The Experimental Study Of Relevant Factors Of Impact On Fracture Healing In Diabetic Rats And BMP-2,IGF-I Intervention In The Treatment

Objective:To investigate the variations of bone mineral density and the expression of BMP-2, IGF-Ⅰ,typeⅠcollagen protein,osteocalcin and alkaline phosphatase in diabete rats in the fracture region and serum during the fracture healing,and to evaluate the effects on promoting fracture healing in diabete rats by using BMP-2,IGF-Ⅰin the fracture region.Then to discuss the relevant factors affected fracture healing and the roles of BMP-2,IGF-Ⅰin promoting fracture healing in diabete rats.Methods:The experiment was divided into two parts.The first part was to investigate the relevant factors affected the fracture healing in diabete rats.60SD rats(male,3 months old,and 250-300g weight) were divided into two groups randomly,30 in each.Group A was experimental group(the diabete rats with fracture),groupB was control group(the rats with fracture,but without diabete). More rats were taken to guarantee that 5 rats used to be examined in each group in each time point for the rats died and the failure model.Streptozotocin was injected into enterocoelia to destory the B cells of islet of Langerhans in group A to induce diabetes models.Group B was given as the control group.The left tibias of all the models were cutten by shears,and then treated with reduction and external fixation.The X-ray of left lower extremity and the bone mineral density of lumbar spine,hip,local bone and callus were taken in 1st,2nd,3rd, 4th,6th,8th week after the surgery to investigate the healing of fracture,the tissue of fracture region was obtained for HE staining.Draw off blood from arteria cruralis and screened the concentration of glucose,calcium,phosphorus,and alkaline phosphatase.BMP-2,IGF-Ⅰin serum was examined by ELISA assay. Radioimmunoassay for OC in serum,immunohistochemical method for BMP-2, IGF-Ⅰ,and OC in local callus,and real-time quantitative RT—PCR for the expression of BMP-2,IGF-Ⅰand typeⅠcollagen protein in local callus were used.And contrasted the difference between the two groups.The second part of the study was to investigate the treatment effects of BMP-2,IGF-Ⅰin the process of fracture healing in diabetes rats.30 rats were divided into two groups randomly as same as part one(A'and B'),15 in each.Group A' was the group of fracture treating by IGF-Ⅰcomplex fibrin glue in diabetes rats,and group B'was by BMP-2 composite fibrin glue.More rats were taken to guarantee that 5 rats used to be examined in each group in each time point for the rats died and the failure model.The X-ray of left lower extremity and the bone mineral density of lumbar spine,hip,local bone and callus were taken in 2nd,4th,6th,week after the surgery to investigate the healing of fracture.The tissue of fracture region was obtained for HE staining.Draw off blood from arteria cruralis and screened the concentration of glucose,calcium, phosphorus,and alkaline phosphatase.Radioimmunoassay for OC in serum, immunohistochemical method for BMP-2,IGF-Ⅰ,and OC callus in local,and real-time quantitative RT—PCR for the expression of typeⅠcollagen protein in local callus were used.And contrast the difference among A',B'and A.SPSS11.5 statitical package was used to deal with all data.Calculate a p-Value for Student's t-Test and for one-factor analysis of variance.Results:In part one,comparation of Group A and B,there were significantly higher increased of blood glucose in the 1st,2nd,3rd,4th,6th,8th week in group A,all over 13.8mmol/L.The content of calcium,phosphoru decreased in group A,and the significantly difference appeared in sixth week.In Serum,secretion of BMP-2,IGF-Ⅰ, alkaline phosphatase,and osteocalcin were significantly decreased and the peak delayed one week later in group A.The callus formation was poor in each time point in group A.Bone mineral density of hip joints,lumbar spine,local bone,and bone callus organization suggested the slight decreased within four weeks,then significant decresed after four weeks,and the significantly difference appeared in eighth week in group A.HE staining under a light microscope suggested the hysteresis of bone formation.Immunohistochemical staining showed the decrease of the expression of BMP-2,IGF-Ⅰ,osteocalcin in group A.Real-time quantitative RT—PCR suggested the expression of BMP-2,IGF-Ⅰand typeⅠcollagen protein in local bone callus was decreased in group A,and the significantly difference appeared in 2rd week,but no significantly difference in expression of BMP-2,IGF-Ⅰin 4th week,and the peak was delayed one week in each group.There were no significantly differences ofⅠcollagen protein between the two groups in the eighth week.Part two,compared with group A.In Serum,the expression of ALP and osteocalcin were increased significantly in group A' and B',and the highest expression of them were appeared in the second week,but there were no significant differences of them in the sixth week.X-ray suggested the callus formation in A' and B' was increased in 2 groups in each time point,and density of local bone and callus were increased slightly. The expression of typeⅠcollagen protein in callus was increased fastest in group A' and B' detected by RT—PCR,and the significant differences appeared in the second week,but no significant differences in sixth week.Conclusion:1) The healing of the fracture in diabetes rats was not satisfactory,even the delayed or non-union occurred.2) During the healing after fracture in rats with Diabetes,the expression of BMP-2 and IGF-Ⅰboth in the serum and in fracture region were decreased early.3) During fracture healing rats with diabetes,the quantity and the function of osteoblast was declined,and the expression of typeⅠcollagen protein was deceased in local bone and bone callus organization.4)Calcium and phosphorus of rats with diabetic are loss obviously,and during the process of fracture healing.the bone mineral density is decreased in local bone and bone callus organization which caused mineralization obstacles in callus.5) The early decrease of BMP-2 and IGF-Ⅰboth in the serum and in fracture region lead to decline both the function and the quantity of osteoblast,and cause the reducing expression of typeⅠcollagen protein at the same time,which was one of the important reasons of poor fracture healing in rats with diabetes.6)Under the same conditions and the same concentration,the fracture healing in rats with diabetic can be improved significantly by using the IGF-Ⅰcomplex fibrin glue or BMP-2 composite fibrin glue.7) But IGF-Ⅰcomplex fibrin glue had more remarkable effect than BMP-2 composite fibrin glue on fracture rats with diabetes.8)fibrin glue complex BMP-2 or IGF-Ⅰin local treatment of fractures in diabetic rats can not effectively improve the bone mineral density of callus and local bone on the fracture.