| Background:Osteoporosis caused by estrogen deficiency is a major problem affecting the quality of life of elderly women.Postmenopausal osteoporosis often leads to accelerated alveolar bone resorption,difficult periodontal tissue regeneration and repair,thus affecting tooth retention and healing of periodontal bone defects.Our previous studies confirmed that bone marrow mesenchymal stem cells(BMSCs)of ovariectomized rats had abnormal functions and decreased osteogenic differentiation,which may be an important reason for the difficult healing of periodontal bone defects in osteoporosis.Therefore,restoring and improving the osteogenic differentiation ability of ovariectomized rat BMSCs is the key to promote periodontal bone regeneration and repair.Periostin(Postn)was found to be one of the most important extracellular matrix proteins for maintaining periodontal tissue integrity.Estrogen may be involved in regulating periodontal tissue metabolism and function by regulating Postn.Mice with the Postn gene knocked out showed signs of osteoporosis and periodontal disease,which directly or indirectly confirmed that Postn may play a key role in regulating bone metabolism and maintaining the stability of the local microenvironment in the periodontal tissue.However,the relationship between Postn and functional abnormalities and osteogenic differentiation of BMSCs in rats with osteoporosis has not been studied.In addition,it is often difficult to provide a healthy growth microenvironment for transplanted stem cells or stem cell polymers due to the inflammatory state of the damaged tissue.Moreover,due to the limitations of their own ability to polymerize and the supply of central nutrients,apoptosis and necrosis may occur in the central cells of cell polymers(cell sheets).Self-assembled polypeptide(RADA 16)is a new kind of scaffold material,which has attracted much attention due to its stability,good biocompatibility and nano-level 3D environment.However,it has not been reported that self-assembled peptide scaffold materials can be combined with the cell membrane to provide a good three-dimensional microenvironment and mechanical support for cell polymers.Therefore,this study intends to explore Postn relationship with osteoporosis rats BMSCs osteogenesis differentiation,hope that through and restore the function of osteoporosis rats BMSCs transplantation BMSCs provide good environment,promote the castrated rats alveolar bone defect repair and regeneration,thus for postmenopausal osteoporosis in patients with periodontal disease stem cell transplantation therapy provides new train of thought and treatment strategies.Objective:To explore the relationship between Postn and osteogenic differentiation of BMSCs in the ovariectomized rat.To observe the effect of Postn up-regulation on the biological behavior of OVX-BMSCs infected by lentivirus.A new type of BMSCs sheet-RADA 16 tissue engineering complex was constructed and used to repair periodontal tissue defects.Methods:1.A rat osteoporosis model was established and verified by micro-CT.BMSCs of sham operation group(SHAM-BMSCs)and BMSCs of ovariectomized group(OVX-BMSCs)were isolated and cultured.BMSCs was identified by flow cytometry and induced osteogenic and adipogenic differentiation.2.Compare the expression levels of Postn in SHAM-BMSCs and OVX-BMSCs.Furthermore,Postn gene carried by lentivirus was transferred into OVX-BMSCs to up-regulate its expression.Osteogenic levels of OVX-BMSCs were explored by experiments including alizarin red and alkaline phosphatase staining,real-time PCR and Western Blot after osteogenic differentiation induction.3.The cell sheet was combined with self-assembled peptides techniques to construct a novel tissue engineering complex BMSCs sheet-RADA 16 scaffold.The morphology of the cell and complex was observed by scanning electron microscopy and laser confocal microscopy.Besides,the proliferation of cells were assessed by CCK8.4.An animal model of osteoporosis alveolar bone defect was established,and the OVX-BMSCs cell sheet/scaffold,Postn-OVX-BMSCs cell sheet/scaffold or scaffold were implanted at the defect relatively.Micro-CT scanning was performed after 3 and 6 weeks.After 6 weeks,HE and Masson staining were performed to investigate the effect of Postn on the repair of alveolar bone defects in rats with osteoporosis.Results:1.Both ovaries of SD rats were removed,and micro-CT scanning and 3d reconstruction of the femur were performed 3 months after surgery.Compared with the SHAM group,the density of femur in OVX group decreased.Trabecular thickness?the number of trabecular bone and the bone volume fraction in OVX group were significantly lower than those in SHAM group,while trabecular spacing was significantly increased,indicating severe osteoporosis in OVX group.2.Flow cytometry results showed that the bone marrow stem cell surface markers CD29 and CD90 were positively expressed;meanwhile,the hematopoietic stem cell surface markers CD34 and CD45 were negatively expressed.Alkaline phosphatase,alizarin red and oil red O staining showed that SHAM-BMSCs and OVX-BMSCs could differentiate into osteoblasts and adipocytes.The osteogenic differentiation ability of OVX-BMSCs was decreased and the adipogenic differentiation ability was increased.3.(1)PCR and Western Blot showed that Postn expression of OVX-BMSCs was significantly lower than that of SHAM-BMSCs(p< 0.05).(2)OVX-BMSCs were transfected with a recombinant retrovirus,and CCK8 assay showed that Postn-OVX-BMSCs showed enhanced proliferation.The osteogenic ability of Postn-OVX-BMSCs was better than that of OVX-BMSCs by alkaline phosphatase and alibi red staining.PCR and Western Blot were used to detect the related osteogenic levels,indicating that the expression levels of osteogenic positive correlation molecules ALP,Runx2 and OCN in Postn-OVX-BMSCs were increased than that of in OVX-BMSCs,while the expression levels of osteogenic negative correlation molecules Sost were decreased.4.The cell sheet was combined with self-assembled peptides techniques to construct a novel tissue engineering complex BMSCs sheet-RADA 16 scaffold.The HE staining?microscopy and confocal laser showed that the cells adhered to the scaffold material and grew well after the sheet was co-cultured with the scaffold material.CCK8 assay showed that the proliferation rate of complex cells increased.5.Rat model of a alveolar bone defect with osteoporosis were constructed and were divided into three groups: control,OVX-BMSCs and Postn-OVX-BMSCs.Micro-CT showed that Postn-OVX-BMSCs group had the fastest healing rate.HE staining results showed that Postn-OVX-BMSCs group had more new blood vessels and osteoblasts,while the control group and OVX-BMSCs group had only a small number of osteoblasts and angiogenesis.Masson staining showed that the control group and OVX-BMSCs group had a large amount of immature bone collagen,while Postn-OVX-BMSCs group had more mature bone.Conclusions:1.Postn expression and osteogenic differentiation of BMSCs in ovariectomized rats were decreased.After the overexpression of Postn in OVX-BMSCs,the osteogenic differentiation and proliferation of OVX-BMSCs were significantly improved.2.A complex can be formed by cell sheets and RADA 16 self-assembling polypeptide gel scaffold.The scaffold material can promote the proliferation and osteogenic differentiation of BMSCs.3.By promoting osteogenesis and angiogenesis,Postn overexpression can accelerate the healing of alveolar bone defects in rats with osteoporosis,which is beneficial to the regeneration of alveolar bone in osteoporosis. |
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