Growth Of Bone Marrow Mesenchymal Stem Cells Research Articles

Insulin-like growth factor binding protein 4 inhibits proliferation of bone marrow mesenchymal stem cells and enhances growth of neurospheres derived from the stem cells.

Insulin-like growth factor binding protein 4 (IGFBP-4) was reported to trigger cellular senescence and reduce cell growth of bone marrow mesenchymal stem cells (BMSCs), but its contribution to neurogenic differentiation of BMSCs remains unknown. In the present study, BMSCs were isolated from the femur and tibia of young rats to investigate effects of IGFBP-4 on BMSC proliferation and growth of neurospheres derived from BMSCs. Bone marrow mesenchymal stem cell proliferation was assessed using CCK-8 after treatment with IGFBP-4 or blockers of IGF-IR and β-catenin. Phosphorylation levels of Akt, Erk, and p38 in BMSCs were analysed by Western blotting. Bone marrow mesenchymal stem cells were induced into neural lineages in NeuroCult medium; the number and the size of BMSC-derived neurospheres were counted after treatment with IGFBP-4 or the blockers. It was shown that addition of IGFBP-4 inhibited BMSC proliferation and immunodepletion of IGFBP-4 increased the proliferation. The blockade of IGF-IR with AG1024 increased BMSC proliferation and reversed IGFBP-4-induced proliferation inhibition; however, blocking of β-catenin with FH535 did not. p-Erk was significantly decreased in IGFBP-4-treated BMSCs. IGFBP-4 promoted the growth of neurospheres derived from BMSCs, as manifested by the increases in the number and the size of the derived neurospheres. Both AG1024 and FH535 inhibited the formation of NeuroCult-induced neurospheres, but FH535 significantly inhibited the growth of neurospheres in NeuroCult medium with EGF, bFGF, and IGFBP-4. The data suggested that IGFBP-4 inhibits BMSC proliferation through IGF-IR pathway and promotes growth of BMSC-derived neurospheres via stabilizing β-catenin.

Effects of Polyacrylonitrile/MoS2 Composite Nanofibers on the Growth Behavior of Bone Marrow Mesenchymal Stem Cells

In recent years, molybdenum disulfide (MoS2) as a typical class of two-dimensional (2D) materials has attracted wide attention because of its various fascinating properties. In this study, we fabricated MoS2 composite nanofibers by electrospinning technology combined with a doping method. The as-prepared MoS2 composite nanofibers exhibited excellent biocompatibility. In addition, the detailed investigation about the response of MoS2 composite nanofibers on bone marrow mesenchymal stem cells (BMSCs) indicated that the obtained MoS2 composite nanofibers could promote BMSC growth behavior, improve BMSC contact with each other, maintain cellular activity, and also provide positive promotion to regulate cellular proliferation. Moreover, the alkaline phosphatase expression significantly increased with increasing MoS2 concentration. Compared with the excellent biocompatibility and natural extracellular-matrix-like structure, we believe that the MoS2 composite nanofibers could provide new insight for the preparat...

Pamidronate Disodium Leads to Bone Necrosis via Suppression of Wnt/β-Catenin Signaling in Human Bone Marrow Mesenchymal Stem Cells In Vitro

Pamidronate disodium-associated bone necrosis is poorly understood at the cellular and molecular levels. This study proposes a pathway leading to the pamidronate disodium-mediated inhibition of osteogenic differentiation of human bone marrow mesenchymal stem cells (BMMSCs) derived from the mandible invitro. Primary human BMMSCs were isolated from the mandible and marrow tissue. A proliferation assay was performed to determine the experimental concentration of pamidronate disodium. Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin red S (ARS) staining were assessed after treatment with pamidronate disodium (0, 0.1, 0.5, 1, 5, 10μg/mL). Quantitative real-time polymerase chain reaction and western blotting specific for Wnt and β-catenin signaling genes or proteins were performed after treatment with pamidronate disodium 0.5μg/mL. Wnt3a was used to observe the osteogenic differentiation of BMMSCs during treatment with pamidronate disodium 0.5μg/mL. As expected, pamidronate disodium 1, 5, and 10μg/ml were unfavorable for BMMSC growth (P<.05), whereas 0.1 and 0.5μg/mL did not affect BMMSC growth (P≥.05). BMMSCs treated with pamidronate disodium 0.5μg/mL had lower ALP activity, ALP staining, and ARS staining (P<.05), and BMMSCs treated with low concentrations (<0.5μg/mL) of pamidronate disodium had the same levels of ALP activity, ALP staining, and ARS staining as the control (0μg/mL). Pamidronate disodium 0.5μg/mL decreased the expression of genes and proteins involved in Wnt and β-catenin signaling. BMMSCs with Wnt3a and pamidronate disodium 0.5μg/mL had higher ALP activity, ALP staining, and ARS staining (P<.05). Pamidronate disodium inhibited Wnt and β-catenin signaling, which controls osteogenic differentiation in BMMSCs. Wnt3a, a Wnt and β-catenin signaling activator, reversed the negative effects caused by pamidronate disodium to salvage the osteogenic defect in BMMSCs.

The effect of mesoporous bioglass on osteogenesis and adipogenesis of osteoporotic BMSCs.

This study evaluated the effect of mesoporous bioglass (MBG) dissolution on the differentiation of bone marrow mesenchymal stem cells (BMSCs) derived from either sham control or ovariectomized (OVX) rats. MBG was fabricated by evaporation-induced self-assembly method. Cell proliferation was tested by Cell Counting Kit-8 assay, and cytoskeletal morphology was observed by fluorescence microscopy. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) staining and activity, Alizarin Red staining, while adipogenic differentiation was assessed by Oil Red-O staining. Quantitative real-time PCR and Western blot analysis were taken to evaluate the expression of runt-related transcription factor 2 (Runx2) and proliferator-activated receptor-γ (PPARγ). We found that MBG dissolution (0, 25, 50, 100, 200 µg/mL) was nontoxic to BMSCs growth. Sham and OVX BMSCs exhibited the highest ALP activity in 50 µg/mL of MBG osteogenic dissolution, except that sham BMSCs in 100 µg/mL showed the highest ALP activity on day 14. Runx2 was significantly upregulated after 100 µg/mL of MBG stimulation in sham and OVX BMSCs for 7 and 14 days, except that 25 µg/mL showed highest upregulation effect on OVX BMSCs at day 7. PPARγ was downregulated after MBG stimulation. The protein level of Runx2 from the sham BMSCs group was significantly upregulated after lower doses (25 and 50 µg/mL) of MBG stimulation, whereas PPARγ was downregulated in the sham and OVX BMSCs group. Thus, both the osteogenic and adipogenic abilities of BMSCs were damaged under OVX condition. Moreover, lower concentration of MBG dissolution can promote osteogenesis but inhibit adipogenesis of the sham and OVX BMSCs. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3004-3014, 2016.

Plasma treatment in conjunction with EGM-2 medium increases endothelial and osteogenic marker expressions of bone marrow mesenchymal stem cells

For many tissue engineering applications, an important goal is to create functional tissues in vitro. In order to make such tissues viable, they have to be vascularized. Bone marrow mesenchymal stem cells (BMSCs) are a promising candidate for vascularization. Growth supplements are commonly used to induce BMSCs differentiation, and further improvements in differentiation conditions can be achieved by modifying the cells’ growth environment, such as pretreating the culture dish with gas plasma to modify the surface functional groups that the cells are seeded on. In this work, we compare the effects of different gas plasmas on the growth and differentiation of BMSCs. We treat the growth dish with different plasmas (CO2, N2, and O2) and induce BMSC differentiation on them with endothelial growth medium-2 (EGM-2). We find that EGM-2 by itself does not have strong effect on endothelial differentiation. However, we find that plasma treatment markedly increases the mRNA expression of endothelial cell markers. Interestingly, we also find that the upregulation of osteogenic markers. The data show that plasma treatment in conjunction with EGM-2 can enhance BMSCs differentiation into both endothelial-like cells and osteogenic-like cells. The effects of EGM-2 on BMSCs differentiation are influenced by the plasma-modified surface of the substrate. Our findings provide a method to enhance EGM-2-based cell differentiation, with consequences for tissue engineering and stem cell biology applications.

An experimental study of mesenchymal stem cells in tissue engineering scaffolds implanted in rabbit corneal lamellae to increase keratoprosthesis biointegration

To complete a preliminary evaluation of the feasibility of implanting the complex of mouse bone marrow mesenchymal stem cells (BMSC) and a tissue engineering scaffold into rabbit corneal lamellae, based on which a solution may be proposed to consolidate the keratoprosthesis and the recipient surface, and to reduce the risk of complications. This experimental study was composed of two parts. (1) In vitro: some mouse BMSC were marked with red fluorescent proteins (RFP) and integrated with a decellularized pig articular cartilage extracellular matrix (ECM) scaffold. The cell survival was observed under a fluorescence microscope at 4 and 8 weeks. The cell distribution was examined by toluidine blue staining. The pore structure and the cell adhesion were observed under a scanning electron microscope. (2) in vivo: the complex of mouse BMSC and a decellularized scaffold was implanted into the lamellar cornea of 8 rabbit eyes with the fellow eyes as the controls. The eyes were sampled for observation using HE staining under a light microscope at 2, 4 and 8 weeks, respectively. The cell survival was examined under a fluorescence microscope, and the intracorneal cell survival at 8 weeks was observed using in vivo imaging. The conditions of ocular anterior segment of all the experimental animals were recorded. (1) Under the scanning electron microscope, the ECM scaffolds showed satisfactory porosity required for the adhesion and growth of cells and tissues, and the cell distribution over the cell-scaffold complex can be observed by toluidine blue staining. (2) Under the immunofluorescence microscope, cell proliferation was observed in vitro and in the interlamellar space (the maximum observation time was 8 weeks) after the RFP-marked mouse BMSC were integrated in vitro with ECM scaffolds. (3) Under the light microscope (HE staining), the stromal cells were detected to increase at each timepoint. A small number of monocytes and some mouse BMSC were observed in the superficial layer of corneal stroma, with sparsely and orderly arranged collagenous fibers and no neovascularization. All the epithelial cells appeared as mononuclear, columnar and undamaged, and the shape of ECM scaffolds, which were fused with the collagens, became unclear. (4) By in vivo imaging, it was found that the mouse BMSC survived for 8 weeks after being integrated with scaffolds and implanted into the interlamellar space of rabbit cornea. (5) After the implantation of cell-scaffold complex, severe postoperative inflammatory reactions, obvious conjunctival congestion and neovascularization were not observed. The corneal tissues surrounding the recipient area were transparent. One week later, mild inflammatory reactions were barely observed, and the cornea was transparent enough to observe the scaffold in the stromal layers. Four weeks later, the scaffolds became thinner. Eight weeks later, the scaffolds became extremely thin with normal vascular system in the corneal limbus. The ECM scaffold is a solid and biocompatible carrier for the growth and proliferation of BMSC. The mouse BMSC can grow and proliferate in the microenvironment of the interlamellar space of cornea.

The SCD – Stem Cell Differentiation ESA Project: Preparatory Work for the Spaceflight Mission

Due to spaceflight, astronauts experience serious, weightlessness-induced bone loss because of an unbalanced process of bone remodeling that involves bone marrow mesenchymal stem cells (BMSCs), as well as osteoblasts, osteocytes, and osteoclasts. The effects of microgravity on osteo-cells have been extensively studied, but it is only recently that consideration has been given to the role of BMSCs. Previous researches indicated that human BMSCs cultured in simulated microgravity (sim-μg) alter their proliferation and differentiation. The spaceflight opportunities for biomedical experiments are rare and suffer from a number of operative constraints that could bias the validity of the experiment itself, but remain a unique opportunity to confirm and explain the effects due to microgravity, that are only partially activated/detectable in simulated conditions. For this reason, we carefully prepared the SCD – STEM CELLS DIFFERENTIATION experiment, selected by the European Space Agency (ESA) and now on the International Space Station (ISS). Here we present the preparatory studies performed on ground to adapt the project to the spaceflight constraints in terms of culture conditions, fixation and storage of human BMSCs in space aiming at satisfying the biological requirements mandatory to retrieve suitable samples for post-flight analyses. We expect to understand better the molecular mechanisms governing human BMSC growth and differentiation hoping to outline new countermeasures against astronaut bone loss.

Effect of low temperature on Schwann-like cell differentiation of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types. The potential of MSCs for differentiation and proliferation depends on the control of various processes. In this study, we have investigated the effects of low temperature on the Schwann-like cell differentiation of human bone marrow mesenchymal stem cells (BM-MSCs). The growth of BM-MSCs decreased at low temperature, but increased Schwann-like cell differentiation. The expression of cell surface antigen decreased CD73, CD105, and CD90, which are known markers of MSCs at 35°C. Also, the levels of matrix RNA [S100, glial fibrillary acidic protein (GFAP), p75, myelin basic protein, P0, and others] at 35°C in the differentiation culture were higher than those at 37°C in the differentiation culture, and S100, p75, and GFAP proteins were expressed more than at 37°C. GFAP and S100 were stained strongly at 35°C by immunohistochemical examination. These data demonstrate that culturing BM-MSCs at reduced temperature can improve differentiation into Schwann-like cells.

Enhanced proliferation of bone marrow mesenchymal stem cells by co-culture with TM4 mouse Sertoli cells: involvement of the EGF/PI3K/AKT pathway.

Bone marrow mesenchymal stem cells (BM-MSCs) are consideredas a promising option in the field of regenerative medicine and tissue engineering. However, little is known about how TM4 mouse Sertoli cells, which are known to enhance stem cells proliferation in co-culture, may influence the proliferation of BM-MSCs andwhich signaling pathways are involved in. To address these questions, an in vitro transwell system was used. We found that TM4 cells could produce soluble factors which enhanced the growth of BM-MSCs without inhibiting the multipotency. Furthermore, cell cycle analysis showed that co-culture with the TM4 cells accelerated the progress of BM-MSCs from the G1 to the S phase. The expression of the phospho-akt, mdm2, as well as pho-CDC2, and cyclin D1 were markedly upregulated in co-cultured BM-MSCs. The observed promoting effect was significantly inhibited by the administration of the PI3K/AKT inhibitor, LY294002. Among the various growth factors produced by TM4 cells, the epithelial growth factor (EGF) stimulated the proliferation of the BM-MSCs more significantly compared with the other growth factors examined in this study. Neutralization of EGF via a blocking antibody significantly limited the promoting growth effect in BM-MSCs. These results suggest that TM4 cells provide a favorable in vitro environment for BM-MSCs growth and imply the involvement of the EGF/PI3K/AKT pathway.

Superparamagnetic iron oxide magnetic nanomaterial-labeled bone marrow mesenchymal stem cells for rat liver repair after hepatectomy

BackgroundSuperparamagnetic iron oxide magnetic nanomaterials (SPIO) are tracers used for treatment of central nervous and cardiovascular system complications in animal models. The present study investigated survival and proliferation of SPIO-labeled bone marrow mesenchymal stem cells (BMSCs) to determine their potential therapeutic value in liver repair. MethodsSurface antigens of BMSCs were measured using flow cytometry. BMSCs viability, growth curve, and SPIO (0–100 μg/mL) labeling rate were evaluated using trypan blue staining, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Prussian blue staining, respectively. SPIO-labeled BMSCs were transplanted via liver or spleen injection in rats undergoing 70% hepatectomy. Distribution of SPIO-labeled BMSCs in liver and spleen, and liver repair were evaluated by magnetic resonance imaging (MRI), and serum alanine aminotransferase, aspartate aminotransferase, albumin, and total bilirubin levels. ResultsCD29+/CD90+/CD45− BMSCs were successfully isolated from rats. Labeling rate of SPIO in 25 μg/mLwas 94.9%. SPIO labeling did not affect BMSCs survival and proliferation. MRI showed that BMSCs colonized in the liver, whether via spleen or liver injection. Serum levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin in the transplanted rats were significantly lower than in the hepatectomy group at days 1, 3, and 7 after hepatectomy (all P < 0.05), whereas serum albumin levels were significantly higher in the transplanted rats on posthepatectomy day 3 (both P < 0.05). These indicators were not significantly different between the spleen and liver injection approaches. ConclusionsBMSCs transplantation via liver or spleen injection could significantly accelerate liver healing. In vivo MRI of SPIO-labeled BMSCs can be used to trace real-time liver healing during clinical treatment after hepatectomy.

Osteogenic differentiation of bone marrow mesenchymal stem cells on the collagen/silk fibroin bi‐template‐induced biomimetic bone substitutes

Biomimetic bone substitutes of collagen-silk fibroin/hydroxyapatite (COL-SF/HA) were synthesized via a bi-template-induced coassembly strategy. Collagen-hydroxyapatite (COL-HA) and silk fibroin-hydroxyapatite (SF-HA) served as controls were prepared with similar method. The osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs) seeded onto the resulting materials was evaluated both in vitro and in vivo. The results suggested that the bi-template-induced biomimetic substitutes were able to support the growth and proliferation of BMSCs. We further demonstrated that BMSCs were stimulated to differentiate into the osteoblast cell lineage by evaluating several specific osteogenic markers including staining of alkaline phosphate (ALP) and calcium nodular and expression of osteogenic genes of osteocalcin (OCN) and osteonectin (ONN). The rat femoral defect model was used to assess the aforementioned biomimetic bone substitutes combined with BMSCs in vivo. Histological analysis indicated that the bi-template material exhibited good biocompatibility and strong ability of the new bone formation in comparison with the control of single-template material in vivo.

Biological responses of human bone marrow mesenchymal stem cells to Sr‐M‐Si (M = Zn, Mg) silicate bioceramics

Strontium (Sr), Zinc (Zn), magnesium (Mg), and silicon (Si) are reported to be essential trace elements for the growth and mineralization of bone. We speculated that the combination of these bioactive elements in bioceramics may be effective to regulate the osteogenic property of bone-forming cells. In this study, two Sr-containing silicate bioceramics, Sr(2)ZnSi(2)O(7) (SZS) and Sr(2)MgSi(2)O(7) (SMS), were prepared. The biological response of human bone marrow mesenchymal stem cells (BMSCs) to the two bioceramics (in the forms of powders and dense ceramic bulks) was systematically studied. In powder form, the effect of powder extracts on the viability and alkaline phosphatase (ALP) activity of BMSCs was investigated. In ceramic disc form, both direct and indirect coculture of BMSCs with ceramic discs were used to investigate their biological response, including attachment, proliferation, ALP activity, and bone-related genes expression. Beta-tricalcium phosphate (β-TCP) and akermanite (Ca(2)MgSi(2)O(7), CMS) were used as control materials. The results showed that the Sr, Zn, and Si (or Sr, Mg, and Si)-containing ionic products from SZS and SMS powders enhanced ALP activity of BMSCs, compared to those from β-TCP. Both SZS and SMS ceramic discs supported the growth of BMSCs, and most importantly, significantly enhanced the ALP activity and bone-related genes expression of BMSCs as compared to β-TCP. The results suggest that the specific combination of bioactive ions (Sr, Zn, Si, e.g.) in bioceramics is a viable way to improve the biological performance of biomaterials, and the form of materials and surface properties were nonnegligible factors to influence cell response.

High-Dose 111In Induces G1 Cell Cycle Arrest and Cell Death in Rat Bone Marrow Mesenchymal Stem Cells

This study was performed to evaluate the effect of (111)In-labeling on the cell growth, cycle and viability of bone marrow mesenchymal stem cells (BMSCs). Rat BMSCs were labeled with various doses of (111)In (0.4-11.1Bq/cell). The growth curve of (111)In-BMSCs was obtained up to 14th day of labeling. The cell cycle was evaluated by 5-bromo-2-deoxyuridine (BrdU) labeling or propidium iodide (PI) staining. Senescent cells were counted under a light microscope after staining with 5-bromo-4-chloro-3-indolyl-D-galactopyranoside. Flow cytometry was performed to measure apoptotic and necrotic fractions after staining with annexin V-FITC and PI. The growth of BMSCs labeled with higher doses of (111)In (4.4 or 11.1Bq/cell) was significantly inhibited from the 3rd day of labeling. Flow cytometry revealed less BrdU-positive BMSCs at 11.1Bq (111)In/cell during all measurement days and G1 arrest at 4.4 and 11.1Bq (111)In/cell. Significant increases in apoptosis and necrosis were also observed at 4.4 (3.04%/1.35%) and 11.1Bq (111)In/cell (9.07%/3.18%) on the 14th day (control = 1.60%/0.39%). However, no cellular senescence was visualized up to the 14th day. A high dose of (111)In-labeling induced cell cycle arrest and death in BMSCs; therefore, it should be used with a careful dosimetry in case of applying it to humans.

Cell proliferation of human bone marrow mesenchymal stem cells on biodegradable microcarriers enhancesin vitrodifferentiation potential

For reasons of provision of highly-specific surface area and three-dimensional culture, microcarrier culture (MC) has garnered great interest for its potential to expand anchorage-dependent stem cells. This study utilizes MC for in vitro expansion of human bone marrow mesenchymal stem cells (BMMSCs) and analyses its effects on BMMSC proliferation and differentiation. Effects of semi-continuous MC compared to control plate culture (PC) and serial bead-to-bead transfer MC (MC bead-T) on human BMMSCs were investigated. Cell population growth kinetics, cell phenotypes and differentiation potential of cells were assayed. Maximum cell density and overall fold increase in cell population growth were similar between PCs and MCs with similar starting conditions, but lag period of BMMSC growth differed substantially between the two; moreover, MC cells exhibited reduced granularity and higher CXCR4 expression. Differentiation of BMMSCs into osteogenic and adipogenic lineages was enhanced after 3 days in MC. However, MC bead-T resulted in changes in cell granularity and lower osteogenic and adipogenic differentiation potential. In comparison to PC, MC supported expansion of BMMSCs in an up-scalable three-dimensional culture system using a semi-continuous process, increasing potential for stem cell homing ability and osteogenic and adipogenic differentiation.

Application of Autologous Cryopreserved Bone Marrow Mesenchymal Stem Cells for Periodontal Regeneration in Dogs

Background: Stem cell-based therapies have shown great promise in regenerative medicine and continue to generate wide interest in future clinical applications. However, the issue of storage and preservation of stem cells, for future clinical applications, still requires extensive investigation. Objective: The purpose of this study was to evaluate the effect of cryopreservation on the regenerative capacity of bone marrow stem cells in periodontal defects in dogs. Materi als and Methods: Bone marrow mesenchymal stem cells (BMSCs) were obtained from 5 beagle dogs. After cryopreservation for 1 month, cell viability, surface adherence ability, alkaline phosphatase activity and mineralized nodule formation were assessed. Twenty-six periodontal fenestration defects (5 × 5 mm) were created at a location 5 mm apical to the cemento-enamel junction in experimental teeth. Cryopreserved BMSCs were transplanted into the defects using a collagen scaffold carrier. Freshly isolated BMSCs and collagen scaffold alone were used as controls. All animals were sacrificed 8 weeks after surgery, and specimens were evaluated by histomorphometry. Results: Cryopreservation had no discernible negative effect on BMSC growth and differentiation in vitro. Both freshly isolated and cryopreserved BMSC transplantations induced significantly better periodontal regeneration with newly formed cementum, alveolar bone and periodontal ligament compared with the application of collagen scaffold alone. Conclusion: Cryopreserved BMSCs showed no altered regenerative capacity compared with freshly isolated BMSCs in the application of periodontal regeneration.

Temporal expression of estrogen receptor alpha in rat bone marrow mesenchymal stem cells

Estrogen responsiveness of bone formation is mediated by the estrogen receptor alpha (ERα) in osteoblastic lineage. As osteoblasts arise from the multipotent bone marrow stromal (mesenchymal) cells, this study was undertaken to observe the ERα in primary female adult rat bone marrow mesenchymal stem cells (BMSCs). The ERα was localized using immunocytochemical analysis in identified primary BMSCs. Then, using real-time PCR analysis, we measured the expression of ERα messenger RNA (mRNA) in BMSCs. ERα transcripts showed different trends between untreated cultures (control group) and osteogenic-induced cultures (treated group). In the control group, ERα mRNA climbed at peak levels at a confluence stage and decreased until day 20, whereas, in the treated group, the ERα mRNA kept climbing from a low level until day 20. Thus, the observed developmental expression of ERα mRNA correlates with progressive BMSCs growth and osteogenic differentiation and BMSCs may be a primary target cell for estrogen in maintaining bone formation.