from SCI poses many obstacles to promoting regeneration of the neural cells annd axons.Subsequent events include the formation of a cystic cavity at the injury site,which is surrounded by a glial scar,composed of mainly reactive astrocytes.The demyelination that occurs after injury produces several inhibitory molecules that contribute to the lack of regeneration.Many strategies for treatment of SCI are developed involving replacing the cells lost to injury with cells derived from an alternative source.The end goal of such treatments is to help restore function that was lost in the injury.There are numerous researches report many strategies that attempt to overcome these negative factors and promote axon regeneration.In recent years,with the tremendous advancement of the cellular transplantation technique and the immunology,the development of the xenograft,such as the stem cells co-transplanted with biomaterial scaffold,brings a promising therapy method for the people who afflicted with SCI.For example,numerous studies have explored the potential of transplants to bridge the gap at the lesion site,including Schwann cells,olfactory ensheathing cells,macrophages,embryonic tissue transplants,stem cells of various sorts transplants,peripheral nerve bridges,artificial scaffolds,and various combinations of transplants and growth factors.Among of them,Cell therapy and scaffold transplantation are investigated most widely,because they can help overcome this barrier through a variety of methods,such as implantation of neurons or stem cells which can differentiate into neurons and secrete factors that promote regeneration of the damaged axons into the cavity and also provide the trophic support necessary for cell migration.But,currently there is no literature claimed that they have got satisfied therapeutic effects on treating the spinal cord injury in animal experimental researches.One of the main reseasons is clear that traditional autografts or allografts and various synthetic implant materials have many limitations such as finite rescorces,ethic issues,immunological rejection,and the structural limits of the artificial scaffolds themselves which cann't mimic the natural structure and spatial distribution of the extracellular matrix of the target tissues.Moreover,axons regenerating across the scaffolds may disperse.How can we overcome these drawbacks?Tissue engineering(TE) which is developing fastly brings promise to the patients.Tissue engineering aims at repairing and restoring damaged tissue function employing three fundamental"tools",namely cells,scaffolds and growth factors which are always simultaneously co-used.At the same time,recent experimental and clinical evidences indicate that the success of any TE approach mainly relies on the delicate and dynamic interplay among these three components and that functional tissue integration and regeneration depends upon their structural integration.Therefore an ideal scaffold should posses a three-dimensional and well defined microstructure with an interconnected porous network,have mechanical properties similar to the target tissues,be biocompatible and bio-resorbable at a controllable degradation and resorption rate as well as provide the control on the delivery of specific bioactive factors to enhance and guide the regeneration process.The co-transplantation can not only be able to brige the both stumps of the spinal cord,promote the regeneration of the neurons,inhibit the formation of the glial scar,but also have the effects on inducing the proliferation,orientational differentiation and migration of the stem cells,induce the orientiational spread of the axons,reconstruct the neural circuit loop,and even to promote the function restoration of the injured spinal cord.But up to now,in all of the studies the motive function of the hindlimbs in the experiemental animals can only get some degrees restoration.Many researches involving the neural growth factors,trophic factors,stem cells and biomaterial scaffolds have been carried out,but we can't yet obtain a further progress in the field of repairing the injured spinal cord.Why?The answer has not been found by now.As we know,comparing with the peripheral nerve,the spinal cord belongs to the central nervous system which has many distinct characteristics such as having delamination and zonation at the same level.All of the scaffolds which have already been researched cannot mimic the three-dimensional(3D) extracellular matrix structure of the spinal cord,this maybe the key reseason for the failure of those researches,because those scaffolds lack the mimic 3D matrix structure,so that may not have the ability to more effectively induce the orientational differentiation and migration of the implanted stem cells.Furthermore,all of the scaffolds which have been described in the literatures lack the structural foundation to rebuild the nerve conduction model of"saltation",as a result,which has no way to amplify the neuroelectricity pulses in the injured spinal cord.As we know,currently there is no literature describing the matrix structure of the spinal cord.So,we proposed the concept of acellular spinal cord scaffold at the first time,wish to find a method to fabricate this kind of scaffold,discover the three-dimensional structure of the extracellular matrix of the spinal cord and to investigate the effects of the acellular spinal cord scaffold co-transplanted with human umbilical cord blood mesenchymal stem cells on treating the complete transected spinal cord in rats.Objective:1.To explore a method for fabricating the acellular spinal cord scaffold and to observe the construction features of the scaffold.2.To search for a method to elevate the achievement ratio of isolating and culturing the human umbilical cord blood mesenchymal stem cells.3.To observe the cytotoxicity and cytocompatibility of the acellular spinal cord scaffold with human umbilical cord blood mesenchymal stem cells.4.To investigate the mechanisms of treating the complete transected spinal cord by the acellular spinal cord scaffold co-transplanted with human umbilical cord blood mesenchymal stem cells in rats through observing the plasticity effects of the acellular spinal cord scaffold on the neural cells and the axons.Methods:1.To fabricate the acellular spinal cord scaffold. 1.1 Materials:Adult female rats(Sprague-Dawley;195-230g) were sacrificed under deep chloral hydrate(400mg/kg;ip) anesthesia.Rats were perfused transcardialy with saline.The spinal cord was extracted from the vertebra at the C1-T12 level.1.2 Pretreatment and chemical extraction:Under operative microscopy,the fat and a part of dural matter were cut before the extraction procedure. Numerous segments of spinal cord obtained from rats were divided randomly into A group(frequency of vibration=80r/min),B group(frequency of vibration=120r/min) and C group(frequency of vibration=160r/min).The spinal cord was delt with solution of Triton X-100 and with solution of sodium deoxycholate at room temperature.Then washed with distilled water,delt with HE staining and observed under light microscope.Scanning electron microscope was used to observe the ultramicrostructure.The fabricated acellular spinal cord scaffolds were stored in 0.01mol/L PBS (pH7.4).2.The collection of the human umbilical cord blood(UCB):2.1 UCB was obtained from placentas from full-term scheduled(included cesarean section) births at the Nangfang Hospital.2.2 About a volume of 20-120ml UCB(added with 20U/ml heparin for anticoagulation) was obtained by cannulation of the umbilical vein close to the placenta under asepsis condition.2.3 UCB is stored at 4℃and isolated within six hours.3.To isolate the mononuclear cells from the UCB and to culture and purify the human umbilical cord blood mesenchymal stem cells(hUCB-SCs).3.1 Four methods for isolating the mononuclear cells(MNCs) from the UCB, include methyl cellulose settling process(MCSP)(A group),density gradient centrifugation(DGC)(B group),methyl cellulose settling process united with density gradient centrifugation(MUD)(C group),methyl cellulose settling process united with modified density gradient centrifugation(MUMD)(D group).3.1.1 Methyl cellulose settling process(MCSP)(A group):UCB were diluted 1:1 with 5g/L methyl cellulose aqueous solution and were settled at 4℃alone.MNCs were obtained from diluted UCB by methyl cellulose settling process.Each flask was seeded with 6 to 7×106 MNCs in 5 ml of DMEM/F12 medium containing 10%FBS.The cells were incubated for 24 hours and the non-adherent cells were removed with medium replacement.After that,every 2 days,cells and medium were demidepleted.3.1.2 Density gradient centrifugation(DGC)(B group):UCB were overlayed gently onto the Ficoll-Hypaque solution,and then were centrifugated at 1500r/min,4℃,for 20 min.MNCs enrichment was performed by density gradient centrifugation in the third liquid layer,then collected the third liquid layer.Each flask was seeded with 6 to 7×106 MNCs in 5 ml of DMEM/F12 medium containing 10%FBS,5ng/mL GM-CSF,5ng/mL EGF,0.1 U/mL penicillin,0.1μg/mL streptomycin.The cells were incubated for 24 hours at 37℃in an incubator with a 5%CO2 atmosphere and the non-adherent cells were removed with medium replacement.After that,every 2 days,cells and medium were demidepleted and cultured.3.1.3 Methyl cellulose settling process united with density gradient centrifugation (MUD)(C group):After the methyl cellulose settling process,the supernatant was collected,and then were overlayed onto the Ficoll-Hypaque solution,centrifugated at 1500r/min,4℃,for 20 min.MNCs enrichment was performed by density gradient centrifugation in the third liquid layer,then collected the third liquid layer.Each flask was seeded with 6 to 7×106 MNCs in 5 ml of DMEM/F12 medium containing 10%FBS,5ng/mL GM-CSF,5ng/mL EGF,0.1 U/mL penicillin,0.1μg/mL streptomycin.The cells were incubated for 24 hours and the non-adherent cells were removed with medium replacement.After that,every 2 days,cells and medium were demidepleted.3.1.4 Methyl cellulose settling process united with modified density gradient centrifugation(MUMD)(D group):After the methyl cellulose settling process,the supernatant was collected,and then were overlayed onto the Ficoll-Hypaque solution,centrifugated at 1500r/min,4℃,for 20 min.MNCs enrichment was performed by density gradient centrifugation in the third liquid layer,then collected the upper three liquid layers.Each flask was seeded with 6 to 7×106 MNCs in 5 ml of DMEM/F12 medium containing 10%FBS,5ng/mL GM-CSF,5ng/mL EGF,0.1 U/mL penicillin,0.1μg/mL streptomycin.The cells were incubated for 24 hours and the non-adherent cells were removed with medium replacement.After that,every 2 days,cells and medium were demidepleted.4.To culure and purify the hUCB-SCs in vitro.4.1 To evaluate the viability of the MNCs:Drop a droplet of MNCs suspension on cell counting board,added a drop of 0.5%tapan blue into it,put the cover glass on it.To count 100 cells,and the viability equal to the percent of the cells which were not dyed blue.4.2 To culure the passage hUCB-SCs:About 24 hours later,the medium were substituted wholely,after that,every 3 days,cells and medium were demidepleted.After 7 to 10 days,the cultures became to 80%to 90% confluency,the cells were lifted by incubation with 0.25%trypsin and 0.2% EDTA at 37°C for 3 to 4 min.They were diluted at a ratio of 1:2 or 1:3 and replated and cultured at 37℃in an incubator with a 5%CO2 atmosphere.4.3 Hoechst33258 staining for the nucleus of the hUCB-SCs.4.4 To identify the CD system antigen phenotype of the hUCB-SCs by flow cytometry.5.To investigate the cytocompatibility and the degradation of the acellular spinal cord scaffold by co-culturing with hUCB-SCs in vitro,observe the microstructure of the co-cultured compounds and to investigate the survival,distribution and migration of the implanted hUCB-SCs with scanning electron microscope.6.Animal experiment:At first,to establish the acute transected spinal cord injury model at T9 level in rats.Then two hundred and ninety nine Sprague-Dawley (SD) female rats were divided randomly into A group(control group,n=33),B1 group(spinal cord transection ordinarily and PBS injection,n=57),B2 group (spinal cord transection under operative microscope and PBS injection,n=66),C group(acellular spinal ocrd scaffold transplantation simply,n=38),D1 group(spinal cord transection ordinarily and hUCB-SCs transplantation,n=29) and D2 group(spinal cord transection under operative microscope and hUCB-SCs transplantation by using micromanipulation,n=38),E group(spinal cord transection under operative microscope,and acellular spinal cord scaffold co-transplanted with hUCB-SCs by using micromanipulation,n=38).From 1 week to 12 week post-operation,a behavioral testing was performed weekly upon each hindlimb for all animals according to the BBB scoring system.At the 12th week,all animals were sacrificed and the spinal cords were taken out for morphological observation.7.To investigate the effects of the acellular spinal cord scaffold co-transplanted with hUCB-SCs on treating the transected spinal cord in rats.A behavioral test was performed to measure the functional recovery of the hindlimbs according to the BBB scores system in rats.The scale used for measuring hindlimb function including the use of individual joints,coordinated joint movement,coordinated limb movement,weight-bearing,and other functions.The BBB scores system were performed at 7 days after operation and the rats were subsequently tested weekly for 12 weeks.8.Tissue preparation and Immunohistochemistry staining:Animals were sacrificed under deep chloral hydrate(400mg/kg;ip) anesthesia at 12 weeks after operation.Rats were perfused transcardialy with saline,followed by 4% paraformaldehyde.The entire spinal cord included the injured region was extracted from the vertebra and then immersed in 4%paraformaldehyde overnight.The spinal cord segments of C1-T2 and T7-T12 were embedded in paraffin.Cl-T2 and T7-T12 were cut for coronal sections(5μm).The sections were blocked with 0.1M PBS with 1%BSA for 1h.Pimary antibodies(1:100 dilution) specific for neural cells,mouse anti-NSE,rabbit anti-NF 200 were diluted in 0.1M PBS containing 1%BSA and applied overnight at 4℃.Appropriate goat anti-mouse or anti-rabbit secondary antibodies were diluted 1:200 in 0.1 M PBS containing 1%BSA and applied individually for 1 to 2 h at room temperature.The sections were observed to investigate the number,morphology and spatial distribution of the positive cells under a microscope and took photographs.9.To trace the corticospinal tract(CST) with a fluorescent neural tracer fluorescent-gold(FG).Using the precise micro injector,a total volume of 400nl of FG was injected stereotactically into the sciatic nerve for tracing the CST axon regeneration in complete transected spinal cord region in rats. Results:1.Acellular spinal cord scaffold can be fabricated by chemical extraction.The three-dimensional(3D) structure of the acellular spinal cord scaffold are kept intact,in which there are bundles of extracellular matrix(ECM) fiber which aligned lengthways were interlaced with transversal matrix fibers.The native 3D structure of the ECM in the scaffold maybe provide the structural foundation for inducing effectively the neurons and axons to directionally grow,migrate than the synthetic polymer scaffold,and for rebuilding the neuroelectricity conduction circuit loop to improve the functional restoration of the injured spinal cord finally.2.Every one of the four methods,include MCSP,DGC,MUD and MUMD method,can isolate successfully the MNCs from the UCB,but the achievement ratio of the DGC is lower than other methods because it is interfered by more interference factors.Among the four methods,the achievement ratio in MUMD group is the highest,because it can more thoroughly reclaim the adherent stem cells on the surface of the centrifuge tube than the other methods during the isolating procedure for improving the achievement ratio of culturing hUCB-SCs in vitro.3.The residual chemical extraction agent is cytotoxic and fatal to the co-cultured hUCB-SCs.Preincubation can eliminate the residual chemical extraction agent within the acellular spinal cord scaffold to provide suitable microenvironment for the surviving,differentiating and migrating of the hUCB-SCs.4.Acellular spinal cord scaffold transplantation simply can't improve the motive function of the complete transected spinal cord in rats.The hUCB-SCs transplantation simply or co-transplanted with acellular spinal cord scaffold can improve effectively the motive function of the complete transected spinal cord in rats,but there is no difference between the two groups statistically.The dural matter of the implanted acellular spinal cord scaffod which was micro-sutured with the recipient can stop effeciently the ingrowth of the connective tissues from outside of the spinal cord and inhibit the glial scar formation in the region of the injured site.The acellular spinal cord scaffold can provide suitable microenvironment for inducing effectively the neurons and axons to directionally grow and migrate.The acellular spinal cord scaffold co-transplanted with hUCB-SCs can promote the rebuild of the structure of the injured spinal cord and improve the motive function of the hindlimbs in rats finally.Conclusions1.Acellular spinal cord scaffold can be fabricated by chemical extraction.The native 3D structure of the ECM in the scaffold maybe provide the structural foundation for inducing effectively the neurons and axons to directionally grow,migrate than the synthetic polymer scaffold,and for rebuilding the neuroelectricity conduction circuit loop to improve the functional restoration of the injured spinal cord finally.2.The method of MUMD can more thoroughly reclaim the adherent stem cells on the surface of the centrifuge tubes for improving the achievement ratio of culturing the hUCB-SCs in vitro.3.The preincubated acellular spinal cord scaffold has favourable cytocompatibility and can provide suitable microenvironment for the hUCB-SCs to survive, directionally differentiate and migrate.4.Acellular spinal cord scaffold is able to bridge the both stumps of the injured spinal cord by means of"alete butt joint",stop the in-migrating of the peripheral tissues around the spinal cord,guide the directional growth and migration of the neural cells and axons,and can promote the functional recovery of the hindlimbs in the complete transected spinal cord rats when it is co-transplanted with human umbilical cord blood mesenchymal stem cells.
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