The Expression Of Estrogen Receptors And The Effect Of Estrogen On Osteogenic Differentiation In Human Periodontal Ligament Stem Cells

Estrogen deficiency is important in the pathogenesis of postmenopausal osteoporosis. Previous studies show that the bone mass in the mandible and skeletal bone mass had an evident correlation in postmenopausal women with osteoporosis, and the osteoporotic alveolar bone would appear in postmenopausal women. Estrogen replacement treatment (ERT) can increase the estrogen level in postmenopausal women and decrease the bone loss, gingival blooding and attachment loss. Estrogen exerts its influence by binding to specific receptors, estrogen receptor (ER). ER-αand ER-βhave been detected in periodontal ligament (PDL) cells, and it has been reported that estrogen could stimulate bone formation capacity in cultured PDL cells by increasing alkaline phosphatase activity, the production of osteocalcin and the formation of mineralized nodules.Periodontal ligament stem cells (PDLSCs) are progenitors of PDL cells, and the application of PDLSCs may be effective for periodontal regenerative therapy as for their ability to form different tissue types and self-renew. However, considering the effects of estrogen on bone-forming capability in cultured human PDL cells, few studies have been conducted in PDLSCs. The aims of the present study were to determine ERs expression in human periodontal ligament stem cells and to investigate the effects that estrogen on osteogenic differentiation of PDLSCs in vitro.In the present study, PDLSCs were isolated by immunomagnetic method. The flow cytometry and immunocytochemistry procedure were used to disclose the cell cycle and surface marker of the PDLSCs. And osteoinduction and adipoinduction were done to conform the multidirectional differentiation ability of PDLSCs as meanwhille. We investigated ERs expression in PDLSCs by immunocytochemistry, ERs mRNA expression by RT-PCR and ERs protein expression by Western Blot analysis. Furthermore, we evaluated the effects of estrogen on ALP activity and collagen synthesis in the osteogenic differentiation of PDLSCs.We harvested the following results:1. The isolated PDLSCs have orbicular-ovate nuclei and irregular morphous, which are similar to fibroblast-like cells. The cell growth curve indicated that PDLSCs had a low proliferation rate.2. The isolated PDLSCs have the ability to form multi-cell clusters and cell life cycle which similar to that of stem cells. Analysis of surface epitopes of PDLSCs by FACS showed CD44 and CD146 were of high expression, in the contrary, CD34 and CD45 were of low expression. In the meanwhile, STRO-1 and Vimentin were both positively stained. Furthermore, after adipogenetic induction, PDLSCs developed into oil red O-positive lipid-laden fat cells as well as small round alizarin red-positive nodules were also formed in the osteogenic induction cultures.3. Immunocytochemistry showed that both ER-αand ER-βwere positively stained in the nuclei of PDLSCs. Western blot analysis and RT-PCR procedure indicated that the expressions of ERs (both ER-αand ER-β) in PDL cells were in a lower level compared with that in the PDLSCs. In PDLSCs groups, the expression of ER-αwas higher than that of ER-β, and the expressions of ERs in 17-βestradiol treated group were higher than that of untreated group.4. Comparing to the group of control, the DNA synthesis of PDLSCs depressed in the first three days, and increased apparently in the following time while treated with 17-βestradiol. In the estrogen treated groups, the DNA synthesis was of a dose dependent manner.5. When PDLSCs were treated with 17-βestradiol, the ALP expression increased from day 3. Furthermore, the ALP levels were higher in the estrogen treated groups than control group, and in the estrogen treated groups, the ALP activity was of a dose dependent manner.6. When PDLSCs were treated with 17-βestradiol, compared with control groups, the synthesis of typeⅠcollagen increased more apparently in the estrogen treated groups, and the synthesis was of a dose dependent manner.Conclusion:1. Immunomagnetic method is an effective way to isolate and purify the PDLSCs. The acquired cells showed the characteristics of stem cells.2. Both ER-αand ER-βare expressed in PDLSCs. The expression of ER-αis higher than ER-β, which indicates that estrogen effects on PDLSCs are probably mainly displayed via ER-α. When PDLSCs were treated with estrogen while osteogenic induction, the expression of ER-αand ER-βincreased, this signified that estrogen may modulate the osteogenic differentiation of PDLSCs through ERs.3. Estrogen could accelerate the DNA synthesis and osteogenic differentiation of PDLSCs, which indicates that the estrogen effects on periodontal reconstruction may be displayed through PDLSCs.