The Neural Repair Effects Of SA/Col/SDF-1 Hydrogel Scaffold Loaded With BMSCs On A Rat Model Of Traumatic Brain Injury

Traumatic brain injury?TBI?is a nervous system injury disease caused by external forces,which was accompanied by temporary or permanent dysfunction in terms of cognition,physical function,and psychology.Stem cell transplantation and tissue engineering technology have brought promising hope for the treatment of TBI,but the loss of stem cells at the transplant site and low survival rate are the main obstacles to stem cell therapy.Tissue engineering technology is essential for improving stem cell transplantation.Noticeably,injectable hydrogels are widely used in tissue engineering with high water content and porous structure.Sodium Alginate?SA?has good biocompatibility and is a common drug carrier.Collagen?Col?can promote the migration and survival of exogenous stem cells in the brain injury site and improve the stem cell treatment effect.The three-dimensional hydrogel scaffold prepared by mixing SA and Col can reduce cell loss,improve the survival microenvironment of stem cells,and promote nerve repair and functional reconstruction.Stromal cell derived factor-1?SDF-1?/and its receptor CXCR4 are key factors in the activation,homing and differentiation of stem cell.However,the expression of CXCR4 on the cell membrane of normal mesenchymal stem cells?MSCs?is very low,which limits the migration of MSCs targeting SDF-1 in vivo.On the other hand,SDF-1expression is increased at the injury site,unfortunately TBI can also activate the expression of metalloproteinases 2 and 9?MMP2?MMP9?,which can degrade SDF-1 leading to neurodegeneration.In order to prolong the half-life of SDF-1 or increase the availability of SDF-1,it is necessary to continuously express or secrete SDF-1 at the injury site.Further studies have shown that controlling the release of SDF-1 in different biological materials can stimulate the homing of stem cells.However,the effect of SA/Col hydrogel scaffold with sustained SDF-1 release on the survival,migration and differentiation of bone marrow-derived mesenchymal stem cells?BMSCs?and the nerve repair efficiency to TBI remains unclear.ObjectivesIn this study,an injectable SA/Col hydrogel scaffold was prepared by using SA and Col.SA/Col/SDF-1 neural scaffold with sustained release of SDF-1 was prepared by doping exogenous SDF-1 protein.Then,BMSCs were embedded into SA/Col/SDF-1 scaffold.Finally,the cell and biological compatibility of BMSCs/SA/Col/SDF-1neural scaffold,and the safety and efficacy of scaffold transplantationon TBI model were systematically studied.MethodPart I:Preparation,optimization and characterization of SA/Col hydrogelSA,SA₄ColI₁ and SA₂ColI₁ composite hydrogels were prepared by SA-CaCO₃-GDL system.The internal morphology was observed by SEM;Gelation time was determined by the tube tilt method;the degradation rate and water content of the gel were measured by freeze drying and weighing method.SA/Col hydrogel was injected subcutaneously into SD rats to detect the degradation rate of hydrogel in vivo,HE staining was used to detect inflammation in the transplant site,and evaluate the biocompatibility of hydrogel.The elastic modulus and physicochemical properties of hydrogels were characterized by rotatory rheometer and XRD.CCK-8,AO-EB double fluorescent staining and laser confocal microscopy were used to detect the proliferation and survival of BMSCs in SA/Col hydrogel on 1,3,5 and 7 day.Part II:Preparation of SA/Col/SDF-1 complex hydrogel and its effect on proliferation and migration of BMSCsThe effects of SDF-1 on the proliferation,migration and neural differentiation of BMSCs were detected by CCK-8,Transwell assay,Immunofluorescence and qRT-PCR.The release SDF-1 in SA/Col/SDF-1hydrogel at 1 to 30 days was detected by ELISA.Transwell was used to detect the activity of SDF-1 to the migration of BMSCs.CCK-8,AO-EB double fluorescent staining and laser confocal microscopy were used to detect the effects of SA/Col/SDF-1 hydrogel on proliferation and survival of BMSCs in 1,3,5 and 7 day.Part III:Neuroprotective effects of SA/Col/SDF-1 hydrogel transplantation loaded with BMSCs on TBI ratsModified Feeney weight-drop method was adopted to generate TBI model from free hammer fall injury,then rats were randomly divided into TBI group,BMSCs group,BMSCs/SA/Col group,BMSCs/SA/Col/SDF-1 group?BMSCs were labeled with DiI fluorescence?.Rats were examined for changes in body weight,wire-hanging time,and mNSS scores.Morris water maze and new object recognition were used to test the recovery of learning,memory and cognitive ability.Behavioral experiments were used to evaluate the difference of anxiety and depression in four groups of rats,including sucrose preference test,forced swimming test,tail suspension test,open field test and novelty suppressed feeding Test.The expression of apoptosis protein was detected by Western Blot.The number of apoptotic cells at the injured area was detected by TUNEL staining.ELISA assay and immunofluorescence were used to detect the inflammatory response.The brain injury volume and edema were examined by CV staining and dry and wet weight method.The survival and migration of BMSCs at the injury sites were detected by DiI staining.Western Blot was used to analyze the expression of SDF-1/CXCR4 and FAK/PI3K/AKT pathway associated protein.ResultsPart I:Preparation,optimization and characterization of SA/Col hydrogelThree hydrogels prepared by SA-CaCO₃-GDL system were translucent and could be shaped according to the mold.As the increase of Col concentration,the pore size,gel time and moisture content were decreased,while the degradation rate was increased gradually?P<0.05?.The composition of hydrogel was clear and the elasticity was ranging from 200 Pa to 1000 Pa,meanwhile SA₂ColI₁ exhibited a stable modulus value compared to SA₄ColI₁ and SA.The subcutaneous SA/Col hydrogel has good biocompatibility with no inflammatory reaction,and was consistent with degradation rate in vivo.The proliferative capacity and survival rate of BMSCs in the hydrogel was increased along with Col concentration?P<0.05?.After comprehensively comparing the various properties,we finally selected the SA to Col ratio was 2:1 for subsequent experiments.Part II:Preparation of SA/Col/SDF-1 complex hydrogel and its effect on proliferation and migration of BMSCsCompared with NC group,SDF-1 at 100,150 and 200 ng/mL could promote the survival and migration of BMSCs?P<0.05?.After induction of 150 ng/mL treatment group for 48h,the migrated BMSCs and differentiation efficiency were increased significantly?P<0.01?.The release of SDF-1 in SA/Col/SDF-1 hydrogel exhibited a short term of brust release?with the first day for 40%?followed by a long term sustained and stable release?the following 29 days?.The results of ELISA showed that released SDF-1 on the 3rd,12th and 21st day could significantly promote the migration of BMSCs?P<0.05?.SA/Col/SDF-1 hydrogel with 150 ng/mL SDF-1 concentration significantly promoted the migration of BMSCs in vitro?P<0.05?.In addition,BMSCs grew well in SA/Col/SDF-1 hydrogel with good biocompatibility?P<0.05?.Part III:Neuroprotective effects of SA/Col/SDF-1 hydrogel transplantation loaded with BMSCs on TBI ratsCompared with the TBI group and BMSCs group,the body weight was increased significantly?P<0.05?,the hanging time was prolonged?P<0.05?,and the mNSS score was significantly decreased in the BMSCs/SA/Col and BMSCs/SA/Col/SDF-1group.The cognitive function was significantly improved,meanwhile anxiety and depression were significantly relieved in the BMSCs/SA/Col/SDF-1 group?P<0.05?.In addition,BMSCs/SA/Col/SDF-1 transplantation significantly reduced the number of apoptosis cells,inflammation response,brain damage volume and edema,while increasing the antioxidant level at the injury site?P<0.05?.The survival and migration rate of BMSCs at the injured site were significantly promoted,and the number of EdU/NeuN positive cells and the expression of neurotrophic factors?BDNF,NGF?were significantly increased?P<0.05?.Compared with the other groups,the expressions of SDF-1,CXCR4,p-FAK/FAK,p-PI3K/PI3K,and p-AKT/AKT were significantly increased in the BMSCs/SA/Col/SDF-1 group?P<0.05?.Conclusion1.SA/Col injectable hydrogel exhibts highly water content,stable degradation capacity and elastic modulus,which is beneficial to the survival and proliferation of BMSCs.2.SDF-1 can significantly promote the survival and migration of BMSCs,and significantly enhance the neurogenic differentiation efficiency of BMSCs.SA/Col/SDF-1 hydrogel could continuously and stably release SDF-1 and promote the proliferation and migration of BMSCs.3.SA/Col/SDF-1 hydrogel transplantation loaded with BMSCs can significantly improve the neurological function of TBI rats,reduce the volume of brain damage,improve the microenvironment of the injured site,and promote the migration of BMSCs and neurogenesis.Moreover,the mechanism is related to the activation of FAK/PI3K/AKT pathway mediated by SDF-1/CXCR4.