Experimental Study Of Extracorporeal Shock Wave Combined With Autologous Bone Marrow Mesenchymal Stem Cell Transplantation In The Treatment Of Bone Defects

Objective:To explore the feasibility and effectiveness of autologous bone marrow mesenchymal stem cells(BMSCs)transplantation supplemented with specific parameters of extracorporeal shock wave(ESWT)for the treatment of segmental bone defects.Methods:(1)36 New Zealand white rabbits were randomly divided into 4 groups,group A for BMSCs/ESWT,group B for ESWT,group C for BMSCs,and group D for control;(2)Bone marrow taken from experimental rabbits were isolated and cultured with autologous bone marrow MSCs by differential apposition method to observe their morphology and identify the phenotype to obtain BMSCs;(3)rabbit mid-superior radius segment was prepared to make radial bone defect model;(4)P3-generation BMSCs were transplanted into the bone defect area and given extracorporeal shock waves;(5)X-ray was used to observe the healing of the bone defect at 3,6 and 9 weeks after treatment,and the Lane-Sandhu method was used to score each group radiologically;(6)Western blotting of muscle tissue around the bone defect in each group of animals at3,6 and 9 weeks after treatment.Western blotting of muscle tissue around the bone defects in each group at 3,6,and 9 weeks after treatment to detect the expression of Runx2 and OCN proteins;(7)Histological staining and immunohistochemistry were used to observe the repair of bone defects and the expression of VEGF proteins after 9 weeks,and the corresponding histological scores were assigned to analyze the repair of bone defects by quantitative comparison.Results:(1)The cultured rabbit autologous bone marrow mesenchymal stem cells were isolated and purified by differential wall centrifugation to observe their morphology and identify their phenotype,which was elongated,polygonal,and spindle-shaped,and the results of flow cytometric analysis: BMSCs expressed high CD73,CD90,CD105(97.3 %,97.0 %,96.7 %)and low CD34,CD45(1.03 %,2.06 %),consistent with the characteristics of BMSCs,and sufficient numbers of P3-generation BMSCs were obtained for transplantation;(2)Differential comparison of bone defect repair areas using Lane-Sandhu scores: the comparison of X-ray scores at week 3,6 and 9 in all four groups was significantly different(P<0.05),and the X-ray scores in groups B,C and D at all three-time points The X-ray scores of groups B,C and D were significantly lower than those of group A(P<0.05),and the X-ray scores of groups B and C were significantly higher than those of group D(P<0.05).(3)Western blotting results: the comparison of Runx2 and OCN protein levels at weeks3,6,and 9 in the four groups were significantly different(P<0.05),and the Runx2 and OCN protein levels in groups B,C,and D were significantly lower than those in group A at the three-time points(P<0.05),and the Runx2 and OCN protein levels in groups B and C were significantly higher than those in group D(P<0.05).And within the three groups of groups B,C,and D,Runx2 protein was significantly lowest at week 3,significantly increased with time,and was highest at week 9.In contrast,group A was significantly lowest at week 3 and reached a significant maximum at week 6.Within all three groups,groups A,B,and D,OCN protein was significantly lowest at week 3,significantly elevated over time,and highest at week 9.In contrast,in group C,it was significantly lowest at week 6 and reached a significant maximum at week 9.(4)Comparative histopathological results: There was a significant difference in the comparison of HE staining scores of pathological tissues in bone defect areas in groups A,B,C and D(P<0.05).HE staining scores in group A were significantly higher than those in groups B,C,and D.(5)VEGF expression: there was a significant difference in the comparison of OD values(P<0.05),which represented that the VEGF expression in group A was significantly higher than that in groups B,C and D;the VEGF expression in groups B and C was significantly higher than that in group D;no significant difference was seen in the VEGF expression between groups B and C.Conclusion:(1)Extracorporeal shock wave combined with autologous bone marrow mesenchymal stem cell therapy can promote the expression of osteogenic-related proteins and neointima formation in skeletal muscle tissue;(2)Extracorporeal shock wave combined with autologous bone marrow mesenchymal stem cells has a positive effect on the healing of bone defects.