Osteogenic Differentiation Of hBMSCs Research Articles

Calcium phosphate-phosphorylated adenosine hybrid microspheres for anti-osteosarcoma drug delivery and osteogenic differentiation.

Biocompatibility, biodegradability and bioactivity are significantly important in practical applications of various biomaterials for bone tissue engineering. Herein, we develop a functional inorganic-organic hybrid system of calcium phosphate-phosphorylated adenosine (CPPA). Both calcium phosphate and phosphorylated adenosine molecules in CPPA are fundamental components in mammalians and play important roles in biological metabolism. In this work, we report our three leading research qualities: (1) CPPA hybrid microspheres with hollow and porous structure are synthesized by a facile one-step microwave-assisted solvothermal method; (2) CPPA hybrid microspheres show high doxorubicin loading capacity and pH-responsive drug release properties, and demonstrate positive therapeutic effects on six osteosarcoma cell lines invitro and a mouse model of 143B osteosarcoma subcutaneous tumor invivo; (3) CPPA hybrid microspheres are favorable to promote osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs) by activating the AMPK pathway, with satisfactory evidences from cellular alkaline phosphatase staining, alizarin red staining, real time PCR and western analysis. The as-prepared CPPA hybrid microspheres are promising in anti-osteosarcoma and bone regeneration, which simultaneously display excellent properties on drug delivery and osteogenic differentiation of hBMSCs.

Gremlin2 Suppression Increases the BMP-2-Induced Osteogenesis of Human Bone Marrow-Derived Mesenchymal Stem Cells Via the BMP-2/Smad/Runx2 Signaling Pathway

Osteoblasts are essential for maintaining skeletal architecture and modulating bone microenvironment homeostasis. From numerous associated investigations, the BMP-2 pathway has been well-defined as a vital positive modulator of bone homeostasis. Gremlin2 (Grem2) is a bone morphogenetic protein (BMP) antagonists. However, the effect of Grem2 on the BMP-2-induced osteogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs) remains ambiguous. This study aimed to analyze the procedure in vitro and in vivo. The differentiation of hBMSCs was assessed by determining the expression levels of several osteoblastic genes, as well as the enzymatic activity and calcification of alkaline phosphatase. We found that Grem2 expression was upregulated by BMP-2 within the range of 0-1 μg/mL, and significant increases were evident at 48, 72, and 96 h after BMP-2 treatment. Si-Grem2 increased the BMP-2-induced osteogenic differentiation of hBMSCs, whereas overexpression of Grem2 had the opposite trend. The result was confirmed using a defective femur model. We also discovered that the BMP-2/Smad/Runx2 pathway played an important role in the process. This study showed that si-Grem2 increased the BMP-2-induced osteogenic differentiation of hBMSCs via the BMP-2/Smad/Runx2 pathway. J. Cell. Biochem. 118: 286-297, 2017. © 2016 Wiley Periodicals, Inc.

Exosomes/tricalcium phosphate combination scaffolds can enhance bone regeneration by activating the PI3K/Akt signaling pathway.

BackgroundRecently, accumulating evidence has shown that exosomes, the naturally secreted nanocarriers of cells, can exert therapeutic effects in various disease models in the absence of parent cells. However, application of exosomes in bone defect repair and regeneration has been rarely reported, and little is known regarding their underlying mechanisms.MethodsExosomes derived from human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSC-Exos) were combined with tricalcium phosphate (β-TCP) to repair critical-sized calvarial bone defects, and the efficacy was assessed by histological examination. We evaluated the in vitro effects of hiPSC-MSC-Exos on the proliferation, migration, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) by cell-counting, scratch assays, and qRT-PCR, respectively. Gene expression profiling and bioinformatics analyses were also used to identify the underlying mechanisms in the repair.ResultsWe found that the exosome/β-TCP combination scaffolds could enhance osteogenesis as compared to pure β-TCP scaffolds. In vitro assays showed that the exosomes could release from β-TCP and could be internalized by hBMSCs. In addition, the internalization of exosomes into hBMSCs could profoundly enhance the proliferation, migration, and osteogenic differentiation of hBMSCs. Furthermore, gene expression profiling and bioinformatics analyses demonstrated that exosome/β-TCP combination scaffolds significantly altered the expression of a network of genes involved in the PI3K/Akt signaling pathway. Functional studies further confirmed that the PI3K/Akt signaling pathway was the critical mediator during the exosome-induced osteogenic responses of hBMSCs.ConclusionsWe propose that the exosomes can enhance the osteoinductivity of β-TCP through activating the PI3K/Akt signaling pathway of hBMSCs, which means that the exosome/β-TCP combination scaffolds possess better osteogenesis activity than pure β-TCP scaffolds. These results indicate that naturally secreted nanocarriers-exosomes can be used as a bioactive material to improve the bioactivity of the biomaterials, and that hiPS-MSC-Exos combined with β-TCP scaffolds can be potentially used for repairing bone defects.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0391-3) contains supplementary material, which is available to authorized users.

Monitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probes

Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can be observed in a non-destructive manner. In addition, the osteogenic hBMSCs can be prospectively identified and obtained based on the relative intracellular fluorescence of Sox9 in relation to Runx2 using fluorescence activated cell sorting. Relatively homogeneous cell populations with high osteogenic potential can be isolated from the original heterogeneous osteogenically induced hBMSCs within the first week of induction. This offers a more detailed analysis of the effectiveness of new therapeutics both at the individual cell level and the response of the population as a whole. By identifying and isolating differentiating cells at early time points, prospective analysis of differentiation is also possible, which will lead to a greater understanding of MSC differentiation.

Biocompatibility of nano-hydroxyapatite/polyamide 66 composite with human bone mesenchymal stem cells

Objective To explore the biocompatibility of nano-hydroxyapatite/polyamide 66 (nHA/PA66) with human bone mesenchymal stem cells (hBMSCs) after osteogenic induction. Methods After hBMSCs were isolated and cultured in vitro, the experiment was conducted in 3 groups. Group A were hBMSCs subjected to no osteogenic induction, group B hBMSCs subjected to osteogenic induction, and group C was the composite of nHA/PA66 with hBMSCs subjected to osteogenic induction. Adhesion of the cells onto the nHA/PA66 in group C was observed by electron microscope scanning. Growth and proliferation of the cells in groups B and C were detected by MTT test at 1, 2 and 3 weeks. The ability of osteogenic differentiation of hBMSCs in vitro was analyzed by alkaline phosphatase (ALP) activity and alizarin red staining. The ability of osteogenic differentiation of hBMSCs on nHA/PA66 was tested by ALP activity. Results Electron microscope scanning showed that the cells spread and attached well on the surface of the composite scaffold in group C; the proliferation of the cells in groups B and C showed no significant difference (P>0.05) . These suggested that the proliferation of hBMSCs was not affected by nHA/PA66. The number of mineralized nodules in group B was significantly larger than in group A (P 0.05) . These indicated that the hBMSCs were capable of osteogenic differentiation which was not affected by nHA/PA66. In groups B and C, the ALP activity of the cells at 12 days was significantly higher than at 6 days, indicating the ALP activity increased with increased induction time (P<0.05). Conclusion nHA/PA66 can be used as a carrier of hBMSCs in bone tissue engineering because hBMSCs can well adhere to, proliferate, and differentiate into bone on nHA/PA66 scaffolds. Key words: Bone marrow cells; Hydroxyapatite; Polyamides; Biocompatible materials

MiR-33a-5p modulates TNF-α-inhibited osteogenic differentiation by targeting SATB2 expression in hBMSCs.

miRNAs play a number of roles in bone, including mediating the pathological effects of inflammation. Here, we found that miR-33a-5p expression was significantly increased after TNF-α treatment during BMP-2-induced osteogenic differentiation of hBMSCs. Luciferase reporter assays and western blotting demonstrated that special AT-rich sequence-binding protein 2 (SATB2) is a target of miR-33a-5p. Moreover, we show that BMP-2 induces SATB2 expression by interacting with SATB2 directly via the BMP-2-RUNX2 pathway. However, TNF-α first decreases SATB2 expression by inhibiting miR-33a-5p degradation. We thus conclude that miR-33a-5p plays a central role in this complex regulatory network. These findings will help to understand the regulatory role of miR-33a-5p in the inflammatory process.

Inhibition of miR-222-3p activity promoted osteogenic differentiation of hBMSCs by regulating Smad5-RUNX2 signal axis

miRNAs are recently found playing important roles in osteogenesis. In this study, we identified that miR-222-3p decreased during osteogenic differentiation of human mesenchymal stem cells (hBMSCs) using Quantitative Real-Time Reverse Transcription PCR (qRT-PCR). Furthermore, we investigated the effect of miR-222-3p on osteogenic differentiation of hBMSCs. Inhibition of miR-222-3p function in hBMSCs using infection of lentiviruses carrying miR-222-3p specific inhibitor promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Whereas, overexpression of miR-222-3p inhibited osteoblast differentiation of hBMSCs in vitro. Moreover, Smad5 and RUNX2, which are the critical transcription factors in osteogenic differentiation, were predicted to be targets of miR-222-3p by bioinformatic analysis. Overexpression of miR-222-3p in hBMSCs significantly suppressed the protein levels of Smad5 and RUNX2, while inhibition of miR-222-3p increased their protein levels. Furthermore, inhibition of miR-222-3p increased phosphorylation of Smad1/5/8, which regulated the expression of osteogenic genes. Our findings suggest that suppression of miR-222-3p activity promoted osteogenic differentiation hBMSCs through regulating Smad5-RUNX2 signaling axis.

Silencing of Long Non-Coding RNA NONHSAT009968 Ameliorates the Staphylococcal Protein A-Inhibited Osteogenic Differentiation in Human Bone Mesenchymal Stem Cells

Background/Aims: Osteomyelitis is defined as an inflammation of the bones and bone marrow. The inflammatory microenvironment attenuates the osteogenic differentiation capacity of stem cells and inhibits osteoblast-mediated bone formation, leading to net bone loss. However, the whole expression profile, function and side effect of long non-coding RNAs (lncRNAs) on osteogenic differentiation of stem cells in an inflammatory microenvironment of osteomyelitis are not known. Methods: In the present study, human bone mesenchymal stem cells (hBMSCs) were treated with different concentrations of Staphylococcal protein A (SpA) to trigger an inflammatory microenvironment in vitro to partly duplicate the inflammatory microenvironment of osteomyelitis, which was confirmed using ELISA for detecting the inflammatory cytokines. The complete expression profiles of lncRNAs and mRNA during osteogenic differentiation of hBMSCs in an inflammatory microenvironment triggered by SpA were analyzed using a lncRNA microarray. LncRNA expression levels were verified by quantitative reverse transcription PCR analysis (qRT-PCR). The expression of NONHSAT009968 in hB-MSCs was silenced by infection with lentivirus expressing NONHSAT009968-shRNA. The expression of Runx2, OCN, OPN, COL1A1, and alkaline phosphatase (ALP) activity was detected by western blot. Alizarin red staining and ALP activity detection were carried out. Results: The results of ELISA showed that SpA treatment induced secretion of inflammatory cytokines IL-1A, IL-6, and TNFA. The results of alizarin red staining and ALP detection showed that SpA treatment suppressed the osteogenic differentiation of hBMSCs. A total of 2033 lncRNAs were found with aberrant expression in SpA-treated hBMSCs compared to controls. Among these lncRNAs, 641 were down-regulated and 1392 were up-regulated. Based on the results of qRT-PCR, lncRNA NONHSAT009968 was chosen for further investigation. The results of alizarin red staining, ALP activity detection, and western blot detection of Runx2, OCN, OPN, COL1A1, and ALP indicated that NONHSAT009968 silencing ameliorates SpA-inhibited osteogenic differentiation in hBMSCs. Conclusion: Our present study provides a basis for future analyses of the role of lncRNAs in osteoblastic differentiation in an inflammatory environment triggered by SpA, and lncRNA NONHSAT009968 might be a new target for promoting osteoblast formation.

Preameloblast-Derived Factors Mediate Osteoblast Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Runx2-Osterix-BSP Signaling.

Epithelial-mesenchymal interaction occurs during development of various tissues, including teeth and bone. Recently, a preameloblast-conditioned medium (PA-CM) from mouse apical bud cells (ABCs), a type of dental epithelial cell, was found to induce odontogenic differentiation of dental pulp stem cells and promote dentin formation. The aims of the present study were to investigate the effects of PA-CM on human bone marrow mesenchymal stem cells (hBMSCs) in vitro, and to investigate the bone regenerative capacity in vivo through epithelial-mesenchymal interactions of developmental osteogenesis. Coculturing with ABCs and PA-CM treatment upregulated osteoblast differentiation markers of hBMSCs compared to cells cultured alone. PA-CM accelerated mineralized nodule formation and also increased bone sialoprotein promoter activity in hBMSCs. PA-CM facilitated the migration of hBMSCs, but did not significantly influence proliferation. PA-CM promoted bone formation of hBMSCs in vivo. Radiographic and histologic findings showed that PA-CM induced the bony regeneration at calvarial defects in rat. Taken together, these data show that PA-CM enhances the migration and osteogenic differentiation of hBMSCs in vitro and induces bone formation in vivo.

Silibinin alleviates high glucose-suppressed osteogenic differentiation of human bone marrow stromal cells via antioxidant effect and PI3K/Akt signaling

High glucose is one of the possible causes for osteoporosis and fracture in diabetes mellitus. Our previous study showed that silibinin can increase osteogenic effect by stimulating osteogenic genes expression in human bone marrow stem cells (hBMSCs). However, no study has yet investigated the effect of silibinin on osteogenic differentiation of hBMSCs cultured with high glucose. The aim of this study was to evaluate the influence of high glucose on osteogenic differentiation of hBMSCs and to determine if silibinin can alleviate those effects. In this study, the hBMSCs were cultured in an osteogenic medium with physiological (normal glucose, NG, 5.5mM) or diabetic (high glucose, HG, 30mM). The effects of silibinin on HG-induced osteogenic differentiation were evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction. HG-induced oxidative damage was also assessed. Western blot were performed to examine the role of PI3K/Akt pathway. We demonstrated that HG suppressed osteogenic differentiation of hBMSCs, manifested by a decrease in expression of osteogenic markers and an increase of oxidative damage markers including reactive oxygen species and lipid peroxide (MDA). Remarkably, all of the observed oxidative damage and osteogenic dysfunction induced by HG were inhibited by silibinin. Furthermore, the PI3K/Akt pathway was activated by silibinin. These results demonstrate that silibinin may attenuate HG-mediated hBMSCs dysfunction through antioxidant effect and modulation of PI3K/Akt pathway, suggesting that silibinin may be a superior drug candidate for the treatment of diabetes related bone diseases.

Secreted klotho protein attenuates osteogenic differentiation of human bone marrow mesenchymal stem cells in vitro via inactivation of the FGFR1/ERK signaling pathway

Increasing evidence indicates that the osteogenic differentiation of mesenchymal stem cells (MSCs) is related to bone formation, heterotopic ossification, and even vascular calcification. Therefore, it is essential to understand the microenvironment that regulates these processes. The Klotho gene plays an important role in tissue mineralization, and its secreted protein functions as a hormone. We investigated the effects of secreted Klotho protein on the osteogenesis of human bone marrow MSC (hBMSCs). To this end, the cells received osteogenic medium with or without Klotho protein. The results showed that osteoblast-specific gene expression and mineral deposition were decreased when MSCs were incubated with Klotho. Klotho reduced the expression of fibroblast growth factor receptor 1 (FGFR1) and phosphorylated extracellular signal-regulated kinase 1/2. However, both MEK and FGFR1 inhibitors delayed bone mineral formation more than Klotho. These data suggest that secreted Klotho protein attenuates the osteogenic differentiation of hBMSCs in vitro through FGFR1/ERK signaling.

Uric Acid Promotes Osteogenic Differentiation and Inhibits Adipogenic Differentiation of Human Bone Mesenchymal Stem Cells.

To investigate the effect of uric acid on the osteogenic and adipogenic differentiation of human bone mesenchymal stem cells (hBMSCs). The hBMSCs were isolated from bone marrow of six healthy donors. Cell morphology was observed by microscopy and cell surface markers (CD44 and CD34) of hBMSCs were analyzed by immunofluorescence. Cell morphology and immunofluorescence analysis showed that hBMSCs were successfully isolated from bone marrow. The number of hBMSCs in uric acid groups was higher than that in the control group on day 3, 4, and 5. Alizarin red staining showed that number of calcium nodules in uric acid groups was more than that of the control group. Oil red-O staining showed that the number of red fat vacuoles decreased with the increased concentration of uric acid. In summary, uric acid could promote the proliferation and osteogenic differentiation of hBMSCs while inhibit adipogenic differentiation of hBMSCs.

The osteogenic or adipogenic lineage commitment of human mesenchymal stem cells is determined by protein kinase C delta.

BackgroundMesenchymal stem cells (MSCs) have the potential to differentiate into specialized cell lineages such as osteoblasts and adipocytes in vitro. There exists a reciprocal relationship between osteogenic and adipogenic differentiation of MSCs that an osteogenic phenotype occurs at the expense of an adipogenic phenotype and vice versa, which in turn influence one another’s phenotype through negative feedback loops. Thus, it is important to understand what signaling molecules modulate the lineage commitment of MSCs. Protein kinase C (PKC) plays a central role in cellular signal transduction for mediating diverse biological functions, and dysregulation of PKC activity is involved in various metabolic diseases including cancer, diabetes, and heart disease. Although the role of individual PKC isoforms has been investigated in various fields, the potential role of PKC in bone metabolism is not completely understood. In this study, we investigated the potential role of PKCδ in osteogenic lineage commitment of human bone marrow-derived mesenchymal stem cells (hBMSCs).ResultsWe observed that expression and phosphorylation of PKCδ were increased during osteogenic differentiation of hBMSCs. Pharmacological inhibition and genetic ablation of PKCδ in hBMSCs resulted in a significant attenuation of osteogenic differentiation as evidenced by reduced ALP activity and ECM mineralization, as well as down-regulation of the expression of osteoblast-specific genes. These effects were also accompanied by induction of adipogenic differentiation and up-regulation of the expression of adipocyte-specific genes involved in lipid synthesis in osteogenic induction of hBMSCs. Additionally, the activation of AMPK, which is a key cellular energy sensor, induced osteogenesis of hBMSCs. However, the inhibition of AMPK activity by compound C did not affect the activation of PKCδ at all, indicating that there is no direct correlation between AMPK and PKCδ in osteogenesis of hBMSCs.ConclusionsThese results suggest that PKCδ is a critical regulator for the balance between osteogenesis and adipogenesis of hBMSCs and thus has a potential novel therapeutic target for the treatment of metabolic bone diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-014-0042-4) contains supplementary material, which is available to authorized users.

Instructive nanofibrous scaffold comprising runt-related transcription factor 2 gene delivery for bone tissue engineering.

Inducer molecules capable of regulating mesenchymal stem cell differentiation into specific lineages have proven effective in basic science and in preclinical studies. Runt-related transcription factor 2 (RUNX2) is considered to be the central gene involved in the osteoblast phenotype induction, which may be advantageous for inducing bone tissue regeneration. This work envisions the development of a platform for gene delivery, combining liposomes as gene delivery devices, with electrospun nanofiber mesh (NFM) as a tissue engineering scaffold. pDNA-loaded liposomes were immobilized at the surface of functionalized polycaprolactone (PCL) NFM. Human bone-marrow-derived mesenchymal stem cells (hBMSCs) cultured on RUNX2-loaded liposomes immobilized at the surface of electrospun PCL NFM showed enhanced levels of metabolic activity and total protein synthesis. RUNX2-loaded liposomes immobilized at the surface of electrospun PCL NFMs induce a long-term gene expression of eGFP and RUNX2 by cultured hBMSCs. Furthermore, osteogenic differentiation of hBMSCs was also achieved by the overexpression of other osteogenic markers in medium free of osteogenic supplementation. These findings demonstrate that surface immobilization of RUNX2 plasmid onto elestrospun PCL NFM can produce long-term gene expression in vitro, which may be employed to enhance the osteoinductive properties of scaffolds used for bone tissue engineering strategies.

Myricetin enhances osteogenic differentiation through the activation of canonical Wnt/β-catenin signaling in human bone marrow stromal cells

Myricetina flavonoid compound, has been reported to possess antioxidative, antiproliferative and anti-inflammatory effects. However, no study has yet investigated the effect of myricetin on osteogenic differentiation of human bone marrow stem cells (hBMSCs). This study was designed to investigate the effects of myricetin on osteogenic differentiation of hBMSCs in vitro. Cell viability was analyzed by MTT and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity assay, Alizarin red S dye, real time-polymerase chain reaction (RT-PCR) and Western blot analysis. We found that the ALP activity and the mineralization of hBMSCs were enhanced by treatment with myricetin. Myricetin increased the mRNA expressions of Osteocalcin (OCN), Collagen type I (COL-I), ALP and Runt-related transcription factor 2 (RUNX2). Additionally, we found that myricetin activated the Wnt/β-catenin pathway and increased the expression of several downstream genes including T-cell factor-1(TCF-1) and lymphoid enhancer factor-1 (LEF-1). Depletion of β-catenin almost completely blocked the positive role of myricetin on osteogenic differentiation. Taken together, our findings suggest that myricetin enhanced osteogenic differentiation of hBMSCs by activating the Wnt/β-catenin signaling. The study may aid in the development of a therapeutic approach utilizing myricetin for the enhancement of bone health and prevention of osteoporosis.

A Novel Single Pulsed Electromagnetic Field Stimulates Osteogenesis of Bone Marrow Mesenchymal Stem Cells and Bone Repair

Pulsed electromagnetic field (PEMF) has been successfully applied to accelerate fracture repair since 1979. Recent studies suggest that PEMF might be used as a nonoperative treatment for the early stages of osteonecrosis. However, PEMF treatment requires a minimum of ten hours per day for the duration of the treatment. In this study, we modified the protocol of the single-pulsed electromagnetic field (SPEMF) that only requires a 3-minute daily treatment. In the in vitro study, cell proliferation and osteogenic differentiation was evaluated in the hBMSCs. In the in vivo study, new bone formation and revascularization were evaluated in the necrotic bone graft. Results from the in vitro study showed no significant cytotoxic effects on the hBMSCs after 5 days of SPEMF treatment (1 Tesla, 30 pulses per day). hBMSC proliferation was enhanced in the SPEMF-treated groups after 2 and 4 days of treatment. The osteogenic differentiation of hBMSCs was significantly increased in the SPEMF-treated groups after 3–7 days of treatment. Mineralization also increased after 10, 15, 20, and 25 days of treatment in SPEMF-treated groups compared to the control group. The 7-day short-course treatment achieved similar effects on proliferation and osteogenesis as the 25-day treatment. Results from the in vivo study also demonstrated that both the 7-day and 25-day treatments of SPEMF increased callus formation around the necrotic bone and also increased new vessel formation and osteocyte numbers in the grafted necrotic bone at the 2nd and 4th weeks after surgery. In conclusion, the newly developed SPEMF accelerates osteogenic differentiation of cultured hBMSCs and enhances bone repair, neo-vascularization, and cell growth in necrotic bone in mice. The potential clinical advantage of the SPEMF is the short daily application and the shorter treatment course. We suggest that SPEMF may be used to treat fractures and the early stages of osteonecrosis.

MicroRNA-125b suppresses the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

The regressive biological function of human bone marrow‑derived mesenchymal stem cells (hBMSCs) is one of the key factors resulting in the decrease of bone mass in senile osteoporosis. MicroRNAs (miRs) are non‑coding small RNAs involved in various gene regulation processes. Whether any miR(s) are involved in the progression of osteoporosis by regulating the biological function of hBMSCs remains to be elucidated. The present study aimed to compare the expression levels of miR‑125b in hBMSCs derived from senile osteoporotic patients with that of control (normal) subjects. A significantly upregulated expression of miR‑125b in osteoporotic hBMSCs was detected. To elucidate the biological function of miR‑125b in senile osteoporosis, the effects of miR‑125b expression on proliferation and osteogenic differentiation of hBMSCs were assessed using gain‑ and loss‑of‑function studies. It was evident that the overexpression of a miR‑125b mimic was able to suppress the proliferative and osteogenic differentiation of senile hBMSCs. In contrast, repression of the function of miR‑125b by transfection of an miR‑125b inhibitor promoted the proliferation and osteogenic differentiation of hBMSCs. Furthermore, the potential target gene of miR‑125b, osterix (Osx), was examined. The results of the present study strongly suggested that miR‑125b may regulate osteogenic differentiation of hBMSCs through the modulation of Osx expression.

Exogenous polyamines promote osteogenic differentiation by reciprocally regulating osteogenic and adipogenic gene expression

Polyamines are naturally occurring organic polycations that are ubiquitous in all organisms, and are essential for cell proliferation and differentiation. Although polyamines are involved in various cellular processes, their roles in stem cell differentiation are relatively unexplored. In this study, we found that exogenous polyamines, putrescine, spermidine, and spermine, promoted osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) without inducing cell death or apoptosis. Alkaline phosphatase (ALP) activity and the mRNA level of osteogenic genes, including Runx2, ALP, osteopontin, and osteocalcin, were up-regulated by exogenous polyamines. When hBMSCs were cultured at high cell density favoring adipocyte formation, exogenous polyamines resulted in down-regulation of adipogenic genes such as PPARγ, aP2, and adipsin. Extracellular matrix mineralization, a marker for osteoblast maturation, was enhanced in the presence of exogenous polyamines, while lipid accumulation, an indication of adipogenic differentiation, was attenuated. Exogenous polyamines increased the mRNA expression of polyamine-modulated factor 1 (PMF-1) and its downstream effector, spermidine/spermine N(1)-acetyltransferase (SSAT), while that of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was suppressed. These results lead to possible connections between polyamine metabolism and osteogenic differentiation pathways. To summarize, this study provides evidence for the involvement of polyamines in osteogenic differentiation of hBMSCs, and is the first to demonstrate that osteogenic and adipogenic differentiation are reciprocally regulated by exogenous polyamines.

Novel markers of osteogenic and adipogenic differentiation of human bone marrow stromal cells identified using a quantitative proteomics approach

Today, the tool that is most commonly used to evaluate the osteogenic differentiation of bone marrow stromal cells (BMSCs) in vitro is the demonstration of the expression of multiple relevant markers, such as ALP, RUNX2 and OCN. However, as yet, there is no single surface marker or panel of markers which clearly defines human BMSCs (hBMSCs) differentiating towards the osteogenic lineage. The aim of this study was therefore to examine this issue.Stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was utilized to investigate differently expressed surface markers in osteogenically differentiated and undifferentiated hBMSCs. Labeled membrane proteins were analyzed by mass spectrometry (MS) and 52 proteins with an expression ratio above 2, between osteogenically differentiated and undifferentiated cells, were identified. Subsequent validation, by flow cytometry and ELISA, of the SILAC expression ratios for a number of these proteins and investigations of the lineage specificity of three candidate markers were performed.The surface markers, CD10 and CD92, demonstrated significantly increased expression in hBMSCs differentiated towards the osteogenic and adipogenic lineages. In addition, there was a slight increase in CD10 expression during chondrogenic differentiation. Furthermore, the expression of the intracellular protein, crystalline-αB (CRYaB), was only significantly increased in osteogenically differentiated hBMSCs and not affected during differentiation towards the chondrogenic or adipogenic lineages.It has been concluded from the present results that CD10 and CD92 are potential markers of osteogenic and adipogenic differentiation and that CRYaB is a potential novel osteogenic marker specifically expressed during the osteogenic differentiation of hBMSCs in vitro.

Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling

Silibinin is the major active constituent of the natural compound silymarin; several studies suggest that silibinin possesses antihepatotoxic properties and anticancer effects against carcinoma cells. However, no study has yet investigated the effect of silibinin on osteogenic differentiation of human bone marrow stem cells (hBMSCs). The aim of this study was to evaluate the effect of silibinin on osteogenic differentiation of hBMSCs. In this study, the hBMSCs were cultured in an osteogenic medium with 0, 1, 10 or 20μmol/l silibinin respectively. hBMSCs viability was analyzed by cell number quantification assay and cells osteogenic differentiation was evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction (RT-PCR). We found that silibinin promoted ALP activity in hBMSCs without affecting their proliferation. The mineralization of hBMSCs was enhanced by treatment with silibinin. Silibinin also increased the mRNA expressions of Collagen type I (COL-I), ALP, Osteocalcin (OCN), Osterix, bone morphogenetic protein-2 (BMP-2) and Runt-related transcription factor 2 (RUNX2). The BMP antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated silibinin-promoted ALP activity. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by silibinin treatment. These results indicate that silibinin enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and RUNX2 pathways. Thus, silibinin may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs.