Calcitonin Induces Collagen Synthesis And Osteoblastic Differentiation In Human Periodontal Ligament Stem Cells

ObjectivePeriodontitis is caused by the microorganisms in dental plaque and periodontal tissue that supports the chronic infectious diseases.After the onset of the disease can break the dynamic balance of alveolar bone resorption and repair,causing the absorption of alveolar bone is greater than the repair of alveolar bone,resulting in alveolar bone height was reduced.The purpose of periodontal therapy is not only to control the inflammation,but also to regenerate the damaged periodontal tissue to form a new attachment.Periodontal ligament stem cells(PDLSCs)are in orientation in the periodontal tissue stem cells,have multilineage differentiation potential and periodontal regeneration,repair processes play an important role in periodontal tissue wound.The study showed that the high expression of periodontal ligament stem will secrete extracellular matrix(ECM)and osteogenic differentiation promote alveolar bone regeneration,which is the cellular basis of alveolar bone repair.Calcitonin family members can activate intracellular signaling pathway by binding to the same type of calcitonin receptor and the biological properties of different cell.The mechanism of calcitonin(CT)in the process of bone healing is to promote bone cell proliferation,secretion and synthesis of extracellular matrix and promote the calcified matrix and promote bone cells into bone cells,but also by promoting the expression of OPG,reduce the expression of RANKL,inhibition of osteoclast generation.But until now,it has not been reported that the role of CT in the regulation of PDLSCs osteogenic differentiation,and its role in alveolar bone regeneration.This study was to investigate the effect of CT on collagen synthesis and osteogenic differentiation of PDLSCs and its possible molecular mechanism.MethodsPart one: 60 patients were divided into two groups: chronic periodontitis group and healthy control group.The gingival index,probing depth and clinical attachment level were recorded.Collect the gingival crevicular fluid by quantitative enzyme linked immunosorbent assay(ELISA)detection in gingival crevicular fluid in CT adsorption,TGF-?1,BMPs content.Compared CT normal and periodontitis in gingival crevicular fluid,TGF?1,the expression levels of BMPs and statistical analysis of CT and TGF?1,BMPs expression levels of correlation,and CT/TGF?1/BMPs expression correlation with clinical parameters in chronic periodontitis patients.Part two: We took the healthy premolars from adolescent for orthodontic reason,later primary culture of PDLSCs was collected by the method of enzyme digestion and tissue adhesion.The sorting of STRO1 positive periodontal ligament cells using immunomagnetic beads technique to obtain periodontal ligament stem cells.The ability of clone formation of PDLSCs was detected by clone formation assay.By inducing culture medium,respectively,by von Kossa staining,Alizarin red S and Oil red O staining was used to detect the periodontal ligament stem cells with multiple differentiation ability of.To construct a recombinant adenovirus Ad.CT containing CT gene reading frame,and infect human periodontal ligament stem cells.The expression level of human I /III type PDLSCs collagen /OCN/ALP was detected by quantitative PCR assay and Western-blot assay.The effect of CT on the calcification of human PDLSCs fibers was discussed.Part three: Ad.CT infected human PDLSCs,TGF?1 expression levels were measured by quantitative PCR and Western-blot,and the effect of CT on the expression level of TGF?1 in human PDLSCs was observed.Synthesis of siRNA fragment specific for TGF?1.Expression of TGF?1siRNA specific inhibitor TGF?1 in human PDLSCs in Ad.CT infected human PDLSCs.The expression level of /III type I collagen in human PDLSCs was detected by quantitative PCR assay and Western-blot assay.To investigate whether CT can affect the collagen synthesis of PDLSCs by regulating the expression of TGF?1.Part four: Ad.CT infected human PDLSCs,BMPs expression levels were measured by quantitative PCR and Western-blot,and the effect of CT on the expression level of BMPs in human PDLSCs was observed.Ad.CT infection of human PDLSCs at the same time to give the BMPs pathway specific inhibitor Noggin treatment.The expression levels of osteogenic differentiation related markers OCN/ALP in human PDLSCs were detected by quantitative PCR assay and Western-blot assay.To investigate whether CT regulates the expression of BMPs and then to affect the osteogenic differentiation of PDLSCs.Results1.BMP2/4/7 and TGF-?1 expression level are significantly higher in periodotitis patients and BMP2 and TGF-?1 expression are association with CT expression The results of statistical analysis showed that the expression level of CT expression was positively correlated with TGF-?1/BMP2/4/7 in patients with chronic periodontitis.The expression levels of CT in patients with gingival index,probing depth,clinical attachment level of negative correlation,no correlation with age and gender.2.We successfully isolated and identified PDLSCs and using STRO1 positive periodontal ligament cells by immunomagnetic separation.Flow cytometry showed that the proportion of STRO1+/CD146+ cells in the sorted cells was 96.2%.The results of cell clone formation assay showed that the STRO1+/CD146+ cell clone formation rate was 65.7±9.1%,which was significantly higher than that of the non sorted periodontal ligament cells(27.1±5.2%,P<0.01).Kossa von staining appeared mineralized nodules after osteogenic induction of PDLSCs.After PDLSCs was induced to be cultured,the oil red O staining showed the lipid droplets.After PDLSCs into cartilage induction culture,the O positive staining of the red.After Ad.CT infection of human periodontal ligament stem cells 48 hr,the mRNA and protein expression levels were significantly increased in the I type /III collagen type /OCN/ALP.3.Ad.CT infected PDLSCs 48 hr,the detection of TGF-?1 mRNA and protein expression levels were significantly increased.TGF-?1siRNA transfection of human periodontal ligament stem cells after 48 hr,can significantly inhibit the expression of TGF-?1 mRNA and protein.Ad.CT/ TGF-?1siRNA treatment of humanPDLSCs,the expression of type I collagen type /III compared with Ad.CT treatment alone significantly decreased.4.Ad.CT infection of human PDLSCs after 48 hr,detected the expression level of BMP2/4 mRNA and protein were significantly increased,but BMP7 expression level showed no significant change.Noggin treatment of human PDLSCs after 48 hr can significantly inhibit the activity of BMPs.Ad.CT/Noggin treatment of human PDLSCs,the expression level of OCN/ALP was significantly lower than that of Ad.CT alone treatment.Conclusion1.CT in periodontitis gingival crevicular fluid of patients with high expression and and in patients with gingival index,exploration of probing depth,clinical attachment levels negatively correlated,suggesting that CT is involved in the occurrence and development of periodontitis and high expression of CT may be protective factors on the onset of periodontitis,the effects might be related to the regulation of periodontal tissue injury repair.The expression level of CT in gingival sulcus fluid was positively correlated with the expression of TGF-?1/BMP2/4/7,suggesting that CT may promote the repair of periodontal tissue damage by regulating the TGF-?1 and BMPs signaling pathway.2.Primary cultured human STRO+ PDLSCs into the ability to clone was significantly higher than that of periodontal ligament cells.In addition STRO+ periodontal ligament cells have multipotent differentiation ability and prompt people STRO+ periodontal ligament cells with human periodontal ligament stem cell properties.Overexpression of CT increases collagen I/III expression and induces osteoblastic differentiation in human PDLSCs,suggesting CT inducesd collagen synthesis and osteoblastic differentiation in human PDLSCs.3.The over expression of CT can significantly increase the expression level of TGF-?1 in human PDLSCs.Inhibition of TGF-?1 expression can significantly inhibit the expression of type I and type III collagen induced by CT in human PDLSCs,therefore we proposed that CT could increase collagen expression through TGF-?1 mediated pathway in human PDLSCs.4.The over expression of CT can significantly increase the expression level of BMP2/4 in human PDLSCs.Inhibition of BMP2/4 expression can significantly inhibit the expression of OCN/ALP induced by CT in human PDLSCs,therefore we proposed that CT could increase collagen expression through BMP2/4 mediated pathway in human PDLSCs.