| Estrogen deficiency, which is important in the pathogenesis of postmenopausal osteoporosis, has received increasing attention in the studies related to the periodontal diseases in postmenopausal women, such as the alveolar bone resorption and clinical attachment loss. Accumulating evidences have demonstrated that osteoporotic women exhibited a higher frequency of bone turnover, as observed in the femur. In the ovariectomized rat model, estrogen induced similar activation of bone resorption with increased. Although there is no doubt that estrogen has a broad influence in the biology of various human tissues, the etiology of estrogen-associated periodontal diseases remains an enigma. Therefore, we hypothesize that estrogen may play an important role in the maintenance and regeneration of periodontal tissue.The periodontal ligament is the connective tissue located between the alveolar bone and root surface of the tooth. Periodontal ligament cells (PDLcs) play an important part in maintaining the integrity of the periodontal tissue. PDLcs are capable of differentiating into osteogenic cells and exhibit osteoblastic phenotypes, such as the production of bone-like matrix proteins, high alkaline phosphatase (ALP) activity, and the formation of calcified nodules. It is well known that estrogen affects the biological properties of osteogenic cells such as the production of osteocalcin and ALP activity. However, less information is available regarding the effect of estrogen on PDLcs. Better understanding the mechanisms of estrogen on periodontium would facilitate our effort in the maintenance of the health of postmenopausal periodontal tissue.The sex hormones exert their influence on target tissues by binding to specific receptors that belong to a superfamily of ligand-activated transcription factors that regulate cell growth, differentiation and development. Two estrogen receptor (ER) subtypes, designated ER-alpha and ER-beta, have been identified and these receptors show a tissue specific expression pattern. When investigating the effects of estrogen on PDLc, it is critical to exam whether ER is expressed in PDLc. Since human osteoblasts express ERs, it is possible that PDLcs also do so. Since it was quite controversial that whether PDLcs express ERs in previous reports, more evidences are needed to assess the expression and functional significance of ER in PDLcs.In the present study, we established animal model by bilateral ovariectomy (OVX) and examined the effect of estrogen deficiency on the alveolar bone metabolism and the change of serum estrogen; investingated the ERs expression in periodontal ligament of rat by histoimmunochemistry. By the culture of human periodontal ligament cells ,we investigated ERs expression in human PDLc by cytoimmunochemistry, ERs mRNA expression by RT-PCR and ERs protein expression by western blotting, and further evaluated the influence of estrogen on DNA and collagen synthesis and the potential action of estrogen on bone-forming in PDLc.We found that:(1) Significant alveolar bone loss was detected in OVX group, indicating the success of the animal model. Estrogen replacement treatment (ERT) clearly attenuated the bone loss by the deficiency of estrogen induced by OVX. In rats of OVX + vehicle, as expected, the average serum 17-beta estradiol level was significant lower than that in OVX + 17-beta estradiol and sham.(2) By histoimmunochemistry, ERαand ERβwere abundantly expressed in periodontal ligament, and ERβwas much more than ERα(p<0.01). ERs were localized in both nuclei and cytoplasms, which indicates estrogen may play its role by different mechanisms in periodontium.(3)After successful culture of primary human periodontal ligament cells, both ERαand ERβprotein expressions were found in the cells by specific antibody by cytoimmunochemistry.(4)ERs mRNA was examined by RT-PCR. The expected PCR products of ER-alpha in 287bp and ER-beta in 659bp respectively were obtained from the primary cultured PDLcs. We found that the PDLcs did express both ER subunit ER-alpha and -beta although the intensities of the HER-alpha and -beta bands were much weaker than those observed in positive control MCF-7 cells. The results suggested that ER mRNA expression in PDLcs is in relative low level.(5)The protein expressions of ERs were also determined by Western Blot analysis. Similar expressions in low levels of ERs were detected compared to the positive control of MCF-7 cell extract. However, no difference was detected in ERs expression after 17-beta estradiol treatment.(6)When PDLcs were incubated in the presence of 17-beta estradiol, neither periodontal ligament cell DNA synthesis nor collagen synthesis was affected by E2 treatment for 24 hours.(7) Both ALP activity and osteocalcin production were increased by 17-beta estrodiol in a dose-dependent manner. Estrogen receptor antagonist ICI 182780 (10-6mol/L) completely blocks the effect of 17-beta estradiol. The results suggested that PDLcs mediates estrogen bone-sparing action through ER, therefore contributes to the mechanisms of estrogen deficiency alveolar bone loss.In conclusion:(1)Our results suggested that estrogen deficiency induced by ovariectomy significantly accelerated alveolar bone loss whereas ERT could efficiently reverse the process,confirmed that there existed obvious correlation between estrogen level and periodontal health.(2) both ER-alpha and ER-beta were expressed in PDLcs providing the bases that the ERs may be involved in the pathology mechanism of alveolar bone loss in postmenopausal women.(3) There was no effect on periodontal ligament cell DNA synthesis and collagen synthesis by treatment with E2. The genes regulated by the estrogen–ER complex in periodontal ligament cells remain to be identified these genes are probably not associated with periodontal ligamentcell proliferation and collagen formation.(4) estrogen could exhibit obvious positive modulation on ALP activity and osteocalcin production in PDLcs,confirmed that estrogen could significantly promote the osteogenic capability of human PDLcs(5)Our current study suggested that estrogen played an important role in periodontal diseases. Further studies will be necessary to delineate the actions of estrogen on periodontal ligament cells in detail.
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