Study The Effect Of Osteogenic Repair Ability Of Co-culture Of BMSCs And HUVECs Combined With HA-TCP Scaffolds On The Calvarial Defect In Rats

Objective: To establish the model of critical calvarial defects in SD rats and implanted with pure HA-TCP scaffold and the scaffold with seed cells,then the healing status of the defected bone tissue,structure of new bone,the changes of the bone mineral density and the expressions of ALP,Col-I and the transcription levels of Runx2,OCN in the microenvironment of bone reconstruction were observed,to primarily study the osteogenesis repair ability of tissue engineering bone constructed by co-cultured BMSCs and HUVECs combined with HA-TCP scaffold on the critical calvarial defects in SD rats,to establish a pre-basic research for exploring an effective treatment method to repair the critical defect of maxillofacial bone.Methods: The critcal calvarial defect model of SD rat was established,then the subjects were randomly divided into four groups according to the type of the implanted scaffolds materials:(1)group A as the control group,pure HA-TCP scaffolds;(2)group B as the co-culture group,BMSCs + HUVECs + HA-TCP scaffolds;(3)group C as the BMSCs group,BMSCs + HA-TCP scaffolds;(4)group D as the HUVECs group,HUVECs + HA-TCP scaffolds.At the 4th,8th and 12 th weeks respectively postoperative,the morphological changes of the calvarial bone defects and the dynamic changes of bone mass were observed through Micro-CT scanner.Tissue-engineered bone and its surrounding tissues were collected from the corresponding rats,then HE and Masson staining were used to observe the changes of tissue morphology and composition during the healing period of the calvarial defects.Immunohistochemical staining was used to observe the positive-staining of Col-I of the tissue engineering bone during the period of calvarial defect healing in rats,and the expression of Col-I in the immunohistochemical sections was quantified using image-pro-plus 6.0 image software.Activity of ALP was detected by the ALP activity kit,and the dynamic changes of the transcription levels of Runx2 and OCN were detected by q PCR during the healing period.The data was analyzed by SPSS16.0software,One-way analysis of variance(ANOVA)was used to compare the differences between groups and the statistical significance was tested through Tukey HSD.Results: The present study established the critical calvarial defect model of SD rats successfully,the success rate was 98%.The analyzed results of Micro-CT scan showed that the bone formation rate of group B was the fastest,which the bone mineral density was significantly higher than the group C and D either,the bone healing rate of group A was the slowest and the value of bone mineral density of it was the lowest,also(P <0.05).The bone mineral density values of each group increased by time,while the bone mineral density increased fastest in group B.The results of the HE and Masson staining showed that the three-dimensional space of group B was gradually replaced by new bone compared with group A,C and D,there are more irregular trabecular bone and new blood vessels distributed around or in the scaffold material,which are thicker than the other groups,the rate of formation new bone and the maturation of callus was fastest in the group B;The results of immunohistochemical staining of Col-I showed that the positive-staining area of Col-I was smaller and the staining color was lighter in the group A at the4 th,8th and 12 th week respectively.The positive-staining area of Col-I in the group B was the largest,and the staining color was the deepest,the average optical density values were highest in its sections,The difference is statistically significant compared with the C group and the D group(P <0.05),while the positive-staining area of Col-I decreased slightly in each group during the research.The results of ALP protein activity showed that the activity of ALP increased by time and peaked at the 12 th week in group B,the corresponding values increased gently in group C and D during the first 8 weeks and then decreased at the 12 th week,while the values increased slowly in group A at each time point,and the activity of ALP in group B was significantly higher than that in group A,C and D(P <0.05).The results of q PCR test showed that group B could promote the transcription levels of Runx2 and OCN in the bone reconstruction microenvironment,which was better than group A,C and D.conclusion: Through establishing the model of critical calvarial defect of SD rats and implanted with HA-TCP scaffolds with or without seed cells.The changes of the calvarial defect tissue structure,the ALP activity in the newborn bone tissue,the expression of Col-I and the transcription levels of Runx2,OCN were observed.To explore the repair effects of the tissue engineering bone constructed by the composite scaffold technique on the critical calvarial defect in rats,the results of the present study were summarized as follows: co-cultured of BMSCs and HUVECs combined with HA-TCP scaffold tissue engineering bone can accelerate the healing of callus formation,increase the density of new bone tissue and obtain good growth of bone tissue;It can induce BMSCs to differentiate into osteoblasts and can promote the expressions of ALP,Col-I and the transcription levels of Runx2,OCN in the microenvironment of bone reconstruction.It might induce the formation of new blood vessels inside composite scaffolds;It can repair the critical calvarial defects of SD rats successfully.