| Background:Spinal cord injury(SCI)is a serious central nervous system trauma.In recent years,tissue engineering provides a new way for the treatment of SCI,composing of scaffold,seed cell and neurotrophic factors additional,contributing to the recovery of spinal cord function.The project has an important role in tissue repair and the scaffold provide a suitable microenvironment of mechanical support for seeded cell adhesion,growth and so on.Currently,it is natural degradation or synthetic polymer or composite materials that used for spinal cord scaffold,such as: hyaluronic acid,chitosan,PLGA silk protein-chitosan composite scaffold materials.These materials have their own advantages,but they don't have a unique three-dimensional network structure of the spinal cord,limiting in the repair of spinal cord reconstruction.In previous studies,we decellularized rat spinal cord to retain collagen,fibronectin,laminin and other ECM three-dimensional structure,construct a new acellular spinal cord scaffold.But the early methods of acellular were not stable,mainly displaying something that you can't reserve acellular extracellular matrix incompletely or acellular completely.In addition,acellular spinal cord scaffold also suffer from the shortage problem of neurotrophic factor.Therefore,we will be the basis of previous research to study the next step: firstly,we optimize the methods of acellular spinal cord so that it is vital stable for preparation of acellular spinal cord scaffold.Then,we use chemical crosslinking method to combine neurotrophin-3(NT-3),so that NT-3 crosslinked acellular spinal cord scaffold have a biologically active role in sustained release drug delivery.Lastly,after seeded cells,the NT-3 crosslinked acellular spinal cord scaffold can promote cell adhesion and proliferation.After the above research,we will provide a new solution for research SCI restored in tissue engineering.Objiective:To optimized acellular spinal cord scaffold and construct NT-3 crosslinked acellular spinal cord scaffold delivery system,analysis of the sustained release drug delivery system and the influence of BMSCs attached,proliferation.Methods:1.Using traditional and optimization methods respectively for preparing rat acellular spinal cord scaffolds,using visual inspection,scanning electron microscopy,light microscopy,freeze-dried,weighed,enzymatic techniques and DAPI staining comparatively to study gross morphology,microstructure,excellent rate,porosity rate,moisture content,enzymatic rate,BMSCs adhesion and other characteristics in different scaffolds.2.Using ELISA to detect sustained release NT-3 from NT-3 crosslinked acellular spinal cord scaffold,the use of dorsal root ganglion from E18 rat embryos of pregnant mice to verify the biological activity for the sustained release of NT-3,using the BCA protein assay decomposition method to detection release entirety protein of NT-3 crosslinked acellular spinal cord scaffold.3.Resuscitated,cultured and passaged to obtain P3 generation of BMSCs,compounded into acellular spinal cord scaffolds and NT-3 crosslinking acellular spinal cord scaffolds,after co-cultured for 4 hours,using DAPI to detect BMSCs in different scaffolds adhesion;co-cultured for 24 hours,48 hours,by CCK-8 to detect BMSCs proliferation in different scaffolds at different points time.Results:1.Scaffold optimization group produce "excellent" rating of 80% assessed by HE staining.the scaffolds which ones score are "excellent" by HE,stained DAPI and adserved by confocal microscopy.The internal structure of scaffolds is acellular thoroughly,score being "excellent".A three-dimensional network structure are showed by electron microscope scan which average pore diameter is 31.02 um.A water content of(228.14 ± 19.39)%,porosity(71.82 ± 2.10)%.The enzymatic stability of 4,8,12,16,20-hour period is differently(8.268 ± 1.229)%,(16.16 ± 1.568)%,(23.5 ± 2.252)%,(28.582 ± 2.243)%,(35.904 ± 1.911)%.The scaffolds of optimization group show acellular thoroughly,high production efficiency,appropriate aperture as quite as the traditional group scaffolds,but its water content,porosity,resistance to enzymolysis is superior to traditional group one.2.NT-3 crosslinked acellular spinal cord scaffold,which combine acellular spinal cord scaffold with NT-3 by EDC crosslinking,was measured by NT-3 ELISA kit in 1,4,7,14,21,28,35 day,the NT-3 sustained concentration being(480.52 ± 69.88)pg / ml,(111.43 ± 5.34)pg / ml,(66.11 ±17.99)pg/ml,(121.35±7.85)pg/ml,(61.34±9.88)pg/ml,(36.22±16.37)pg/ml,(29.95±12.95)pg/ml.After doubled NT-3 concentration,the method of back root ganglion is validated sustained biological activity of NT-3and the first day of sustained-release NT-3 solution can promote the(72.00 ± 8.19)root neurite growth,the fourth day of(32.67 ± 8.14),the seventh days of(10.33 ± 2.52),the fourteenth days of(31.67 ± 8.96),the twenty frist days(8.33 ± 3.79),the twenty eighth days(5.00 ± 1.00),thirty-five days(9.00 ± 2.00),negative control(9.00 ± 2.00),positive control(93.67 ± 33.23).The twenty frist,twenty-eighten,thirty-five days results are lower than the negative control group.Immersed in the medium,NT-3 crosslinked acellular spinal cord scaffolds are measured the whole release proteins by BCA Protein Assay kit and proteins in different time-release 1,4,7,14,21,28 days were lower than 0.2ug,proving NT-3 crosslinked acellular spinal cord scaffolds added to the medium,are retained for 28 days in 37 ? 5% CO2 incubator.3.NT-3 crosslinked acellular spinal cord scaffold seed into rat bone marrow mesenchymal stem cells in the concentration of 3.7 × 105 / ml up to 40 ml and after co-culture to four hours,the number of adhered cells are(109 ± 5),a superior to(46 ± 19)of cross-linked scaffold.A concentration of 1.0 × 105 / ml,rat bone marrow mesenchymal stem cells suspension up to 10 ul is inserted into scaffolds and co-cultured 24 hours and 48 hours,NT-3 crosslinked acellular spinal cord scaffold are conducive to the proliferation of rat bone marrow mesenchymal stem cells.Conclusions:1.We optimize acellular spinal cord scaffold successfully,can more thoroughly remove cellular components within the spinal cord and ECM are maintain relatively intact three-dimensional structure.The indicators of optimization group such as good rate,porosity,water content,enzymatic rate are ahead of the traditional.2.Using ELISA kit,NT-3 sustained release of NT-3 crosslinked acellular spinal cord scaffold up to 35 days;sustained release NT-3 are verified biologically active via dorsal root ganglia.Using the BCA protein assay,NT-3 crosslinked acellular spinal cord scaffold,added to the medium,retain 28 days at 37 ? 5% CO2 incubator.3.NT-3 crosslinked acellular spinal cord scaffold,compared to acellular spinal cord scaffold,can promote BMSCs adhesion,proliferation. |
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