Fractures Of Three Rule On Rabbits Fracture Healing And Vascular Endothelial Growth Factor Gene Expression

Three-period treatment theory of bone fracture is the guideline of the fracture treatment of TCM. It divides the process of fracture healing into three periods, and uses different types of prescriptions according to various of symptoms and signs in different periods. In the first period of fracture healing, the remedy that can invigorate the circulation of blood and clear off the haematoma is used. In the second period of fracture healing, most of the ingredients of the prescription are the medicines that can accelerate the growth of bone. According to the basic theory of TCM, the functions of "kidney", "liver" and "spleen" are related to the growth of bone. So in the last period of fracture healing, invigorative drugs which can enhance the functions of "kidney", "liver"and "spleen" play an important role.Chinese medicine has being used to treat bone fracture since over three thousand years ago. And many theories about treating fracture have been developed. In these theories, three-period treatment is the most unique one. But up to now there are few clinic and experiment research focused on this subject. Many doctors doubted whether it was useful to apply this theory to clinic. Even some experts suggested that there was no need to treat fracture with different prescriptions in different stages. It is in a quite confused situation in clinic. In our early study of three-period treatment theory, we found that TCM treatment can bring forward the expression summit of FGF2, and prolong the period of higher status of FGF2 expression. The FGF2 expression of three-period group had the tendency to be greater than that of the one-period one. But there are some shortages in the early experience. To study the mechanism of the three-period treatment of TCM further, we designed this experiment. A 3mm wild bone defection was made at the lower one third of radius of rabbit as the fracture healing model. The histological changes at the callus in different time points after operation were observed. Immunohistochemistry and in situ hybridization were used to examine the expression of Vascular Endothelial Growth Factor (VEGF) and VEGF mRNA in different tissues of callus in different time points after operation.Materials and methods.140 male and healthy rabbits were made 3mm wide bone defection at lower one third part of both radius as fracture healing model. There was no internal or external fixation exerted. Then those rabbits were seperated into four groups randomly, they are model control group(MCG), one-period treatment group(OTG), three-period treatment group(TTG) and positive medicine treatment group(PTG). Every group were seperated into seven subgroups randomly, 5 rabbits per subgroup. All rabbits were bred separately with normal forage and fresh water.According to the three-period treatment theory of bone fracture and the histological changes in radius fracture healing of rabbit, the process of fracture healing is divided into three periods. The first period is from the 1st day to the 7th day after operation. The second period is from the 8^th to 28^th day after operation. The days after the 29^th day is the third stage of the fracture healing. Rabbits of TTG were feed ZhiShang 1# prescription in the first period of fracture healing. And in the second period of fracture healing, ZhiShang 2# prescription was given. Since the 29th day after operation, ZhiShang 3# prescription was used as the substitute of ZhiShang 2# prescription. Fracture healing process of Rabbits in both one-period group and positive medicine group were not divided into different period, and those rabbits were given single prescription or drug after operation. Rabbits of OTG were feed by the combinated prescription of the ZhiShang1# prescription, ZhiShang 2# prescription and ZhiShang 3# prescription, which is called YiQi prescription. And the rabbits of PTG were feed by guzhecuoshangsan, a Chinese patent medicine which is used to treat bone fracture for decades years. Those in model control group were given no prescription or drug but distilled water as same dose as that of other groups.At day3, 6, 9, 14,28,42 and 56, one subgroup rabbits were selected from every group randomly and were killed by aeroembolism respectively. Their radius were taken out and put into 40g·L^-1 PFA to be stabilized for 24 hours. Then the left one was taken as the research object. After decalcificated by 10%EDTA-Na₂(with 1‰DEPC), all specimen were made into pathological sections. The histological changes were observed by HE stain. Immunohistochemistry and in situ hybridization were performed to examinate the VEGF and VEGF mRNA expression. NIKON Pathological Image Acquisition System was used to take the photos of the tissue near the fracture at high power lens, such as diaphysis cells, outer periosteum, endosteum of bone, and so on. The Mean Optical Density(MOD) of the cells which is positive stained was measured by Image Pro Plus 5．0．ResultThe histological changes of the fracture were influenced by TCM.Considering the histological scores, all the scores of the three medicine treatment groups have the tendency that is higher than that of MCG at the most of time points after operation. The average score of all time points shows that the average score of TTG is the highest among the four groups, and has significant difference when compared with that of MCG. The scores of OTG and PTG have the tendency of being higher than that of MCG too. The p=0．079 and 0．059, it is very close to the bounder of significant difference. If we could increase the number of rabbits the difference would be more wide. From day 9 to 42, the scores of TTG are higher than are the other three groups at most of time point. It illustrate that treating fracture with three-period treatment can improve the quality of callus at the intermediate and late stage of fracture healing.The VEGF expression in diaphysis cells of every group at different time points in fracture healing. The diaphysis cells are very active and the remodeling of cortical plate is very tense during the healing process. The VEGF and VEGF mRNA expression of three medicine treatment group is higher than that of MCG. The expression of TTG is higher than that of the other groups at the early and late stage of healing. It means that TCM can improve the angiogenesis of diaphysis, accelerate the remodeling of Haversian lamella. Three-period treatment has been more effective in the early and late stage of healing. The VEGF expression in outer periosteum different time points in fracture healing. The outer periosteum is very active during fracture healing. From the 6^th day on, the outer periosteum became thick. The mesenchymal cell splitted into osteoblast, and became osteoid tissue gradually. The expression of VEGF and VEGF mRNA mainly appears in the mesenchymal cell, Vascular Endothelial cell and the osteoclast between the outer periosteum and cortical plate. In our experience, the VEGF and VEGF mRNA expression generally declines as time gose by. All medicine treatment groups show a higher expression of VEGF and VEGF mRNA than the MCG at the most of time points after operation. The TTG shows higher expression during day 6 to 42 than the other three groups. It illustrates that TCM can improve the expression of VEGF and VEGF mRNA in the outer periosteum, and three-period treatment has been more effective in the intermediate and late stage of fracture healing.The VEGF expression in endosteum of bone at different time points in fracture healing. From the 3 day after operation, the endosteum of bone became thick. At day 6, there are some osteoid tissues appearing under the endosteum of bone. And the trabeculas of bone in the medullary canal appear much earlier than that of outer periosteum. The endosteum of bone acts a most important role in the healing of fracture. From the expression of VEGF and VEGF mRNA, the three medicine treatment groups show higher expression than MCG at most of time points after operation. The TTG show higher expression in the 2 weeks after operation than the other two medicine treatment groups. It means that TCM can improve the expression of VEGF and VEGF mRNA in the endosteum of bone during the course of fracture healing. The three-period treatment shows crucial effectiveness at the early and intermediate stage of fracture healing.The VEGF expression in haematoma at different time points in fracture healing. The haematoma is very important in the fracture healing. There are obvious expression of VEGF and VEGF mRNA in the inflammatory cell and reticular fibers of haematoma. From the 6 day after operation, the reticular fibers which expression VEGF and VEGF mRNA begin with calcium deposit. The result shows that all the three medicine treatment have the effect to improve the expression of VEGF and VEGF mRNA in the haematoma, and the TTG has the largest effect.The VEGF expression in fibrous callus at different time points in fracture healing. The fibrous callus is the link between haematoma and trabecula of bone. The Vascular Endothelial cell, inflammatory cell and connective tissue appear VEGF and VEGF mRNA expression. The result shows that all the three medicine treatment have the effect to improve the expression of VEGF and VEGF mRNA in the fibrous callus, and the TTG has more effect on the VEGF mRNA expression.The VEGF expression in soft callus at different time points in fracture healing. From day 9 to 42, there are large area of hyaline cartilage under the outer periosteum. In the endochylema and the base of cartilage ceill VEGF and VEGF mRNA expression can be noticed. The result shows that all the three medicine treatment have the effect to improve the expression of VEGF and VEGF mRNA in the soft callus. The three medicine treatment groups have the similiar effect on VEGF and VEGF mRNA expression in soft callus. But at day 28 and 42, the VEGF and VEGF mRNA expression of OTG is lower than the other three groups.The VEGF expression in bone trabecula at different time points in fracture healing. In some bone cell of bone trabecula there is VEGF and VEGF mRNA expression. The result of VEGF and VEGF mRNA expression in the bone trabecula shows that the three medicine treatment have the effect to improve the expression of VEGF and VEGF mRNA in the bone trabecula during the healing process of fracture, and TTG shows higher expression than the other two medicine treatment groups at the early stage and late stage of fracture healing.The VEGF expression in intertrabecula tissue at different time points in fracture healing. The intertrabecula tissue is very important to the remodeling of trabecula. From day 14 to 56, the Vascular Endothelial cell, osteoblast and osteoclast express VEGF and VEGF mRNA. The result shows that the three medicine treatment groups have the effect to improve the expression of VEGF and VEGF mRNA in the intertrabecula tissue during the healing process of fracture, the TTG has more predominant effect on the intertrabecula tissue at the late stage of fracture healing.The VEGF expression in bone marrow at different time points in fracture healing. The bone marrow has the capacity of inducting bone formation. The VEGF and VEGF mRNA expression in bone marrow can be found during the whole process of fracture healing. The three medicine treatment groups show higher expression of VEGF and VEGF mRNA in bone marrow than the MCG at most of time points after operation, the TTG have more predominant effect on bone marrow tissue at the early and intermediate stage of fracture healing.Conclusion1) TCM can promote the VEGF and VEGF mRNA expression at all tissues near the fracture during fracture healing. It could be one of the mechanisms that TCM promote the fracture healing.2) Different Chinese medicines don't have the same effect on VEGF expression at different stage of fracture healing. Treating fracture in stages has more predominant effect on the expression of VEGF and VEGF mRNA than that treating fracture with single prescription or drug. It is necessary to treat fracture in stages.3) The three-period treatment principle of fracture uses suitable prescription based on pathological changes at different stage of fracture healing, promotes the expression of VEGF at relevant tissues. The three-period treatment principle could be the acceptable method of treating fracture in stages.New Creation1) Explore the VEGF expression variation in different tissues during the fracture healing.2) Explore the influence that three-period treatment effect on VEGF expression during the fracture healing using Immunohistochemistry and in situ hybridization. Investigate the mechanism that three-period treatment promote the fracture healing.