A Study On Spinal Cord Injury Repair By BMSCs Stimulated With LIPUS

?Objective?Spinal cord injury is a serious traumatic orthopedic disease,with high incidence and disability rate.There is no effective cure for SCI so far.The research team has found the ability of BMSCs for promoting the recoverment of SCI.However,the low biological activity of BMSCs in vitro,made it research focus optimizing BMSCs.This study intendeds to stimulate BMSCs with different intensity of ultrasound,using the self-built LIPUS intervention system.Morphological structure of BMSCs was observed and its proliferation activity was detected to determine the optimal LIPUS parameter.Then the cell apoptosis and the expression of neurotrophic factors were detected to explore the affect of LIPUS on BMSCs.LIPUS-BMSCs were futher transplanted into the epicenter of injured spinal cord in rats.The motor function evaluation,tissue construction and the local neurotrophic factors expression were detecetd to find out how do LIPUS promote BMSCs'ability of promoting the recoverment of SCI.?Method?1.Experiment in vitroExtraction and culture of BMSCs:BMSCs were isolated from Wistar rats and purified through the method of whole bone marrow adherent,BMSCs were then cultured and amplified in vitro.CD29,CD90,CD34 and CD45 were detected by flow cytometry for cell identification.Establishment of ultrasonic equipment:The self-built LIPUS intervention system included power supply,signal generator,signal amplifier and ultrasonic transducer,while the ultrasonic oscilloscope was used to detect the ultrasonic waveform and intensity.Then BMSCs were stimulated by LIPUS with different ultrasonic intensity.The optimal LIPUS parameter for promoting BMSCs depended on the change of the proliferation of BMSCs after being stimulated.Detection of BMSCs biological activitycell:The TUNEL method was adopted to detect the apoptosis of LIPUS-BMSCs and WB was employed to detect the expression of neurotrophic factor.2.Experiment in vivoSCI model establishment and rats experimental grouping:Thirty-six Wistar rats were randomly divided into 4 groups:sham group,control group,BMSCs group and LIPUS-BMSCs group.The model of spinal cord contusion injury was employed.As for BMSCs group and LIPUS-BMSCs group,the BMSCs and LIPUS-BMSCs were employed transplanted 7 days after spinal cord injury.The control group received equal DMEM/F12 with no cells,and the sham group experienced the same operation without spinal cord contusion or receiving any substance.Evaluation of the cell transplantation effect:BBB locomotor rating scale was implemented on the rats of sham group,control group,BMSCs group and LIPUS-BMSCs group,to evaluate the motor function of the hind limbs.All rats were sacrificed to isolate the spinal tissue mass.HE staining,immunofluorescence and ELISA were employed to explore the changes in the spinal cord tissue and the NTFs expression.?Result?1.LIPUS intervention system:This study succeeded to construct LIPUS intervention system which could stimulate BMSCs cells cultured in vitro.50mW/cm~2was determined as the optimal intensity via detecting the proliferation activity changes of BMSCs.2.Biological characteristics of BMSCs:The extracted original BMSCs cells,with a good condition,was successfully identificated,it was confirmed that the BMSCs purity was higher than 95%.The LIPUS stimulation was able to improve the proliferation activity BMSCs.2.The apoptosis rate of BMSCs hardly changed after LIPUS stimulation.However,it was verified that the LIPUS stimulation could promote the expression of neurotrophic factors in BMSCs,especially the BDNF and NGF.3.Behavioral assessment:21 days after cell transplantation,BBB scores in BMSCs group and LIPUS-BMSC group were significantly higher than that in the control group.And in the 28th day,significant difference was observed between the BMSCs group and LIPUS-BMSCs group.LIPUS-BMSCs improved the recovery of hind limb motor function in rats in an even better fashion than BMSCs.4.Histological observation:Histological results verified that in groups received cell transplantation,especially in the LIPUS-BMSCs group,the syringomyelia was reduced,the histomorphological restoration of grey matter was promoted,the survival of nerve fiber was increased,axon sprouting and axon regeneration was activated,and reactive glial cell hyperplasia was decreased.ELISA test confirmed that transplanted LIPUS-BMSCs increased the expression of BDNF and NGF in the SCI center,which performed better than BMSCs.?Conclusion?In this study,experiments in vitro verifyed that stimulation by the self-built LIPUS intervention system was able to increase the proliferation activity and the NTFs expression.It can produce a lot of optimized LIPUS-BMSCs as the seed cells.After transplantation of BMSCs,the motor function,local spinal cord tissue condition and local NTFs expression at the centre of the injured spinal cord was improved.The BMSCs did contribute to the recoverment of SCI,while the effect of LIPUS-BMSCs was obviously better than that of BMSCs.It was proved that the LIPUS,as an in vitro stimulation method,could improve the BMSCs'ability of promoting SCI regeneration.This study provided a new method for optimizing seed cells for cell transplantation treatment of SCI.