Experimental Study Of CGRP-Releasing Borosilicate Bioactive Glass Scaffold In Regulating Vascularization On Bone Defects

Objective: To detect the vascularization characteristics of the stent in vitro,and to test the angiogenesis and osteogenesis performance of the stent implanted in rabbit skull defects in vivo,and jointly study the borosilicate that can release calcitonin gene-related peptide(CGRP)The 1.5-Borosilicate bioactive glass(1.5BBG)composite scaffold(1.5BBG+CGRP)regulates the ability of vascularization to repair critical bone defect of rabbit skull.Methods:(1)Cytology experiment: Human umbilical vein endothelial cells(HUVECs)were planted on 1.5BBG+CGRP composite scaffold,and grouped according to the experiment: 1.5BBG+CGRP group,1.5BBG group,CGRP group,blank medium group to prepare soak The extract was co-cultured with HUVECs cells.SEM scan,CCK-8 method,scratch healing experiment,blood vessel formation experiment,RT-PCR method and ELISA method to detect the biocompatibility and in vitro vascularization characteristics of the composite scaffold.(2)Animal experiment: Establish a rabbit skull round critical(diameter 15mm)bone defect repair model,and group according to the experiment: blank no implantation group,1.5BBG stent implantation group,autologous bone implantation group and 1.5BBG+CGRP composite stent implantation In the group,the blank group was treated with bone defect without implantation,and 1.5BBG scaffold/autogenous bone with or without CGRP was implanted into the bone defect.The rabbit heart,liver,and kidney tissue sections were taken from the rabbit’s heart,liver,and kidney tissues and stained with HE to observe whether the composite scaffold had material toxicity in the first and third months.One month after operation,a skull specimen was taken and performed CD31 immunohistochemistry to detect the vascularization ability of the composite scaffold in the bone defect area.Micro-CT scan,HE staining and fluorescence staining were performed to detect the composite scaffold in the bone defect area after 3 months.Bone capacity.Results:(1)Cytology experiment: SEM observed that cells can adhere and grow well on the surface of 1.5BBG+CGRP composite scaffold.CCK-8experiment results: The difference in cell proliferation absorbance(OD)values of each group on the 1st day was not statistically significant(P>0.05).The OD value of the cells in the 1.5BBG+CGRP group was higher than that in the1.5BBG group,CGRP group and blank medium group on the 3rd to 5th day.The difference was statistically significant(P < 0.05).The cells in each group proliferated significantly.On the 7th day,there was no significant difference in the OD value of cell proliferation in each group(P>0.05).Cell migration experiment results: the 1.5BBG+CGRP group had the strongest cell migration ability at 24 h,the cell migration ability of the CGRP group was second only to the 1.5BBG+CGRP group,and the blank medium group had the worst cell migration ability,which was similar to the 1.5BBG scaffold group.In vitro angiogenesis experiment results: the number of luminal-like structures in the1.5BBG+CGRP group was the largest and showed a complete mesh,the number of luminal-like structures in the CGRP group was less than that in the1.5BBG+CGRP group,and the blank medium group had a small number of slender tubes the cavity formation is similar to the 1.5BBG stent group.RT-PCR experiment results: the expression of each gene in the 1.5BBG+CGRP group was significantly higher than that in the 1.5BBG group and the blank medium group,and the difference was statistically significant(P<0.001).There was a statistically significant difference in gene expression between the CGRP group and the blank medium group(P<0.05).There was no statistically significant difference in gene expression between the 1.5BBG group and the blank medium group(P>0.05).ELISA test results: the VEGF secretion in the 1.5BBG+CGRP group was significantly higher than that in the 1.5BBG group and the blank medium group,and the difference was statistically significant(P<0.01).The secretion of VEGF in the CGRP group was higher than that in the 1.5BBG group and the blank medium group,and the difference was statistically significant(P<0.05).There was no significant difference in VEGF secretion between the 1.5BBG group and the blank medium group(P>0.05).(2)Zoological experiment: There was no inflammation,tumor or necrosis under the microscope observation of heart,liver and kidney tissue in each group of rabbits in the first and third months after operation.CD31 immunohistochemical test results: The number of positive expression structures of CD31 in the bone defect area in the 1.5BBG+CGRP composite scaffold implantation group was more than that in the 1.5BBG scaffold implantation group,while no positive cell expression was seen in the blank non-implantation group.Micro-CT scan,HE staining and fluorescent staining results: no bone tissue structure was formed in the bone defect area of the blank no implantation group.In the 1.5BBG+CGRP composite scaffold implantation group,the scaffold was tightly connected to the host bone,partially fused,and a large amount of bone tissue grew in the scaffold pores.There was a clear boundary between the stent and the host bone in the1.5BBG stent implantation group,and the stent pores saw part of the new bone grow in.The bone defect area of the autogenous bone implantation group was repaired by the even thickness of the autogenous skull,and the skull and the surrounding bones had obvious bone fusion.Conclusion:(1)The 1.5BBG+CGRP composite scaffold studied in this experiment has good biocompatibility,and the released CGRP endows itself with certain angiogenic properties.(2)The 1.5BBG+CGRP composite scaffold is implanted in the critical bone defect area of the rabbit skull Later,the composite scaffold can effectively promote vascular regeneration and bone tissue repair in the critical(15mm)bone defect area of the rabbit’s skull.