The Study Of Periostin Regulates Osteogenic Differentiation In Bone Marrow Mesenchymal Stem Cells From Ovariectomized Rats

The main type of primary osteoporosis is Postmenopausal osteoporosis(PMO).It is a kind of metabolic bone disease caused by the decrease of estrogen level in postmenopausal women,leading to more bone resorption and less bone formation.Alveolar bone,as a part of body bone,will also be absorbed when osteoporosis occurs in our body.Therefore,postmenopausal osteoporosis with periodontal disease has attracted the oral worker’s attention.BMSCs derived from bone tissue,they are kind of Mesenchymal stem cell can differentiate into different cells,such as adipocytes,chondrocytes or osteoblasts.BMSCs from bone marrow can enter the periodontal ligament cavity through vascular channels,it is good for periodontal tissue regeneration that BMSCs have the ability to differentiate into PDLSCs.So,BMSCs is important for bone and periodontal tissue.Some study found significant defects in the osteogenic differentiation and proliferation of PMO-BMSCs,and cell’s function defects is the main cause of osteoporosis.To explore the related factors and mechanisms of abnormal osteogenic differentiation of PMO BMSCs,and restore the osteogenic ability of cells has become the focus of this study,so as to provide some reference for improving the treatment level of bone tissue defect reparation.Postn(periostin)is one of the extracellular matrix proteins that specifically expressed in periosteum and periodontal ligament,it can maintain the integrity of tissue structure and the stability of internal environment.Postn is the basis of highly complex extracellular network structure,it have many function,such as regulate cell adhesion,cell differentiation and construction of cell matrix.The deletion mutation of periostin can directly lead to osteoporosis and periodontal structure’s disorder,It has been found that the effect of oestrogen on osteogenic differentiation of BMSCs is mediated by periostin.Under inflammatory microenvironment,the expression of Periostin in osteoblasts and periodontal ligament cells decreased,which directly or indirectly confirmed that Periostin may play a key role in regulating systemic bone metabolism and maintaining the stability of local periodontal microenvironment.Therefore,We hypothesize that periostin may be involved in the osteogenic differentiation of PMO-BMSCs,and that provides a new direction to explore the abnormal osteogenic differentiation of bone marrow Mesenchymal stem cell in postmenopausal osteoporosis.In this study,First,we will get BMSCs from healthy and osteoporosis bone tiusse,and then making sure the different expression of periostin in their process of osteogenic differentiation,and then by up and down regulating the expression of periostin.We want to demonstrated that periostin is involved in osteogenic differentiation of BMSCs.After verified the regulatory effect of Periostin in this process,we observed that changes of the expression of various molecules in ILK/Akt/Gsk3β pathway by regulating the expression of Periostin.So,Its regulatory effect on bone differentiation of BMSCs may be mediated by binding to integrin receptors,regulating the intracellular integrin chain kinase(ILK),and re phosphorylating its downstream molecules Akt and Gsk3βPart I Isolation and culture identification of BMSCs from healthy and ovariectomized rats and difference of biological behaviors Objective1.Isolation,culture and identification of Sham-BMSCs and OVX-BMSCs;2.Identify the different expression of periostin in the process of osteogenic differentiation,and different osteogenic ability in two groups,periostin correlates with osteogenic differentiation of BMSCs.Methods1.Sham-BMSCs and OVX-BMSCs were isolated and cultured.The expression of stem cell makers were detected by flow cytometry.The ability of multi-directional differentiation was identified by osteogenic and adipogenic induction.2.Osteogenic induction was performed on Sham-BMSC and OVX-BMSCs,and cells were examined by Alkaline phosphatase staining and Alizarin Red staining in Day 0,7,14 and 21 to compare the osteogenic ability of the two groups.3.Osteogenic induction was performed on Sham-BMSC and OVX-BMSCs,and cells were examined by q PCR and WB in day 0、7、14、21to detecte the expression of periostin in two groups.Results1.The bone morphology parameters were analyzed by micro CT bone scan to determine the success of the establishment of the animal model of postmenopausal osteoporosis.Sham-BMSC and OVX-BMSCs can be successfully isolated and cultured,and strongly expressed the mesenchymal stem cell markers CD29 and CD90,negatively expressed the hematopoietic markers CD34 and CD45.2.The results of ALP staining and alizarin red staining showed that the osteogenic ability of OVX-BMSCs was significantly weaker than sham-BMSCs.3.The expression of Periostin in Sham-BMSC and OVX-BMSCs were increased on Day 0、7 and 14,and decreased on Day 21.The expression of Periostin was significantly different between the two groups,and that was correlated with the osteogenetic gene Runx2.Conclusions1.The animal model of postmenopausal osteoporosis in rats was successfully established,sham-BMSCs and OVX-BMSCs can be successfully isolated and cultured.The osteogenic ability of OVX-BMSCs was significantly weaker than that of sham-BMSCs2.With osteogenic induction,the expression of periodin in the two groups was higher than that before osteogenic induction,the expression of periostin reached the peak on day14 and decreased on day 21 in the two groups.The expression of Periostin was significantly different between sham-BMSCs and OVX-BMSCs after 14 days of osteogenic induction.The expression of Periostin was significantly increased in sham BMSCs with strong osteogenic ability.Part II The effect of periostin in the osteogenesis of sham-BMSCs and OVX-BMSCs ObjectiveIdentify the effect of periostin in the osteogenesis of sham-BMSCs and OVX-BMSCs in vitro and vivo study.Methods1.Periostin plasmids for periostin knock down(sh periostin)and periostin lentiviruses for periostin overexpression(lv periostin)were constructed,and transfected into cell to identify MOI and the most effective down-regulation plasmid.2.ALP staining and enzyme activity analysis were used to identify the effect of osteogenesis,as well as q PCR and WB detection of the expression of osteogenic genes BSP,OCN and Runx2 in cells after change the expression of Periostin.3.The cells were transfected by lentivirus and plasmid,and then were mixed with bone filling materials and implanted into nude mice.The nude mice were fed for 4 weeks,and were killed to obtain materials.The tissue masses were stained with HE and Masson,and the expressions of osteogenic genes ALP and Runx2 were detected by q PCR and WB.Results1.BMSCs were successfully transfected,and the MOI of perostin lentivirus was 70.The most effective plasmid to down regulate perostin was POSTN-rat-1034.2.In vitro,up regulation of Periostin expression can restore the osteogenic differentiation ability of OVX BMSCs.Conversely,down regulation of Periostin can inhibit the osteogenic differentiation ability of sham BMSCs.The expression of osteogenic genes Bsp,Ocn and Runx2 in cells changes positively with Periostin.Periostin expression is high,cell osteogenic ability is strong,and osteogenic gene expression is high,and vice versa.3.HE staining and Masson staining of tissue blocks from nude mice showed that up regulation of Periostin expression could promote OVX BMSCs to form more bone glue tissues;Conversely,down regulation of Periostin inhibited the formation of collagen tissue in sham BMSCs.The expression of Alp and Runx2 was detected by grinding tissue blocks,which was consistent with that in vivo.The expression of osteogenic gene changed positively with periodin.ConclusionsIn showed the consistent role in vitro and vivo study,up regulation of Periostin can improve the osteogenic differentiation of OVX-BMSCs,while down regulation of Periostin can inhibit the osteogenic differentiation of sham-BMSCs.Part III Periostin regulates osteogenic differentiation via ILK integrin signaling pathway ObjectiveTo investigate the effect of periostin in osteogenic differentiation of rat BMSCs.Whether Periostin regulates the osteogenic differentiation of cells through ILK / Akt /Gsk3β pathway.Methods1.After osteogenic induction of sham BMSCs and OVX BMSCs for 14 days,the cells of the two groups were stained with periostin and ILK double label fluorescence.2.Overexpression lentivirus was transfected into OVX BMSCs,up regulation of Periostin expression,and then added the ILK inhibitor,detected the periostin and ILK /Akt / Gsk3β in different groups by q PCR and WB.3.After up regulation the expression of periostin,and added ILK inhibitor,then Alizarin red staining,EDU proliferation test,Transwell migration test and apoptosis rate of different groups of cells were detected to observe the changes of osteogenic ability,proliferation ability,migration ability and apoptosis rate.Results1.The results of double label fluorescence staining showed that the optical density of periostin in sham BMSCs group was significantly higher than OVX BMSCs group.The analysis of ILK staining results was consistent with the trend of periostin,the osteogenic differentiation of cells is associated with ILK.2.After up regulation the expression of periostin,and added inhibitor of ILK,and then detected the expression of periostin and ILK / Akt / Gsk3β.The results of q PCR and WB were showed that the expression of ILK increased with the expression of periostin,after added ILK inhibitor,the ILK was decreased,and the expression level of periostin also decreased,so ILK changed positively with the expression of periostin,and ILK also had a negative feedback regulation effect on periostin.Up regulation of periostin and added inhibition of ILK,Akt and Gsk3β in different groups was no significant difference,but the expression level of p-Akt and p-Gsk3β was consistent with the expression level of periostin.3.After down regulation the expression of periostin,and added ILK agonists,and then detected the expression of periostin and ILK / Akt / Gsk3β.The results of q PCR and WB were showed that the expression of ILK decreased with the expression of periostin,after added ILK agonists,the ILK was increased,and the expression level of periostin also increased,so ILK changed positively with the expression of periostin,and ILK also had a negative feedback regulation effect on periostin.Down regulation of periostin and added agonists of ILK,Akt and Gsk3β in different groups was no significant difference,but the expression level of p-Akt and Gsk3β was consistent with the expression level of periostin.We get reverse and similar results,compared with the up-regulated part.Conclusions1.Periostin has a positive regulatory relationship with ILK,it plays an important role in the process of BMSCs bone differentiation through ILK/p-Akt/p-Gsk3β aix.At the same time,ILK also has a negative feedback regulation effect on periostin.2.Periostin plays an important role in the process of BMSCs bone differentiation through ILK/p-Akt/p-Gsk3β aix,Part of reason may be that it plays a regulatory role by affecting cell proliferation,differentiation and migration.