Effects On BFGF、TGF-β、VEGF、BMP-2Expression Of The Tissues Of Periosteum In Different Period Treatment Of Tcm For The Fractures In Rabbit

1.BackgroundThree period treatment(TPT) is characteristic of Traditional Chinese Medine (TCM) for fractures. During the Sui and Tang dynasties, Lin DaoRen inherit and develop treatment for fracture in the experience of previous,and proposed Lin’s seven-step traditional method for fracture injury, creating the first fracture differential period treatment of TCM. Orthopedics scientist summed up the three period treatment for fractures.Three period treatment principle is the guiding principle of TCM of bone fractures.TPT of TCM for cure bone fracture has a long history and certain curative effect,but lack of scientific experimental evidence.Although clinical practice has confirmed its efficacy, but very few studies for it and only limited to the initial observation of clinical efficacy. In recent years, some scholars in the clinical work, they use one period treatment which is combine mid-period and post-period of fractures, achieved a certain effect, thus proposed TPT of TCM fracture point of view. Some scholars consider not to the staging (one period treatment) of the treatment of fractures in clinical and experimental research, also achieved a certain effect. Thus, the three period treatment has much controversy in fracture of TCM, so there are serious chaos appear three period treatment,two period treatment(WPT) and one period treatment(OPT), therefore the fracture of three period treatment faces serious challenges in the status of the guiding principles with fracture of TCM,which severely restricts the development of Chinese medicine treatment of fractures. But previous studies mostly preliminary observation of clinical efficacy, and are comparison between the control group and blank, so only prompted WPT and OPT also has a role to promote fracture healing, and can not explain the WPT and OPT are better than the TPT. Experimental study has not been reported in the literature about TPT of TCM for fracture to explain the effect and mechanism of the system.We have study the callus healing process with SD rats in previous studies, and found WPT not only can significantly improve the expression of Basic fibroblast growth factor (bFGF) in fracture healing process,WPT on the bFGF expression was significantly stronger than the TPT and OPT in the process of fracture healing.Stage treatment group was significantly better than the treatment group with time and quality of callus formation in rabbits callus healing process, and expression of Vascular endothelial growth factors (VEGF) in TPT groups was significantly superior to OPT with outside the periosteum,the periosteum and bone marrow tissue of the fracture, and expression of VEGFmRNA have same results.The function of bFGF promote angiogenesis, induct of embryonic development, promote nerve growth and so on. And VEGF is recognized as the strongest pro-angiogenic formation factor, it can promote the formation of new blood vessels, maintain the cartilage cell survival, accelerate bone turnover, and pro-angiogenic of other factors can be achieved in whole or part through the VEGF. Two growth factors are the more important factor for vascular reconstruction.And another important bone factor is Bone morphogenetic proteins (BMPs) during the bone healing process, which has induced osteogenesis, first mesenchymal quality cell chemotaxis, aggregation, differentiate into cartilage and bone, and ultimately become the bone marrow, it is recognized as the strongest bone induced differentiation factor, has an extremely important role in regulating fracture healing, more experimental research on BMP-2. In addition, Transforming growth factor-β (TGF-β) can promote periosteal mesenchymal cell proliferation and differentiation, promote the proliferation of osteoblasts, stimulate collagen type II, bone connected to the synthesis of the hormone and osteopontin, TGF-β in the play an important role in the metabolic process of the bone tissue. Therefore, in order to further explore the molecular biological level mechanism of TCM treatment for fracture, a reasonable way to study fracture stage, the experimental period treatment of Chinese medicine on bFGF,TGF-β,VEGF and BMP-2growth factor analyzed simultaneously.2.ObjectiveWe study the gene expression, cytokine expression and its distribution pattern on bFGF, TGF-p, VEGF and BMP-2,as well as bFGF mRNA,TGF-p mRNA, VEGFmRNA and BMP-2mRNA on the rabbit fracture which in the bilateral radial bone in the lower1/3a pronator teres remote3mm transverse fracture,and staging from the molecular level hierarchy in healing rabbit radial bone defects, and clarify TCM fractures treatment effect and mechanism in fracture healing from the organ, cell and molecular level system, to determine the best staging and clarify the stage treatment principles of Chinese medicine fracture, to provide more scientific and theoretical basis guiding treatment for TCM fractures treatment from the perspective of modern medicine. Promote Chinese medicine treatment of fractures to achieve towards standardization and modernization,and to create conditions for Chinese medicine treatment of fractures into the world.3Materials and methods:3.1the object of study and the establish a fracture model174male and healthy rabbits from New Zealand were made3mm transverse fracture with a saw at the lower1/3of pronation ended circular muscle, there was not for any fixation.Those rabbits were remove6as a control group randomly by computer.Then the remaining168rabbits were seperated into four groups randomly,42rabbits in each group.They were TPT group(TTG), WPT group(WTG), OPT group (OTG), and model control group(MCG).Each group was seperated into seven subgroups randomly,6rabbits per subgroup.There was3th,6th,9th,14th,28th,42th,56th day group which at after operation.3.2Intervention methods3.2.1Medicine given Method:The process of rabbit radial fracture healing is divided into three periods:The first period is from1st day to7th day after operation, it is hematoma evolving period.The second is8th to28th day after the operation, it is the original callus formating.The day after28th day is the third period, and it si shaping alterations of bone.The prescription of medine of our study were No.1ZhiShang prescription,No.2ZhiShang prescription,and No.3ZhiShang prescription,that were both selected from the uniform textbooks "The bonesetting medicine of TCM".TTG:TTG divided into three phases in the process of fracture healing,rabbits of TTG were feed No.1ZhiShang prescription at first period, and in second period of fracture healing,No.2ZhiShang prescription was given.The third period of fracture healing,use No.3ZhiShang prescription.WTG:WTG divided into two phase in the process of fracture healing,rabbits of WTG were feed No.1ZhiShang prescription at first period, and combinated prescription of No.2ZhiShang prescription and No.3ZhiShang prescription in second and third period.OTG:OTG not to stage,and rabbits of OTG were feed combinated prescription ofNo.1ZhiShang prescription,No.2ZhiShang prescription and No.3ZhiShang prescription in whole fracture healing process.MCG:MCG were not given any prescription or drug but distilled water as same dose as other group.and twice one day. Control Group:Control Group were given water as same dose as other group,and twice one day.3.3Collection and handling of specimensAt day of3,6,9,14,28,42,56,one subgroup rabbits were selected from every groups and killed by air embolism.Their left radius were taken out and make the specimens by tchique process like stabilized and decalcificated,then all specimens were made into pathological sections,and then were observed by HE stain. Immunohistochemistry and in situ hybridization were performed to examinate the bFGF、TGF-β、VEGF、BMP-2expression with the fracture center and near the two ends of the same position, and analysis positive cells taken nine high-power field use USA3Y color image analysis system and Pixelpro Ver4.0image analysis software.3.4Statistical analysisExperimental data statistical analysis by SPSS20.0statistical software, comparing the difference between the fracture healing process in each organization MOD value group by the general linear model repeated measurement process and apply LSD method of differences between each time point group.The results wirte as mean±standard deviation (χ±s),test level a=0.05.4results4.1MOD vlue of bFGF and bFGFmRNA expression in bone marrow of groups at different time points during fracture healing4.1.1The bFGF expression of outer periosteum resultsPairwise comparisons between the average MOD value of groups, WTG and TTG were significantly higher than the MCG (P<0.01); OTG is higher than that in the MCG (P<0.05); including the highest group was WTG, higher than TTG (P<0.05), also higher thanOTG(P<0.01). At9th day after operation, TTG reached the peak, but the obvious staging group was significantly highest, WTG was higher than MCG(P<0.01); WTG was higher than OTG(P<0.05),and TTG was higher than MCG(P<0.05). Overall, the staging group maintained a high level, and significantly higher than MCG and the non-staging group.4.1.2The outer periosteum bFGFmRNA expression resultsPairwise comparisons between the average MOD value of groups, WTG and TTG were significantly higher than the MCG(P<0.01); OTG was higher than MCG(P<0.05), including WTG was highest, and higher than MCG(P<0.05);and higher than TTG(P<0.05), also higher than OTG(P<0.01); TTG was higher than OTG(P<0.05).At9th day after surgery, the WTG reached a peak,and higher than OTG(P<0.05). Overall, the staging group regardless of the peak of the peak average higher than not staging group and MCG.4.1.3The bFGF expression of periosteum resultsPairwise comparisons between the average MOD value of groups,WTG and TTG were significantly higher than MCG(P<0.01); including OTG was higher than that MCG(P<0.05);WTG was the highest value, higher than TTG(P<0.05), also higher than OTG(P<0.01);TTG was higher than OTG(P<0.05).At9th day after operation, WTG was higher than OTG and MCG(P<0.01); TTG was higher than MCG (P<0.05). Overall, the peak level of the staging treatment group was still higher than the non-staging group and model control group.4.1.4The bFGFmRNA Expression of periosteum resultsPairwise comparisons between the average MOD value of groups, WTG and TTG were significantly higher than MCG, and WTG was higher than TTG and OTG (P<0.01);OTG was higher than MCG(P<0.05);TTG was higher than OTG(P<0.05). After9th day, WTG was higher than OTG and MCG(P<0.01); also higher than TTG(P<0.05). Overall, the peak level and the peak of the staging group was higher than one pertiod group and model control group.4.1.5The bFGF expression of marrow cavity resultsPairwise comparisons between the average MOD value of groups, three groups of MOD values were significantly stronger than the model control group, two group than in a group, the difference was significant (P<0.01); two groups above three groupsa group is higher than that in the untreated control group, the three group than a group, there were significant differences (P<0.05). After14days, two groups higher than that in the untreated control group, the difference was very significant (P<0.01); two groups above the three groups, there is a significant difference (P<0.05). Overall, the peak level and the peak period of the staging group were stronger than a group staging.4.1.6The bFGFmRNA expression of marrow cavity resultsThe average MOD value pairwise comparised between two groups, WTG and TTG of MOD values were significantly higher than MCG, WTG was higher than OTG(P<0.01);WTG was higher than TTG, OTG was higher than MCG, there were significant differences (P<0.05).After14th day. TTG was higher than MCG (P<0.01); WTG was higher than MCG(P<0.05). Overall, the peak level and the peak of staging group was higher than not staging group at most of the time points.4.2MOD vlue of TGF-β and TGF-β mRNA expression in bone marrow of groups at different time points during fracture healing4.2.1The TGF-P expression of outer perichondrium resultsThe average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher than MCG (P<0.01);WTG was higher than OTG (P<0.01); WTG above TTG and OTG was higher than MCG, TTG was higher than OTG, there were significant differences(P<0.05). After42nd day,WTG was higher than OTG, TTG, MCG;TTG more than OTG, the differences were very significant (P<0.01). Overall, the staging group maintained a high level, and significantly higher than the model control group and the non-staging group.4.2.2The TGF-βmRNA expression of outer periosteum resultsThe average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher than MCG, WTG was higher than OTG,the difference was significant(P<0.01); WTH was higher than TTG(P<0.05). After42nd day, WTG was higher than OTG and MCG, the differences were very significant(P<0.01);WTG was higher than TTG, TTG was higher than OTG, there were significant difference(P<0.05).Overall,the staging group regardless of the peak of the peak average higher than non-staging group and the model control group.4.2.3The TGF-P expression of endosteal resultsThe average MOD value pairwise comparised between two groups, TTG and WTG of MOD values were significantly higher than the MCG,WTG was higher than OTG, the difference were significant(P<0.01); WTG was higher than TTG, TTG was higher than MCG, there were significant differences(P<0.05). The42nd day after operation, WTG was higher than OTQTTG and MCG(P<0.01);TTG was higher than OTG and MCG(P<0.05). Overall, the peak level of the staging treatment group was still higher than the non-staging group and model control group.4.2.4The TGF-βmRNA expression of the periosteum resultsThe average MOD value pairwise comparised between two groups, TTG and WTG were significantly higher than MCG,WTG was higher than OTG(P<0.01); WTG was higher than TTQTTG was higher than OTG,OTG was higher than MCG, there were significant differences(P<0.05).At42nd day after operation, WTG was higher than OTG, MCG and TTG was higher than OTG, the differences were very significant(P<0.01);WTG was higher than TTG,TTG was higher than OTG and MCG(P<0.05). Overall, the peak level and the peak of the staging group is higher than not staging group and model control group.4.2.5TGF-β expression of backbone cells resultsThe average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher than MCG, WTG was higher than OTG(P<0.01);WTG was higher than TTG,TTG was higher than OTG,OTG was higher than MCG, there were significant differences(P<0.05).At42nd day after operation, WTG was more than OTG and MCG(P<0.01);WTG was higher than TTG,TTG was higher than MCG(P <0.05). Overall, the peak level and the peak period of the staging group were higher than one staging group.4.2.6TGF-PmRNA expression of backbone Cell resultsThe average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher than MCG, and WTG was higher than OTG(P <0.01);WTG was higher than TTQand TTG was higher than OTG, and OTG was higher than MCG,there were significant differences(P<0.05).At42nd day after operation,WTG was higher than MCG and MCG,and TTG was higher than MCG, the differences were very significant(P<0.01);WTG was higher than TTG,and OTG was higher than MCG(P<0.05). Overall, the staging group of peak level and the peak time were higher than one staging group at most of the time points.4.3MOD vlue of VEGF and VEGFmRNA expression in bone marrow of groups at different time points during fracture healing4.3.1The VEGF expression of outer perichondrium resultsThe average MOD value pairwise comparised between two groups, the MOD value of three Chinese medicine treatment groups was significantly higher than MCG(P<0.01). The average MOD of WTG was highest, and WTG was higher than TTG (P<0.05),TTG was higher than OTG(P<0.05),OTG was higher than MCG (P<0.01). At6th day after operation,WTG was highest, compared with OTG and MCG,the difference was very significant(P<0.01);TTG was higher than OTG and MCG(P<0.05). Overall, the staging group maintained a high level and significantly higher than the model control group and the non-staging group.4.3.2The VEGFmRNA expression of outside periosteum resultsThe average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher thanMCG(P<0.01),OTG was significantly higher than MCG(P<0.05). The average MOD of WTG was highest,WTG was higher than TTG(P<0.05),TTG was higher than OTG(P<0.05),OTG was higher than MCG(P <0.05). At6th day after operation,WTG was higher than MCG (P<0.01);WTG was higher than OTG (P<0.05). Overall, the staging group regardless of the peak of time and the peak higher than not staging group and the model control group.4.3.3The VEGF expression of periosteum resultsThe average MOD value pairwise comparised between two groups,OTG,WTG and TTG was significantly higher than MCG(P<0.01); WTG was highest one, higher than TTG(P<0.05);TTG was higher than OTG(P<0.05). At6th day after operation,WTG was higher than MCG(P<0.05);WTG was higher than OTG (P<0.01).Overall,the peak level of the staging treatment group was still higher than the non-staging group and model control group.4.3.4The VEGFmRNA expression of periosteum resultsThe average MOD value pairwise comparised between two groups,WTG and TTG were significantly higher than MCG(P<0.01);and OTG was higher than MCG(P<0.05); and WTG was the highest one,was higher than TTG(P<0.05),and higher than OTG(P<0.01); TTG was higher than OTG(P<0.05). At6th day and9th after operation, WTG was higher than MCG(P<0.01);WTG was higher than OTG (P<0.05);TTG was higher than MCG(P<0.05). Overall, the peak level and the peak of time for staging group were higher than not staging group and model control group.4.3.5The VEGF expression of marrow cavity resultsThe average MOD value pairwise comparised between two groups,OTG,WTG and TTG were significantly higher than MCG(P<0.01); highest value was WTG,was higher than TTG(P<0.05),and was higher than OTG(P<0.01);TTG was higher than OTG(P<0.05).At9th day after operation,WTG was higher than MCG(P<0.05);WTG was higher than OTG,the difference was very significant(P<0.01);TTG was higher than OTG(P<0.05).Overall, the peak level and the peak time of the staging group was higher than not staging group.4.3.6The VEGFmRNA expression of marrow cavity resultsThe average MOD value pairwise comparised between two groups,WTG and TTG were significantly higher than MCG(P<0.01);OTG was higher than MCG(P<0.05);WTG was the highest one, was higher than TTG(P<0.05), also higher than OTG(P<0.01);TTG was higher than OTG(P<0.05). At9th day after operation, WTG was higher than MCG(P<0.05);WTG was higher than OTG(P<0.01);TTG was higher than OTG(P<0.05).Overall, the staging group peak level and the peak time were higher than not staging group at most of the time points.4.4MOD vlue of BMP-2and BMP-2mRNA expression in bone marrow of groups at different time points during fracture healing4.4.1The BMP-2expression of the outer periosteum resultsThe average MOD value pairwise comparised between two groups, MOD value of OTG, WTG and TTG were significantly higher than MCG(P<0.01); and WTG was the highest one,and was higher than TTG(P<0.05),was higher than OTG(P<0.01); TTG was higher thanOTG(P<0.05).At42nd day after operation,WTG and TTG were higher than MCG(P<0.01); OTG was higher than MCG(P<0.05). Overall, the staging group maintained a high level,and significantly higher than the model control group and the non-staging group.4.4.2The BMP-2mRNA express of outer perichondrium resultsThe average MOD value pairwise comparised between two groups, MOD value of OTG, WTG and TTG were significantly higher than MCG(P<0.01); the highest was WTG, and was higher than TTG(P<0.05),and was higher than OTG(P<0.01);TTG was higher than OTG(P<0.01).At42nd day after operation,WTG and TTG were higher than MCG(P<0.01); OTG was higher than MCG(P<0.05). Overall, the staging group regardless of the peak time and the average peak were higher than not staging a group and the model control group.4.4.3The BMP-2expression of backbone cell resultsThe average MOD value pairwise comparised between two groups, MOD value of OTG,WTG and TTG were significantly higher than MCG(P<0.01); and was highest one,was higher than TTG(P<0.05),and was higher than OTG(P<0.01);TTG was higher than OTG(P<0.05).At28th day after surgery,WTG was higher than MCG(P<0.01);WTG and TTG were higher than OTG(P<0.05); WTG was higher than TTG(P<0.05).Overall, the peak level of the staging treatment group was still higher than the non-staging group and model control group.4.4.4The BMP-2mRNA expression of bone cell resultsThe average MOD value pairwise comparised between two groups, the MOD value of the WTG and TTG were significantly higher than the MCG(P<0.01); including WTG was higher than MCG(P<0.05);WTG was the highest one, was higher than TTG(P<0.05), also higher than OTG(P<0.01); TTG was higher than OTG(P<0.05).At14th day after operation, WTG was higher than MCG, OTG and TTG (P<0.01). Overall, the peak level and the peak time of staging group were higher than not staging group and model control group.4.4.5marrow cavity BMP-2expression resultsThe average MOD value pairwise comparised between two groups, the MOD value of WTG and TTG were significantly higher than MCG(P<0.01); including WTG was higher than MCG(P<0.05);WTG was the highest one, was higher than TTG (P<0.05), also was higher than OTG(P<0.01); TTG was higher than OTG (P<0.05).At42nd day after operation,WTG was higher than MCG(P<0.01);WTG was higher than OTG, and TTG was higher thanMCG(P<0.05).Overall, the peak level and the peak time of the staging group were higher than not staging group.4.4.6The BMP-2mRNA expression of marrow cavity resultsThe average MOD value pairwise comparised between two groups,the MOD value of WTG and TTG were significantly higher than MCG(P<0.01);WTG was the highest one,and was higher than TTG(P<0.05);and was higher than OTG(P<0.01); TTG was higher than OTG(P<0.05).At42nd day after operation, WTG and TTG were higher than theMCG(P<0.05). Overall, the peak level and the peak time of the staging group were higher than not staging group at most of the time points.5Conclusion1) TCM can promote the expression of bFGF, TGF-β, VEGF, BMP-2with immunohistochemistry or situ hybridization examination with different organizations at the various parts of each time point in rabbits fracture healing process, it may be one of the mechanisms of TCM to promote fracture healing.2) TPT treatment have to promote the expression of bFGF, TGF-p, VEGF, BMP-2and bFGFmRNA, TGF-p mRNA, VEGFmRNA, BMP-2mRNA in different tissues, and this is not only one of the mechanism of action the three pertiod treatment of fractures to promote fracture healing, but also shows the need for treatment of fractures in stage.3) The WTG of bFGF, TGF-p, VEGF, BMP-2and of bFGFmRNA, TGF-β mRNA, VEGF mRNA, BMP-2mRNA expression are best than the other of groups, were able to play a role in the process of fracture healing, so regardless of the peak level, or peak timehave been fully improved, while two pertiod is the treatment of best method of reasonable fractures staging.6New creation1) Multi-factor comprehensive analysis of the fracture healing process Study in fracture healing process often is the expression of a single factor or two-factor, but studies analysis the organizations of bFGF, TGF-β,VEGF, BMP-2expression quantitative comprehensive have not been reported.2) Multi-gene expression analysis the mechanism of fracture healing staging treatmentBy immunohistochemistry and in situ hybridization studies treatment of fractures installments to promote fracture healing of bFGF, TGF-β, VEGF, BMP-2expression, and explore the mechanisms of fracture healing stage treatment, revealed the fracture installmentstreatment of reasonable installments, not yet reported.