The Effect of Quercetin on the Osteogenesic Differentiation and Angiogenic Factor Expression of Bone Marrow-Derived Mesenchymal Stem Cells

Bone marrow-derived mesenchymal stem cells (BMSCs) are widely used in regenerative medicine in light of their ability to differentiate along the chondrogenic and osteogenic lineages. As a type of traditional Chinese medicine, quercetin has been preliminarily reported to promote osteogenic differentiation in osteoblasts. In the present study, the effects of quercetin on the proliferation, viability, cellular morphology, osteogenic differentiation and angiogenic factor secretion of rat BMSCs (rBMSCs) were examined by MTT assay, fluorescence activated cell sorter (FACS) analysis, real-time quantitative PCR (RT-PCR) analysis, alkaline phosphatase (ALP) activity and calcium deposition assays, and Enzyme-linked immunosorbent assay (ELISA). Moreover, whether mitogen-activated protein kinase (MAPK) signaling pathways were involved in these processes was also explored. The results showed that quercetin significantly enhanced the cell proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in a dose-dependent manner, with a concentration of 2 μM achieving the greatest stimulatory effect. Moreover, the activation of the extracellular signal-regulated protein kinases (ERK) and p38 pathways was observed in quercetin-treated rBMSCs. Furthermore, these induction effects could be repressed by either the ERK inhibitor PD98059 or the p38 inhibitor SB202190, respectively. These data indicated that quercetin could promote the proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in vitro, partially through the ERK and p38 signaling pathways.     

Vibration loading promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells via p38 MAPK signaling pathway

Low magnitude high frequency vibration (LMHFV) exhibits effectively anabolic effects on the bone tissue, and can promote osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. The role of p38 MAPK signaling in LMHFV-induced osteogenesis remains unclear. In this current study, LMHFV loading was applied to BMSCs in vitro, and cell proliferation, alkaline phosphatase (ALP), matrix mineralization, as well as osteogenic genes expression were assayed. The mechanism of mechanical signal transduction was analysed using PCR array, qRT-PCR and Western blot. LMHFV increased cell proliferation in the growth medium, while inhibited proliferation in the osteogenic medium. ALP activity, matrix mineralization and osteogenic genes expression of Runx2, Col-I, ALP, OPN and OC were increased by LMHFV. p38 and MKK6 genes expression, and p38 phosphorylation were promoted in LMHFV-induced osteogenesis. Inhibition of p38 MAPK with SB203580 and targeted p38 siRNA blunted the increased ALP activity and osteogenic genes expression by LMHFV. These findings suggest that LMHFV promotes osteogenic differentiation of BMSCs, and p38 MAPK signaling shows an important function in LMHFV-induced osteogenesis.     

Temporal Expression of Pelp1 during Proliferation and Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells

Background

Osteogenic induction and bone formation are heavily affected by environmental factors, including estrogen, estrogen receptors, and coregulatory proteins, such as the recently reported proline-, glutamic acid-, and leucine-rich protein 1(Pelp1).

Objective

To investigate Pelp1 expression in rat bone mesenchymal stem cells (rBMSCs) during cell proliferation and osteogenic differentiation.

Methods

rBMSCs were cultured in routine and osteogenic differentiation media. Cell proliferation was assessed at days 1, 3, 5, 7, 9, 11, 14, and 21. Pelp1 protein expression in the nucleus and cytoplasm were detected by immunocytochemical analysis. Real-time RT-PCR and western blot were used to detect mRNA and protein expressions of Pelp1, osteocalcin (OCN), and alkaline phosphatase (ALP).

Results

Over 21 days, rBMSCs in routine culture exhibited a 1-2 day lag phase and exponential growth from day 3 to 9, plateauing at day 9, and correlated with temporal mRNA expression of Pelp1, which almost reached baseline levels at day 21. In osteogenic induction cultures, Pelp1 mRNA levels rose at day 9 and steadily increased until day 21, reaching 6.8-fold greater value compared with day 1. Interestingly, Pelp1 mRNA expression in osteogenic cultures exhibited a trend similar to that of OCN expression. Pelp1 knockdown by siRNA transfection inhibited undifferentiated rBMSC proliferation, and bone markers OCN and ALP expressions in rBMSCs cultured in routine and osteogenic differentiation media.

Conclusions

Pelp1 may be a key player in BMSCs proliferation and osteogenic differentiation, meriting further consideration as a target for development of therapies for pathological bone loss conditions, such as menopausal bone loss.     

Effect of Boron on Osteogenic Differentiation of Human Bone Marrow Stromal Cells

Bone marrow stromal cells (BMSCs) have been well established as an ideal source of cell-based therapy for bone tissue engineering applications. Boron (B) is a notable trace element in humans; so far, the effects of boron on the osteogenic differentiation of BMSCs have not been reported. The aim of this study was to evaluate the effects of boron (0, 1, 10,100, and 1,000?ng/ml) on osteogenic differentiation of human BMSCs. In this study, BMSCs proliferation was analyzed by cell counting kit-8 (CCK8) assay, and cell osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity assay, Von Kossa staining, and real-time PCR. The results indicated that the proliferation of BMSCs was no different from the control group when added with B at the concentration of 1, 10, and 100?ng/ml respectively (P?>?0.05); in contrast, 1,000?ng/ml B inhibited the proliferation of BMSCs at days?4, 7, and 14 (P?相似文献   

Sulfated glycosaminoglycans mediate the effects of FGF2 on the osteogenic potential of rat calvarial osteoprogenitor cells

Fibroblast growth factor-2 (FGF2) is a powerful promoter of bone growth. We demonstrate here that brief exposure to FGF2 enhances mineralized nodule formation in cultured rat osteoprogenitor cells due to an expansion of cells that subsequently mineralize. This mitogenic effect is mediated via sulfated glycosaminoglycans (GAGs), FGFR1, and the extracellular signal-regulated kinase (ERK) pathway. The GAGs involved in this stimulation are chondroitin sulfates (CS) rather than heparan sulfates (HS). However, continuous FGF2 treatment reduces alkaline phosphatase (ALP) activity, downregulates collagen Ialpha1 (ColIalpha1) and FGFR3 expression, upregulates the expression and secretion of osteopontin (OPN) and inhibits mineralization. The inhibitory effects of FGF2 on FGFR3 expression and ALP activity are also mediated by the ERK pathway, although the effects of FGF2 on ColIalpha1 and OPN expression are mediated by GAGs and PKC activity. Thus short-term activation of FGF2/FGFR1 promotes osteoprogenitor proliferation and subsequent differentiation, while long-term activation of FGF2 signaling disrupts mineralization by modulating osteogenic marker expression. This study thus establishes the central role of sulfated GAGs in the osteogenic progression of osteoprogenitors.     

A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue

To evaluate the potential of three stem cells for cell therapy and tissue engineering applications, the biological behavior and osteogenic capacity of the newly introduced cord-blood-derived, unrestricted somatic stem cells (USSC) were compared with those of mesenchymal stem cells isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). There was no significant difference between the rates of proliferation of the three stem cells. During osteogenic differentiation, alkaline phosphatase (ALP) activity peaked on day 7 in USSC compared to BM-MSC which showed the maximum value of ALP activity on day 14. However, BM-MSC had the highest ALP activity and mineralization during osteogenic induction. In addition, AT-MSC showed the lowest capacity for mineralization during differentiation and had the lowest ALP activity on days 7 and 14. Although AT-MSC expressed higher levels of collagen type I, osteonectin and BMP-2 in undifferentiated state, but these genes were expressed higher in BM-MSC during differentiation. BM-MSC also expressed higher levels of ALP, osteocalcin and Runx2 during induction. Taking together, BM-MSC showed the highest capacity for osteogenic differentiation and hold promising potential for bone tissue engineering and cell therapy applications.     

Ghrelin联合三氧化二砷对骨髓间充质干细胞增殖与成骨分化的影响

该文主要探究Ghrelin对三氧化二砷(As2O3)导致的骨髓间充质干细胞(BMSCs)增殖和成骨分化的影响。BMSCs设为对照组、As2O3组、Ghrelin组和联合(As2O3+Ghrelin)组。MTT法检测细胞增殖能力;成骨诱导的第7天和第14天,Real-time PCR及Western blot分别检测成骨相关因子OPN、ALP、RUNX2的mRNA及蛋白表达;第21天,茜素红染色分析钙盐沉积情况。结果显示,细胞增殖能力Ghrelin组>对照组>联合组>As2O3组。与对照组比,As2O3组各因子表达均显著下调(P<0.05),Ghrelin组第14天OPN蛋白表达无显著变化,其余因子均上调(P<0.05);联合组与As2O3组比,第14天OPN基因表达和第7天ALP蛋白表达无显著差异,其余均显著上调(P<0.05)。钙盐沉积:Ghrelin组>对照组>联合组>As2O3组。提示0.5μmol/L As2O3抑制BMSCs增殖和成骨分化,600 ng/mL Ghrelin增强细胞增殖和成骨分化;且Ghrelin能减弱As2O3导致的BMSCs增殖和成骨分化抑制作用。     

Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells

Background

Stem cell therapy has emerged as a potential therapeutic option for tissue engineering and regenerative medicine, but many issues remain to be resolved, such as the amount of seed cells, committed differentiation and the efficiency. Several previous studies have focused on the study of chemical inducement microenvironments. In the present study, we investigated the effects of gravity on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into force-sensitive or force-insensitive cells.

Methods and results

Rat BMSCs (rBMSCs) were cultured under hypergravity or simulated microgravity (SMG) conditions with or without inducement medium. The expression levels of the characteristic proteins were measured and analyzed using immunocytochemical, RT-PCR and Western-blot analyses. After treatment with 5-azacytidine and hypergravity, rBMSCs expressed more characteristic proteins of cardiomyocytes such as cTnT, GATA4 and β-MHC; however, fewer such proteins were seen with SMG. After treating rBMSCs with osteogenic inducer and hypergravity, there were marked increases in the expression levels of ColIA1, Cbfa1 and ALP. Reverse results were obtained with SMG. rBMSCs treated with adipogenic inducer and SMG expressed greater levels of PPARgamma. Greater levels of Cbfa1- or cTnT-positive cells were observed under hypergravity without inducer, as shown by FACS analysis. These results indicate that hypergravity induces differentiation of rBMSCs into force-sensitive cells (cardiomyocytes and osteoblasts), whereas SMG induces force-insensitive cells (adipocytes).

Conclusion

Taken together, we conclude that gravity is an important factor affecting the differentiation of rBMSCs; this provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated or undifferentiated cells.     

Comparison between 8‐prenylnarigenin and narigenin concerning their activities on promotion of rat bone marrow stromal cells’ osteogenic differentiation in vitro

A number of recent studies have suggested that flavonols (a class of phytochemical with many biological activities), might exert protective effects against post‐menopausal bone loss. In the present study, we compared naringenin (NG) and 8‐prenylnaringenin (PNG), two major naturally occurring flavonols, on in vitro differentiation of osteoblasts and bone resorbing activity, of rat bone marrow stromal cells (BMSCs). Our results indicated that both compounds, at 10^?6 m , enhanced BMSCs’ differentiation. Then effects of the two compounds at 10^?6 m on ALP activity, osteocalcin secretion and calcium deposition, were compared over a time course. Numbers and areas of colonies stained for ALP (CFU‐F_ALP) expression, and mineralized bone nodules, were histochemically analysed after 12 days and 16 days osteogenic induction, respectively. Expression of BMP‐2, OPG, OSX, RUNX‐2 genes and p38MAPK protein were examined using real‐time PCR and western blotting, respectively. The data presented indicate that PNG, significantly enhanced the rat BMSCs’ differentiation and mineralization through the BMP‐2/p38MAPK/Runx2/Osterix signal pathway, greater than did NG. In conclusion, PNG has a more pronounced ability to enhance osteoblast differentiation and mineralization, than NG.     

Pharmacological inhibition of S6K1 impairs self‐renewal and osteogenic differentiation of bone marrow stromal cells

mTORC1 signaling not only plays important physiological roles in the regulation of proliferation and osteogenic differentiation of BMSCs, but also mediates exogenous Wnt‐induced protein anabolism and osteoblast differentiation. However, the downstream effectors of the mTORC1 signaling in the above processes are still poorly understood. In this study, we explored the specific role of S6K1, one of the major targets of the mTORC1 pathway, in BMSCs self ‐ renewal and osteogenic differentiation. We first found that S6K1 was active in primary mouse bone marrow stromal cells, and further activated upon osteogenic induction. We then determined the effects of S6K1 inhibition by LY2584702 Tosylate, a selective inhibitor of S6K1 (hereafter S6KI), using both primary mouse bone marrow stromal cells and ST2 cells. Colony‐Forming Unit‐Fibroblast (CFU‐F) assays showed that S6KI dramatically reduced the total number of colonies formed in primary BMSCs cultures. Under the basal osteogenic culture condition, S6KI significantly inhibited mRNA expression of osteoblast marker genes (Sp7, Bglap, Ibsp, and Col1a1), ALP activity and matrix mineralization. Upon Wnt3a treatments, S6KI inhibited Wnt3a‐induced osteoblast differentiation and expression of protein anabolism genes in ST2 cells, but to a much lesser degree than rapamycin (a specific inhibitor of mTORC1 signaling). Collectively, our findings have demonstrated that pharmacological inhibition of S6K1 impaired self ‐ renewal and osteogenic differentiation of BMSCs, but only partially suppressed exogenous Wnt3a‐induced osteoblast differentiation and protein anabolism.     

Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells

The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.     

ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice

Osteogenic differentiation refers to the process of bone formation and remodeling, which is controlled by complex molecular mechanisms. Activin A receptor type I (ACVR1) is reported to be associated with osteogenic differentiation. However, the underlying molecular mechanism remains elusive. Therefore, this study evaluates the function of ACVR1 in osteogenic differentiation through the Wnt signaling pathway. The expression of osteocalcin (Oc) and osterix together with osteogenic differentiation and mineralization was examined in ACVR1-knockout (KO) mouse. Furthermore, the Wnt signaling pathway was inhibited in bone marrow stromal cells (BMSCs) of mice to explore the role of the Wnt signaling pathway in osteogenic differentiation by means of alkaline phosphatase (ALP) activity detection and evaluation of mineralized nodules and calcium content. Subsequently, the effect of ACVR1 on the Wnt signaling pathway was assessed by determining the expression of ACVR1, β-catenin, glycogen synthase kinase 3 β (GSK3β), dickkopf-related protein 1 (DKK1), and frizzled class receptor 1 (FZD1). Both their effects on osteogenic differentiation were further evaluated by determination of Oc, osterix, and Runx2 expression. AVCR1 KO mice exhibited increased Oc and osterix expression and promoted bone resorption and formation. ACVR1-knockout was observed to activate the Wnt signaling pathway with an increase of β-catenin and reductions in GSK3β, DKK1, and FZD1. With the inhibited Wnt signaling pathway expression of Oc, osterix, and Runx2 was decreased, and ALP activity, mineralized nodule, and calcium content in cellular matrix were decreased as well, indicating that inactivation of the Wnt signaling pathway reduced the differentiation of BMSCs into osteoclasts. These findings indicate that ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice.     

The Effects of Ce on the Proliferation, Osteogenic Differentiation and Mineralization Function of MC3T3-E1 Cells In Vitro

The effects of Ce on the proliferation, osteogenic differentiation and mineralization function of a murine preosteoblast cell line MC3T3-E1 in vitro were investigated at cell and molecular levels. The results showed that Ce promoted the proliferation, osteogenic differentiation and mineralization function of MC3T3-E1 cells at concentrations of 0.0001, 0.001, 0.01, 0.1 and 1???M, but turned to inhibit the proliferation, osteogenic differentiation and mineralization function at concentrations of 10, 100 and 1000???M. Ce displayed the up-regulation of Runx2, BMP2, ALP, BSP, Col I and OCN genes at concentrations of 0.0001 and 0.1???M; these genes were down-regulated in the MC3T3-E1 cells treated with 1000???M Ce. The expression of BMP2, Runx2 and OCN proteins was promoted by Ce at concentrations of 0.0001 and 0.1???M, but these proteins were down-regulated after 1000???M Ce treatment. The results suggest that Ce likely up-regulates or down-regulates the expression of Runx2, which subsequently up- or down-regulates OB marker genes Col I and BMP2 at early stages and ALP and OCN at later stages of differentiation, thus causing to promote or inhibit the proliferation, osteogenic differentiation and mineralization function of MC3T3-E1 cells.