Effect Of Estrogen Receptors On The Osteogenic Differentiation Of Periodontal Ligament Stem Cells From Ovariectomized Rats

Estrogen has been proved effective in accelerating the bone remodelingprocess. Estrogen deficiency, which typically happens in postmenopause, resultsin higher levels of bone resorption than bone formation and leads to a net loss inbone mass. Nowadays, it is generally believed that osteoporosis can lead to theoccurrence of periodontal disease. Clinical trials in postmenopausal womenhave confirmed an increased prevalence of periodontal disease with lowestrogen levels, even when oral hygiene remains unchanged. Animalexperiments with ovariectomized (OVX) rats have also clarified that estrogendeficiency may result in low mineral density in the mandible. However, theprecise effects of estrogen depletion on periodontal tissues at the molecular levelare unknown, so we would like to provide more valuable clues to discover themechanism underlying the influence of estrogen deficiency on periodontalregeneration. The periodontal ligament (PDL) is a specialized, vascular, and highlycellular connective tissue attaching cementum to the inner wall of alveolar boneand maintaining teeth in situ. It has been demonstrated that human PDL containsa heterogeneous population of cells capable of differentiating into cementoblastsor osteoblasts in vitro. Recently, Seo et al. proved that human periodontalligament cells contained stem cells (periodontal ligament stem cells, PDLSCs)and the application of these stem cells might hold promise as a therapeuticapproach for reconstruction of tissues destroyed by periodontal diseases.Estrogen actively participates in bone metabolism and exhibitsmultifunctional regulation in many types of cells including periodontal ligamentcells (PDLCs). It regulates the function of target cells by utilizing estrogenreceptors (ER). ER exists in two different isoforms: ERα and ERβ. Previousstudies have shown that both ERα and ERβ were expressed in rat bone marrowmesenchymal stem cells (BMSCs) and human PDLCs. Also, while in atransgenic mouse model, it was demonstrated that both ERs play important rolesin the bone metabolism. Therefore, it is necessary to determine the expressionlevels of the two receptors in periodontal tissues, which may help to reveal themechanism under which estrogen exerts its effects.Estrogen deficiency is believed to be one of major reasons forpostmenopausal osteoporosis and one of the risk factors for periodontitis.However, to the best of our knowledge, there is not sufficient informationconcerning the osteogenic differentiation of PDLSCs under estrogen-deficientmicroenvironment. Therefore we harvested PDLSCs from OVX rats andinvestigated the molecular mechanism of their osteogenic differentiation. Ourdata presented herein provide new insights which help us to know effects ofestrogen and its receptors on periodontal tissues at the molecular level. Methods:(1) Animals and osteoporosis modeThree-month-old female Sprague-Dawley rats were divided into two groups(n=6for each): ovariectomized (OVX) and sham-operated rats (Sham). Threemonths later, molars were extracted for cells culture and blood samples of therats from two groups were collected to evaluate estrogen level. Bone mineraldensity (BMD) of the femurs were measured by employing a Lunar DPX-IQDual energy X-ray absorptiometry (DXA).(2) Cell culture and the character of PDLSCsPeriodontal ligament tissue was gently scraped from the surface of themiddle part of the root, minced into1mm3cubes, and placed into6-well culturedishes. Then, PDLSCs were obtained as previously described. Briefly, STRO-1+stem cells were obtained by using immunomagnetic beads according to themanufacturer’s instructions. The character of PDLSCs was evaluated by MTTmethod and Flow cytometry analysis.(3) Effect of ERs on the osteogenic differentiation of periodontal ligament stemcells from ovariectomized ratsThe mRNA level of alkaline phosphatase (ALP), bone sialoprotein (BSP)and estrogen receptor beta (ERβ) measured by real-time PCR, andmineralization assay after osteogenic induction of PDLSCs were evaluated.Furthermore, Furthermore, by means of17β-estradiol (E2) treatment and genemanipulation, we observed the changes of osteogenic differentiation ofPDLSCs.(4) Effect of estrogen receptors on the Notch signaling pathway of periodontalligament stem cells from ovariectomized ratsWe observed the mRNA and protein level of Notch signaling by real-timePCR and western blot. Then, by means of17β-estradiol (E2) treatment and gene manipulation, we observed the changes of Notch signaling on the osteogenicdifferentiation of PDLSCs.Results：(1) Three months later after ovariectomy，the weight of OVX group wereincreased but estrogen level and BMD are decreased.(2) By flow-cytometric analysis we observed PDLSCs exhibited a highexpression profile for the markers STRO-1and CD146compared with PDLCs.Firstly, morphologically, PDLSCs from OVX group seemed to show higherproliferation rate than PDLSCs from Sham group, which was also corroboratedby the results of MTT assay. But osteogenic differentiation including the ALPactivity and mineralization ability decreased in OVX PDLSCs.(3) Real-time PCR analysis presented that the mRNA expressions of ERα andERβ associated with ALP, BSP were all up-regulated when immersed inosteogenic-induced media. However, the expression level of these makers wasdifferent between OVX and Sham groups. In each day, OVX PDLSCs seemedto show lower expression of either ERs or osteogenic makers than Sham cells.The results of genetic manipulation showed that with the down-regulation (>50%reduction) of either ERα or ERβ estrogen-induced ALP activity wasattenuated (>50%reduction) compared with the control group.(4) By means of17β-estradiol (E2) treatment and gene manipulation, we foundE2-treated PDLSCs showed increased expression of Notch1and Jagged1butseemed insensitive to estrogen stimulation after siRNA of ERs was induced intoPDLSCs of two groups.Conclusion:(1) The results indicatied that we have established a rat model of osteoporosis successfully.(2) In our study, we successfully isolated PDLSCs from OVX and Sham rats.(3) Estrogen-deficient microenvironment decreased osteogenic differentiation ofPDLSCs from OVX rats. Both ERα and ERβ were involved in estrogen-inducedosteoblastic differentiation of PDLSCs.(4) Estrogen receptors plays an important role in maintaining osteogenicdifferentiation of PDLSCs, at least in part, by regulating the expression level ofNotch signaling pathway.