Effect Of 17β-estradiol On The Expression Of Periostin Of Bone Marrow Mesenchymal Stem Cells From Ovariectimized Rats

Postmenopausal osteoporosis is caused by estrogen deficiency in postmenopause women,and it is the main risk factor that lead to bone loss of maxilla and mandible.It could result in accelerating absorption of alveolar bone,which could lead to gingival recession,root exposure,loosening or even losing of the teeth.Thus would bring more difficulties and adverse effects to the movement of teeth in orthodontic treatment.Estrogen therapy has been the main treatment method for years,but bone tissue is not the only target of estrogen.Long-term administration of estrogen would cause a series of side effects like endometrial cancer,breast cancer,ovarian cancer,thromboembolism and cholecystitis.How to more effectively promote bone regeneration and the repairmen of periodontal defect gets more and more attention.Bone marrow mesenchymal stem cells(BMMSCs)are a group cells with selfrenewal and multi-directional differentiation potential,and bone marrow stem cell transplants to repair periodontal bone defects has been a hot area of research.The ability of proliferation,apoptosis and osteogenic differentiation of BMMSCs from patients or animals with postmenopausal osteoporosis are all changed.And it may be critical to restore the ability of osteogenic differentiation to repair periodontal bone defects.It is reported that Periostin is secreted by osteoblast and its precursor cells and it could enhance the ability of adhesion,migration,proliferation and osteogenic differentiation of osteoblast,and Periostin plays an important role in regulating the growth and metabolism of bone.But the relationship between estrogen and the expression level of Periostin in BMMSCs is still unknown.In this study,we separated and cultured BMMSCs from both ovariectimized and sham-operated SD rats,then detected the expression level of Periostin m RNA.Besides,we investigated the effect of 17β-estrogen on the expression level of Periostin m RNA in vitro and vivo.Our study could be helpful for seeking new treatment with Periostin to replace the estrogen therapy.Methods:1,Establishment of the animal model of postmenopausal osteoporosisSD rats were randomly divided into OVX group and SHAM group.Rats in OVX group were performed ovariectomy to remove the bilateral ovaries,and those in SHAM group were just cut off moderate amount of adipose tissue surrounding the ovaries.After twelve weeks,separated the femurs of both groups to estimate whether the rat model of postmenopausal osteoporosis was successful or not by H-E staining and Micro-CT scanning.2,Separation and identification of rat bone marrow mesenchymal stem cells(r BMMSCs)Separated and cultured r BMMSCs from the femur and tibia of both groups by method of whole bone marrow adherent.Identification of r BMMSCs was conducted by morphological observation and flow cytometry.3,Comparison of the expression level of Periostin m RNA and the ability of proliferation and osteogenic differentiation in r BMMSCs of both groupsCCK-8 was used to check the proliferation rate.Then rBMMSCs were induced to osteoblasts,and alkaline phosphatase staining and alizarin red staining were performed.Total RNA was extracted by Trizol,and then Real-time PCR was chosen to test the expression level of Periostin of both groups.Besides the expression levels of Runx2、OCN、BSP m RNA were detected at the same time.4,The effect of estrogen on the expression level of Periostin m RNA and the ability of osteogenic differentiation on r BMMSCs of OVX group.(1)In vitro experiment: Stimulated r BMMSCs of OVX group by 17β-E2 in various concentrations to check of optimum concentration by CCK-8,and 17β-E2 in optimal concentration was used to culture the r BMMSCs of OVX group.Then alkaline phosphatase staining and alizarin red staining were performed,and the expression levels of Periostin,Runx2,OCN and BSP m RNA were detected by Real-time PCR.(2)In vivo experiment: There weeks after ovariectomy,rats from OVX group were randomly chosen to treat with 17β-E2 by subcutaneous injection(10μg/Kg),and the injection was performed every 2 days,and continued for 9 weeks.Then H-E staining and Micro-CT scanning were used to observe the microstructure changes of femur.r BMMSCs were separated and cultured by method of whole bone marrow adherent.Alkaline phosphatase staining and alizarin red staining were performed,and the expression levels of Periostin,Runx2,OCN and BSP m RNA were detected by Real-time PCR.Results:1,H-E staining and Micro-CT scanning showed that comparing with SHAM group,amount of bone trabecular number of OVX group reduced significantly,and honeycomb structure of spongy bone was destroyed.BV/TV,Tb.Th and Tb.N all declined,Tb.Sp increased(P<0.05).2,The morphology of most r BMMSCs in monolayer culture was spindle-shape or polygon,and most r BMMSCs were arranged radially or spirally.Results from flow cytometry found BMMSCs cell surface markers(CD29,CD90)with high expression,and the hematopoietic stem cell surface markers(CD34,CD45)with little expression.3,The growth curve of rBMMSCs was the “S” shape,and the proliferative rate of OVX r BMMSCs was inferior to SHAM group.The ability of osteogenic differentiation of OVX group decreased significantly.Besides the expression levels of Periostin,Runx2,OCN and BSP m RNA all declined(P<0.05).4,In vitro experiment: The proliferative rate of OVX r BMMSCs treated with1×10-7mol/L17β-E2 was higher than the others.And after treated OVX r BMMSCs with1×10-7mol/L17β-E2,the ability of osteogenic differentiation was promoted apparently,and the expression levels of Periostin,Runx2,OCN and BSP m RNA were enhanced too(P<0.05).In vivo experiment: H-E staining and Micro-CT scanning showed that the degree of damage of cancellous bone was alleviated after subcutaneous injection with 17β-E2(P<0.05).The ability of osteogenic differentiation was promoted apparently,and the expression levels of Periostin,Runx2,OCN and BSP m RNA were rised too(P<0.05).Conclusion:1,With the method of whole bone marrow adherent could separate and culture r BMMSCs successfully.2,With estrogen deficiency,the ability of proliferation and osteogenic differentiation of r BMMSCs decreased,and expression level of Periostin declined.3,Estrogen treatment could enhance the ability of osteogenic differentiation of rBMMSCs,and promote the expression of Periostin.