Research About The MGF Influence On The Castrated Rats Osteoporotic Fracture Healing

Objective:Osteoporosis(osteoporosis,OP)is the world focus controlling disease of the ageing of the population.Fractures are the serious consequences of OP.And not healing or delayed healing osteoporotic fracture(Osteoporotic fracture,OPF)will seriously affect the health of patients and their quality of life,or even make them maimed or killed.Most of Osteoporotic fracture are comminuted fractures,the bone quality of the fractural site reduces,the strength weakens,and the treatment is quite difficult,so the medical expenses are very alarming.Although in recent years the research of the osteoporosis prevention and treatment has made great achievements, the effective ways of treating the osteoporotic fractures have not been found yet. Therefore,osteoporotic fracture combating has become the hot topic of research nowadays.On the promotion drugs of osteoporotic fracture healing,the estrogen replacement therapy,fluoride,bisphosphonates,calcitonin and calcium preparations are mainly applicated.Although it has made a certain effect,it is still not satisfied,and there is considerable side-effects and whether can be long-term used has been controversial. Chinese medical academics are paying great attention to Chinese medicine promotion of osteoporotic fracture healing progress gradually.The Chinese traditional medicine classified osteoporotic fracture as "bone contaction,bone fratigue" on the vision of clinical symptoms.It is also considered that the bone growth and metabolism had very close relationship with "kidney" in Chinese medicine,depending on the saying that "kidney of fine,fine Health and pulp,pulp Custody Bone".This study is on the basis of the establishment of castration Wistar rat model of osteoporotic fracture,intervents the taking of the Chinese medicine administration of the decoction,uses the immunohistochemistry,molecular biology,and other experimental methods,discusses the biological characteristics of the relevant factor producing Osteoporotic fracture in the fracture healing process in different periods with the lack of estrogen,to clarify the function organization of the MGF in postmenopausal osteoporotic fractures healing process,and provide the experimental basis for the application and the development in the Chinese medical prevention and treatment of osteoporotic fracture healing process. Methods:1.The establishment of an animal model of osteoporotic fractureDevide the 90 6-month female Wistar adult big rats of 260 to 300 grams into 3 groups randomly:30 rats into a castrated contrasting group(Sham),30 into a model group(OVX)and the other 30 into the treatment study group of taking the MGF.A MGF group and an OVX group extract an ovary on the double sides perfectly, and a Sham group only removes the same amount of fat.After raising for 3 months, establish a fracture model on a thighbone in one side by an oppening operation.2.The medicine given way:after the firming operation of the fracture,10mg,/(kg d) of the decoction(offered by the research center of old medicine science of Douser's medical academy of Huazhong University of Science and Technoiogy)is pushed into the stomach towards a MGF group.An OVX group and a Sham group are given the same amount of physiological saline and the course of treatment is 2 weeks,4 weeks and 6 weeks.3.Observation targets and determination ways:3.1 The blood biochemical changes:select five big rats randomly in each group to pick the eyeball and draw blood in the second,forth and sixth week after operation and extract serum from centrifugal 5 min,test the serum E2 and serum BGP with RIA, and test the serum ALP using the Biochemical Analyzer.3.2 The dynamic changes of the IL-6,TGF-β1 in the callus bone in the fracture healing process:make image analysis by using the SABC staining,and the HP IAS-1000 high-resolution color pathological image analysis system.Test the expression of the IL-6,TGF-β1 in the bone callus in the second,forth and sixth week after operation.3.3 The dynamic changes of the BMP-3 and OPG in the callus bone in the fracture healing process:test the expression of the BMP-3mRNA,OPGmRNA in the bone callus in the second,forth and sixth week after operation using the fluorescence quantitative reverse transcription polymerized chain reaction(RT-PCR)method.Results:1.The blood biochemical changes:1.1 The MGF influence on the serum E2 of ovary extracted rats((?)±s)The OVX group is significantly lower than the Sham group and the MGF group in various time periods(P＜0.05);the serum E2 in the MGF group in the second and the forth week is significantly lower than the Sham group(P＞0.05);the serum E2 in the MGF group in the sixth week is significantly higher than the Sham group(P＜0.05); the serum E2 in the OVX group and the MGF group have a significant increasing trend in terms of time(P＜0.05).The Sham group has no statistic differences in terms of time(P＞0.05).1.2 The MGF influence on the serum E2 and the plasma BGP of ovary extracted rats ((?)±s)The Sham group has no significant difference in the three time periods(P＞0.05). The MFG group has discending trend and significant differences in the three time periods(P＜0.05).The serum BGP value in the MFG group is significantly lower than the Sham group in the sixth week(P＜0.05).The serum BGP value in the OVX group has increasing trend and also significant differences in the three time periods(P＜0.05).1.3 The MGF influence on the serum E2 and the plasma ALP of ovary extracted rats ((?)±s)The Sham group in the three time periods has no significant differences(P＞0.05). The OVX group is significantly higher than the Sham group in the second week(P＜0.05).The serum ALP value in the MFG group has a significant descending trend comparatively(P＜0.05).The serum ALP value in the OVX group has a significant increasing trend comparatively(P＜0.05).The serum ALP has the minimum value of each group in the forth week(P＜0.05).2.Histological observation:2.1 The comparison between the Sham group and the OVX group2.1.1 In the second week after fracture,the cartilage bone callus in the OVX group form to take the place of fiber bone callus.The cartilage island appears in the early fiber bone callus and expanses constantly.Most of the cartilage cells surrounding the blood vessels are small circular naive cartilage cells,and as time goes by,most of the cartilage cells surrounding the cartilage callus degenerate and necros,and a small amount of the original(naive)trabecular bones come out.The osteoblast quantity around the original trabecular bones is small and the organization among the trabecular bones is the connective tissues(including the blood cells).There is a large number of neatly arranged osteoblast on the surface of the origina trabecular bone in the Sham group.2.1.2 In the forth week after fracture,the bone callus number decreases in the OVX group,the liber connective tissues decrease gradually and the cartilage bone callus increase gradually.There is mostly bone healing in the fracture side,as well as constant bone callus wrapping in the Sham group.2.1.3 In the sixth week after fracture,the cartilage bone callus peripheral areas are constantly filled with the original trabecular bone(bone knitting)instead,but the cartilage bone callus have not absorbed and mixed yet and there are still some carlitage islands;the early formed original trabecular inside bone cartilage cells degenerate and necros or differentiate,or turn into bone cells;the surface osteoclasts appear at the same time and couple with the osteoblasts,so that the original trabecular bones do not become the mature ones.Compared with the Sham group,the trabecular bone absorbed fast and the number is large in the OVX group,and the distant among the trabecular bones are widened,inside which are full of a large amount of fat cells. At the same time,the fracture sides mostly form bone callus2.2 The comparison between the MGF group and OVX group2.2.1 Two weeks after fracture,the membrane bones are basically similar between the MGF group and the OVX group.The cartilage islands appear in the fiber bone callus and expand gradually,and newly-born trabecular bones appear around the cartilage bone callus.A big amount of liber organiazation,as well as a small amount of liber bone callus form in the OVX group.The cartilage bone callus in the OVX group are more than those of the MGF group.The liber bone callus in the MGF group are more than those of the OVX group.2.2.2 Four weeks after fracture,the bone callus are more and the cartilage cells are less in the MGF Group.No cartilage bone islands exsist.The trabecular bones are uniformed in size and there is no significant broadening in distance.The lamellar bones form,and there are many visible capillaries implantating in the surrounding connective tissues and the bone marrow cavity.The surrounding liber connective tissues decrease gradually in the OVX group and the carlitage bone callus increase gradually.A small amount of mature bone callus appear in the MGF group.2.2.3 Six weeks after fracture,the newly-born trabecular bones around the cartilage bone callus increase gradually in the MGF group.The early formed original trabecular cartilage bone callus turn into bone cells gradually at that time.The original trabecular bones turn into mature trabecular bones constantly.The trabecular bones become mixed and windened,the portion of carlitage bones becomes less, and the cartilage bone callus transmit into mature bone callus gradually.The cartilage bone callus transform into bone cells slowly in the OVX group,and the cartilage bone callus haven't absorbed or mixed yet.Cartilage bone islands still come out,tansparent cartilage bones still exist,and the amount of the cartilage bone callus is still very large.3.Immunohistochemical results3.1 SABC stained histological observation:there is no difference of the cytokine localization of TGF-β1,IL-6 in the fracture healing process in the MGF group and the OVX group.TGF-β1 mainly locats in mesenchymal cells,phagocytes,bone cells, cartilage cells,and so on.IL-6 mainly locats in osteoblast cells,cartilage cells and so on.3.2 The fracture side TGF-β1 expression image analysis results:the TGF-β1 in the fracture side expression has no significant difference in the Sham group in terms of time(P＞0.05);TGF-β1 in the fracture side expression has a significant descending trend in the OVX group in terms of time(P＜0.05);TGF-β1 in the fracture side expression has a significant increasing trend in the MGF group in terms of time(P＜0.05);TGF-β1 in the fracture side expression result is significantly higher than that of the OVX group and the Sham group in the forth and the sixth week.3.3 IL-6 in the fracture side in the expression image analysis results:IL-6 in the fracture side expression has a significant descending trend in the Sham group in terms of time(P＞0.05);IL-6 in the fracture side expression has a significant increasing trend in the OVX group in terms of time(P＜0.05);IL-6 in the fracture side expression has a significant descending trend in the MGF group in terms of time(P＜0.05);IL-6 in the fracture side expression in the MGF group is significantly lower than that of the OVX group and the Sham group in the forth and the sixth week.4.The test results of the Fluorescence quantitative reverse transcription polymerase chain reaction(RT-PCR):4.1 The expression of BMP-3mRNA in the fracture sideRat fracture side BMP-3mRNA expression levels:there is a descending trend in the overall comparison in terms of time and there are significant differences(F=153.19, P＜0.05);the overall comparison in groups is the model group＜experimental group＜control group and there are significant differences(F=262.23,P＜0.05);the interactive comparison in terms of time and groups also has significant differences(F =81.50,P＜0.05);there are significant statistic differences between any two groups (P＜0.05)4.2 The expression of OPGmRNA in the fracture side Rat fracture side OPG mRNA expression levels:there is a descending trend in the overall comparison in terms of time and there are significant differences(F=124.51, P＜0.05);the overall comparison in groups is the model group＜experimental group＜control group and there are significant differences(F=118.42,P＜0.05);the interactive comparison in terms of time and groups also has significant differences(F =16.23,P＜0.05);there are significant statistic differences between any two groups (P＜0.05)Conclusion:MGF has a fracture healing promoting function towards the OPF of the castration rats. The function organizations:maybe improve the E level to promote the bone formation and inhibit the bone resorption to regulate the bone metabolism balance; reduce the gene expression of the fracture side BMP-3 and promote the TGF-β1 synthesis and secretion,stimulate the osteoblasts' generation and differentation to promote the bone formation;reduce the gene expression of the fracture side OPG and reduce the synthesis and secretion of IL-6,inhibit the activity of the osteoclasts to inhibit the bone resorption.Anyway,the MGF promotes the OPF fracture healing in both promoting the bone generation and inhibiting the bone resorption ways.