Osteogenic Differentiation Pathway Research Articles

The Mechanism, Regulatory Effect of Wnt5α/Ca2+ Signaling Pathway on hMSCs Osteogenic Differentiation Under Infection Conditions

Background: The paper aimed to reveal the role and possible regulatory mechanisms of Wnt5 gene and its non-canonical Beta-catenin signaling pathway in osteogenic differentiation and bone repairment of hMSCs under infection environment. Material and Methods : CCK-8 method was employed to detect the effect of SPA on the proliferation of different hMSCs cells. The histological staining of alizarin red was used to observe the differentiation of cells into osteoblasts. The semi-quantitative detection of osteoblast alkaline phosphatase (ALP) was performed. Fluo-2 assay was employed to measure the intracellular calcium concentration of different hMSCs before and after osteogenic differentiation stimulated by SPA. RT-PCR was applied to detect the specific gene fluorescence of osteoblasts differentiation. Western Blotting was employed to determine the differentiation of hMSCs into osteoblasts. Results: Different hMSCs were able to proliferate in vitro under the stimulation of 100 ng/mL SPA. CCK-8 kit was used to detect lentivirus-infected hMSCs and non-transduced hMSCs in vitro. The results showed that all the cells gradually proliferate with time and an increasing rate. The hMSCs treated with different Wnt5 genes were stained with alizarin red after osteogenic differentiation, and the activities of AMSCs ALP increased. The intracellular free calcium concentration increased with the induction time and peaked in the Wnt5 overexpression group. PCR results showed that ALP, Runx-2, Collagen I, OC, Osterix and BSP genes were all increased in contrast with the non-induction Control group. Conclusion: Under the inflammatory condition of SPA virulence factors, non-canonical Wnt signaling pathway is associated with the classical Wnt/ β-catenin signaling pathway in the process of osteogenic differentiation of hMSCs in vitro, which has a certain synergistic effect.

Focal adhesion kinase promotes BMP2-induced osteogenic differentiation of human urinary stem cells via AMPK and Wnt signaling pathways.

Human urine-derived stem cells (hUSCs) serve as favorable candidates for bone transplants due to their efficient proliferative and multipotent differentiation abilities, as well as the capacity to secrete a variety of vasoactive agents to facilitate tissue engineering. The present study aimed to explore the role of focal adhesion kinase (FAK) in bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation of hUSCs and to investigate the underlying mechanism. The degree of osteogenic differentiation and the correlated signals, following BMP2 overexpression and siRNA-mediated silencing of FAK, were determined in vitro. Moreover, hUSCs induced bone formation in a rat model with cranial defects, in vivo. Our findings revealed that alkaline phosphatase production, calcium deposits, osteocalcin and osteopontin expression, and bone formation were upregulated in vitro and in vivo following BMP2-induced osteogenic differentiation, and AMPK and Wnt signaling pathway activation by FAK could effectively regulate BMP2-enhanced osteogenic differentiation of hUSCs. Taken together, these findings indicated that FAK could mediate BMP2-enhanced osteogenic differentiation of hUSCs through activating adenosine 5'-monophosphate-activated protein kinase and Wnt signaling pathways.

Strontium Modulates Osteogenic Activity of Bone Cement Composed of Bioactive Borosilicate Glass Particles by Activating Wnt/Β-Catenin and Bmp Signalling Pathway

Sr (Strontium) facilitates bone regeneration by stimulating osteoblastic activity and inhibiting osteoclastic resorption simultaneously. Our previous study showed that incorporation of Sr into bioactive borate glass cement enhanced the osteogenic capacity in vivo. However, the amount of Sr in the cement to provide an optimal combination of physicochemical properties and capacity to stimulate bone regeneration and the underlying molecular mechanism of this stimulation is yet to be determined. In this study, bone cements composed of bioactive borosilicate glass particles substituted with varying amounts of Sr (0mol% to 12mol% SrO) were created and evaluated in vitro and in vivo. The setting time of the cement increased with Sr substitution of the glass. Upon immersion in PBS, the cement degraded and converted more slowly to HA (hydroxyapatite) with increasing Sr substitution. The released Sr2+ modulated the proliferation, differentiation, and mineralization of hBMSCs in vitro. Osteogenic characteristics was optimal enhanced with cement (designated BG6Sr) composed of particles substituted with 6mol% SrO. When implanted in rabbit femoral condyle defects, BG6Sr cement supported better peri-implant bone formation and bone-implant contact, comparing to cements substituted with 0mol% or 9mol% SrO. The underlying mechanisms are involved in the activation of both Wnt/β-catenin and BMP signal pathway in osteogenic differentiation of hBMSCs. These results indicate that BG6Sr cement has a promising combination of physicochemical properties and biological performance for minimally invasive healing of bone defects.

Notch-1 inhibition reduces proliferation and promotes osteogenic differentiation of bone marrow mesenchymal stem cells.

Low differentiation and high proliferation rates are critical factors affecting bone marrow mesenchymal stem cell (BMSC) tumorigenesis. The present study aimed to investigate the role of the Notch signaling pathway in BMSC proliferation and osteogenic differentiation. Mouse BMSCs were divided into control, vector, Notch1-small interfering (si)RNA, γ-secretase inhibitor, and Notch1-siRNA + γ-secretase inhibitor groups. The siRNA-Notch1, γ-secretase inhibitor, and Notch1-siRNA + γ-secretase inhibitor groups were treated with Notch1 siRNA and/or γ-secretase inhibitor. Following treatment, cell proliferation was evaluated using a Cell Counting Kit-8. Tumor-related factors, including transforming growth factor (TGF)-β1, c-Myc and p53, were detected by reverse transcription-quantitative polymerase chain reaction and western blot analyses. BMSC osteogenic differentiation was induced and the cells were stained with alizarin red at 14 and 21 days. Alkaline phosphatase (AKP) activity was also evaluated. The siRNA-Notch1 and γ-secretase inhibitor both reduced BMSC proliferation and the expression of TGF-β1 and c-Myc and increased the expression of p53. Following the induction of osteogenesis and staining with alizarin red, the level of AKP was significantly higher in cells in the siRNA-Notch1 and γ-secretase inhibitor groups compared with that in the control group. It was found that Notch1 inhibition reduced proliferation and promoted the osteogenic differentiation of BMSCs.

Role of p53 mediated miR-23a/CXCL12 pathway in osteogenic differentiation of bone mesenchymal stem cells on nanostructured titanium surfaces.

Titanium surface modification is widely established and has been proven to improve the osseointegration, but the molecular mechanism remains to be fully elucidated. MicroRNAs serve vital roles in the process of regulating osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). In this study, we report that miR-23a was significantly down-regulated in the osteogenic differentiation process of BMSCs on nanostructured titanium surfaces. Elevated miR-23a inhibited osteogenic differentiation of BMSCs, and decreased miR-23a enhanced this process. In addition, we also observed that CXCL12 was a direct target of miR-23a. Knockdown of CXCL12 inhibited nanotube Ti induced-osteogenic differentiation of BMSCs, similar to the effect of upregulation of miR-23a. Finally, p53 was decreased and it regulated miR-23a/CXCL12 axis during nanotube Ti induced-osteogenic differentiation of BMSCs. Therefore, our findings suggest that by targeting CXCL12, miR-23a serves a vital role in osteogenic differentiation of BMSCs cultured on nanostructured titanium surfaces, which may provide novel clinical treatments for osseointegration.

Regulation of cell deformation induced by RhoA/ROCK signaling pathway in osteogenic differentiation of human mesenchymal stem cells

Objective: To investigate whether the cell deformation induced by RhoA/ROCK signaling pathway plays a regulatory role in the osteogenic differentiation in human mesenchymal stem cells (hMSCs). Methods: The primary hMSCs were cultured until the P3 generation was added to the osteogenic induction solution for 14 days, and the expression levels of RhoA and ROCK-1 proteins were detected by immunofluorescence. Another P3 generation hMSCs cells were divided into induction group, blank plus drug group and medicated group, and the induction group was induced by simple osteogenic induction; the blank drug-added group was added to the osteogenic induction solution and the solvent dimethyl sulfoxide (DMSO) for induction culture; the medicinal group was added with osteogenic induction solution and Y-27632 (RhoA/ROCK signaling pathway inhibitor) dissolved in DMSO for induction culture. The proliferation of the cells was detected by CCK-8 methods. The changes of cytoskeleton in each group were observed by fluorescence microscope. The expression changes of osteogenic genes alkaline phosphatase (ALP), osteocalcin (OCN) and runt-relatedtranscriptionfactor-2 (RUNX-2) in each group were detected by real time polymerase chain reaction (RT-PCR) and ALP staining. The t test was used for data comparison between the two groups. Results: There were significant differences in the expression of RhoA and ROCK-1 protein before and after osteogenic induction of hMSCs (22.7±2.1 vs 14.7±0.6 and 24.3±1.5 vs 20.3±0.6, t=-6.414, -4.243, both P<0.05). Mesenchymal stem cell proliferation on day 1, 5, 9 and day 14 in the medicated group were comparable with those in the induction group (F=0.427, 1.000, 0.298, 1.314, all P>0.05). The cytoskeleton of the medicated group showed obvious spindle-shaped changes, and the cell spreading area became smaller. From the three groups of ALP staining on the 14th day, it can be seen that the color of the medicated group was significantly lighter than that of the induction group. The results of RT-PCR showed that the expression of osteogenic phenotypic genes increased with the induction time in the induction group and the blank drug-treated group, the expression levels of osteogenic phenotype genes ALP, OCN and RUNX-2 in the drug-treated group were gradually decreased with the induction time. The expression of ALP in the drug-treated group was significantly lower than those in the other two groups at 14th day (F=25.891, P=0.001); the expression of OCN in the drug-treated group was significantly lower than those in the other two groups at 11th and 14th days (F=5.773, 25.382, both P<0.05); the expression of RUNX-2 in the drug group was significantly lower than those in the other two groups at 11th and 14th days (F=34.972,10.808, both P<0.05). Conclusion: The RhoA/ROCK signaling pathway may play a role in promoting osteogenic differentiation of hMSCs through mediating cytoskeletal deformation.

Resveratrol improves osteogenic differentiation of senescent bone mesenchymal stem cells through inhibiting endogenous reactive oxygen species production via AMPK activation

ABSTRACTObjective: Resveratrol has been confirmed to improve bone quality and delay osteoporosis, but the mechanisms have not been thoroughly elucidated. In this report, we investigated the osteogenic differentiation effect of resveratrol on senescent bone mesenchymal stem cells (BMSCs) and the involvement of AMP-activated protein kinase (AMPK)/ reactive oxygen species (ROS) signaling pathway.Methods: Cell senescence, viability, and osteogenic differentiation of BMSCs influenced by resveratrol were investigated and ROS production and AMPK expression were detected.Results: Cell senescence, characterized by senescence β-galactosidase staining and senescence-related genes (p16, p21, and p53) expression, was attenuated by resveratrol. Cell viability, extracellular matrix calcification, and osteogenic-related genes expression were significantly enhanced after resveratrol treatment. ROS production in BMSCs was inhibited while AMPK expression was up-regulated by resveratrol. Inhibition of AMPK expression by compound C reduced resveratrol-prompted osteogenesis and ROS production down-regulation.Conclusion: These results provide a potential mechanism involving AMPK activation/ROS inhibition signaling pathway in osteogenic differentiation of BMSCs enhanced by resveratrol. It suggests that development of therapy towards ROS is an effective way for osteoporosis treatment.

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.

A Reciprocal Role of the Smad4-Taz Axis in Osteogenesis and Adipogenesis of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into mature cells of various cell types. Although the differentiation process of MSCs requires lineage‐specific transcription factors, the exact molecular mechanism that determines MSCs differentiation is not clearly addressed. Here, we demonstrate a Smad4‐Taz axis as a new intrinsic regulator for adipo‐osteogenic differentiation of MSCs and show that this function of Smad4 is independent of the transforming growth factor‐β signal. Smad4 directly bound to the Taz protein and facilitated nuclear localization of Taz through its nuclear localization signal. Nuclear retention of Taz by direct binding to Smad4 increased expression of osteogenic genes through enhancing Taz‐runt‐related transcription factor 2 (Runx2) interactions in the C3H10T1/2 MSC cell line and preosteoblastic MC3T3‐E1 cells, whereas it suppressed expression of adipogenic genes through promoting Taz‐peroxisome proliferator‐activated receptor‐γ (PPARγ) interaction in C3H10T1/2 and preadipogenic 3T3‐L1 cells. A reciprocal role of the Smad4 in osteogenic and adipogenic differentiation was also observed in human adipose tissue‐derived stem cells (hASCs). Consequently, Smad4 depletion in C3H10T1/2 and hASCs reduced nuclear retention of Taz and thus caused the decreased interaction with Runx2 or PPARγ, resulting in delayed osteogenesis or enhanced adipogenesis of the MSC. Therefore, these findings provide insight into a novel function of Smad4 to regulate the balance of MSC lineage commitment through reciprocal targeting of the Taz protein in osteogenic and adipogenic differentiation pathways. Stem Cells 2019;37:368–381

Multiform TiO2 nano-network enhances biological response to titanium surface for dental implant applications

Our objective in this study was to evaluate biological responses to a unique multiform nano-network of TiO2 on a titanium (Ti) surface for dental implant applications. A novel multiform TiO2 nano-network was formed on the anodized Ti surface using a simple, rapid electrochemical anodization process. The size of the pores in the nano-network ranged from a few nanometers to a few hundred nanometers. Human bone marrow mesenchymal stem cells (hBMSCs) were used as a model in this study. The multiform nanostructure, which is on the scale of various biological species, was shown to enhance protein adsorption, cell motility, cell proliferation, cell differentiation, and surface bioactivity, as well as stimulate outside-in signals via integrins and the formation of focal adhesion complex. It was also shown to promote hBMSCs toward the osteogenic differentiation pathway and convert the Ti surface from bioinert to bioactive. This is a clear indication that the proposed multilayer TiO2 nano-network has excellent biocompatibility and surface bioactivity. The bioactive TiO2 nano-network structure introduced in this study has considerable potential for applications in implant dentistry.

Tuning the bioactivity of bone morphogenetic protein-2 with surface immobilization strategies

Bone morphogenetic protein-2 (BMP-2) involved therapy is of great potential for bone regeneration. However, its clinical application is restricted due to the undesirable bioactivity and relevant complications in vivo. Immobilization of recombinant BMP-2 (rhBMP-2) is an efficient strategy to mimic natural microenvironment and retain its bioactivity. Herein, we present evidences indicating that osteoinductive capacity of rhBMP-2 can be regulated via variant immobilizing approaches. Three representative superficial immobilizing models were employed to fabricate rhBMP-2-immobilized surfaces including physical adsorption (Au/rhBMP-2), covalent grafting (rhBMP-2-SAM-Au) and heparin binding (Hep-SAM-Au/rhBMP-2) (SAM: self-assembled monolayer). Loading capacity, releasing behavior, osteogenic differentiation and signaling pathways involved, as well as the cellular recognition of rhBMP-2 under various immobilization modes were systematically investigated. As a result, disparate immobilizing approaches not only have effects on loading capacity, but also lead to disparity of osteoinduction at the same dosage. Notably, heparin could reinforce the recognition between rhBMP-2 and its receptors (BMPRs) whereas weaken its binding to its antagonist Noggin. Owing to this “selective” binding feature, the favorable osteoinduction and maximum ectopic bone formation can be achieved with the heparin-binding approach. In particular, manipulation of orientation-mediated BMP-2-cell recognition efficiency may be a potential target to design more therapeutic efficient rhBMP-2 delivery system. Statement of SignificanceBone morphogenetic protein-2 (BMP-2) is crucial in bone regeneration. However, its clinical application is challenged due to its shorten half-life and supra-physiological dose associated complications. In this study, three representative superficial immobilizing patterns were fabricated through physical adsorption, covalent grafting and electrostatic interaction with heparin respectively. We provided evidences indicating an dose-dependent osteoinductive capacity of immobilized BMP-2. Further, a possible mechanism of rhBMP-2-cell recognition at the interface was presented, highlighting the superior effect of heparin on rhBMP-2 bioactivity. Finally, We proposed a dual mechanism of tuning the bioactivity of immobilized rhBMP-2 through surface immobilization approaches: regulation of the saturated loading capacity and orientation-mediated rhBMP-2-cell recognition. These results provide novel insights into designing criterion of efficient delivery vehicle for rhBMP-2.

Role of neuropeptide substance P and the bone morphogenetic protein signaling pathway in osteogenic differentiation of ST2 cells

This study aimed to investigate the role and mechanism of neuropeptide substance P (SP) in ST2 cell (bone mesenchymal stem cells of mice) osteogenic differentiation to provide a basis for the treatment of temporomandibular joint osteoarthritis. Third-generation ST2 cells were cultured with different concentrations of SP (0, 10⁻¹⁰, 10⁻⁸, 10⁻⁶, and 10⁻⁵ mol·L⁻¹). After 24, 48, and 72 h, cell proliferation was detected by CCK-8. The ST2 cells were cultured with 10⁻⁶ mol·L⁻¹ SP for 1, 3, 5, and 7 days. Subsequently, the expression of alkaline phosphatase (ALP), collagen typeⅠ(CollaⅠ), and osteocalcin (OCN) in the culture supernatant was tested by enzyme-linked immunosorbent assay (ELISA). ALP activity was detected by immunofluorescence staining. The ST2 cells were cultured with SP, Noggin (inhibitor of the bone morphogenetic protein signaling pathway), SP+Noggin, and 2% fetal bovine serum, respectively. Finally, the expression of ALP, CollaⅠ, and OCN in the culture supernatant was tested by ELISA. CCK-8 showed that the effect of cell proliferation was most obvious when the SP concentration was 10⁻⁶ mol·L⁻¹ (P<0.01). The ELISA results demonstrated that ALP expression significantly increased at day 5 compared with that in the control group (P<0.01), whereas the expression of CollaⅠand OCN significantly increased at day 7 (P<0.05). Immunofluorescence results showed that ALP activity was strongest at day 5. The expression of ALP, CollaⅠ, and OCN decreased after Noggin addition (P<0.05). SP can promote the proliferation and osteogenic differentiation of ST2 cells, and the bone morphogenetic protein signaling pathway may be involved in this process.

A unique hybrid-structured surface produced by rapid electrochemical anodization enhances bio-corrosion resistance and bone cell responses of \u03b2-type Ti-24Nb-4Zr-8Sn alloy

Ti-24Nb-4Zr-8Sn (Ti2448), a new β-type Ti alloy, consists of nontoxic elements and exhibits a low uniaxial tensile elastic modulus of approximately 45 GPa for biomedical implant applications. Nevertheless, the bio-corrosion resistance and biocompatibility of Ti2448 alloys must be improved for long-term clinical use. In this study, a rapid electrochemical anodization treatment was used on Ti2448 alloys to enhance the bio-corrosion resistance and bone cell responses by altering the surface characteristics. The proposed anodization process produces a unique hybrid oxide layer (thickness 50–120 nm) comprising a mesoporous outer section and a dense inner section. Experiment results show that the dense inner section enhances the bio-corrosion resistance. Moreover, the mesoporous surface topography, which is on a similar scale as various biological species, improves the wettability, protein adsorption, focal adhesion complex formation and bone cell differentiation. Outside-in signals can be triggered through the interaction of integrins with the mesoporous topography to form the focal adhesion complex and to further induce osteogenic differentiation pathway. These results demonstrate that the proposed electrochemical anodization process for Ti2448 alloys with a low uniaxial tensile elastic modulus has the potential for biomedical implant applications.

Effects of tumor necrosis factor-α on osteogenic differentiation and Notch signaling pathway in human periodontal ligament stem cells

To evaluate the effects of tumor necrosis factor-α (TNF-α) on osteogenic differentiation and Notch signaling pathway of periodontal ligament stem cells (PDLSCs) and to investigate the regulatory role of Notch signaling pathway on the osteogenic differentiation of PDLSCs under the influence of TNF-α. PDLSCs were obtained through enzyme digestion and tissue block method. The expression levels of stem cell surface markers CD105, CD90, CD146, CD45, and CD31 were detected by fluorescence activated cell sorter (FACS). PDLSCs were divided into experimental (10 ng·mL⁻¹ TNF-α) and control groups (0 ng·mL⁻¹ TNF-α). The proliferation ability of PDLSCs was detected using cell counting kit-8 (CCK-8). The effect of TNF-α on the osteogenic ability of PDLSCs were tested by measuring alkaline phosphatase (ALP) activity and conducting alizarin red staining and quantitative real-time polymerase chain reaction (PCR). We tested Notch signal pathway receptors Notch1, Notch2, ligand JAG1, JGA2, and downstream gene Hes-1. Changes in DLL1 expression were detected by quantitative real-time PCR. FACS profiling showed that PDLSCs were strongly positive for CD105, CD90, and CD146 but negative for CD45 and CD31. CCK-8 results showed that TNF-α could promote the proliferation of PDLSCs (P<0.05). ALP activity in the experimental group was lower than that in the control group (P<0.05). Alizarin red staining showed that the experimental group had decreased mineralized nodules as compared with the control group. Quantitative real-time PCR results showed that the mRNA expression of osteogenic marker genes cementum attachment protein (CAP), osteopontin (OPN), and Runt-related transcription factor 2 (Runx2) significantly decreased in the experimental group as compared with those in the control group (P<0.05). The expression levels of Notch1, Notch2, JAG1, JGA2 and Hes-1 were significantly decreased (P<0.05), whereas those of Notch3 and DLL1 were increased in Notch signaling pathway-related molecules (P<0.05). TNF-α can promote PDLSCs proliferation and inhibit bone differentiation and Notch signaling pathway expression, indicating that the Notch signaling pathway regulates PDLSCs osteogenic differentiation.

LncRNA KCNQ1OT1 promotes osteogenic differentiation to relieve osteolysis via Wnt/\u03b2-catenin activation

BackgroundResveratrol (RSV) has been reported to stimulate osteoblast differentiation in which Wnt/β-catenin signaling pathway played a crucial role. However, whether and how RSV activated Wnt/β-catenin pathway in osteogenic differentiation still remained elusive.MethodsIn vivo polymethylmethacrylate (PMMA) particle-induced osteolysis (PIO) mouse model and in vitro PMMA particle-stimulated mouse mesenchymal stem cells (mMSCs) experiments were established. Relative expression levels of lncRNA KCNQ1OT1, β-catenin, Runx2, Osterix and osteocalcin were determined using quantitative Real-Time PCR. Western blotting was used to measure β-catenin protein expression. In addition, the alkaline phosphatase activity and mineral deposition level using alizarin red S staining were performed to examine osteogenic differentiation status. The interaction between KCNQ1OT1 and β-catenin was confirmed by RNA pull down assay.ResultsRSV significantly attenuated PIO in vivo and PMMA-particle inhibition of osteogenic differentiation of mMSCs. Moreover, KCNQ1OT1 exerted the similar function in mMSCs by regulating β-catenin. Further study demonstrated that RSV exerted its effect on osteoblastic differentiation by regulating KCNQ1OT1. Consequently, RSV alleviated PMMA-particle inhibition of osteoblastic differentiation via Wnt/β-catenin pathway activation in vivo and in vitro.ConclusionRSV accelerated osteoblast differentiation by regulating lncRNA KCNQ1OT1 via Wnt/β-catenin pathway activation, indicating the functional role of RSV in modulating osteogenesis.

The effect of ascorbic acid on bone cancer cells in vitro

Ascorbic acid (Vitamin C) has long been known for its anti-cancer properties and in the present study the effects of ascorbic acid (AsA) on osteogenic differentiation, apoptosis, and signaling pathways of the human G29 osteosarcoma cell line were studied. The expression of Runt-related transcription factor-2 (RUNX2) and osteocalcin genes were evaluated by real-time polymerase chain reaction (PCR). Osteoblastic maturation was assessed with alkaline phosphatase activity and mineralization with alizarin red deposition, and apoptosis with a caspase-2 apoptotic assay as well as the cell viability via the cytotoxicity assay. The possible role of the MAP kinase pathway (p44/42, p38, and p-JNK signaling pathway) was also studied. Our results showed that RUNX2 and osteocalcin gene expression, mineralization, cell viability, and metabolic activity levels were increased in cells treated with low concentrations of AsA with respect to untreated cells. At higher concentrations, AsA resulted in decreases in these parameters and induced apoptosis of the G292 osteosarcoma cells via downregulation of the MAPK pathway. The findings presented here support the ability of AsA to modulate the viability and differentiation of the G292 type of bone cancer cell with increases or decreases depending on the AsA concentration suggesting a need for further evaluation of the possible use of this vitamin in the regulation of bone cell cancer growth.

RESEARCH PROGRESS OF Hedgehog SIGNALING PATHWAY IN REGULATING BONE FORMATION AND OSTEOGENIC DIFFERENTIATION OF BONE MESENCHYMAL STEM CELLS

To summarize the research progress of the effects and mechanisms of Hedgehog signaling pathway in regulating bone formation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). The related literature concerning the regulations and mechanism of Hedgehog signaling pathway in osteogenic differentiation of BMSCs and bone formation in vivo, in vitro, and ex vivo studies in recent years was analyzed and summarized. The in vitro studies indicate that Hedgehog signaling pathway can promote osteogenic differentiation of BMSCs via activation of key molecules Smoothened (Smo) and Gli1 which are downstream of Hedgehog signaling, and Hedgehog signaling can activate mTORC2-Akt signaling by upregulation of insulin-like growth factor which has similar effects. Hedgehog signaling regulates osteoblast differentiation via activation of Hh-Smo-Ptch1-Gli signaling pathway and inhibition of Hh-Gαi-RhoA stress fibre signaling. Hedgehog signaling can regulate key molecules of osteogenesis Runx2 for promoting osteogenic differentiation and matrix mineralization by synergism of bone morphogenetic protein and Wnt signaling, and promotes bone formation and repair and healing for bone defect and bone graft model in vivo. Hedgehog signaling can regulate bone formation and osteogenic differentiation of BMSCs via activation of Hedgehog signaling and other signaling pathways. Hedgehog signaling pathway may be a potential target for developing treatment for bone related diseases of osteoporosis and fracture healing disorders.

Bone regeneration process driven to human periodontal ligament stem cells cultured onto corticocancellous scaffold

The aim of our research was to develop tissue-engineerized constructs composed by porcine cortico-cancellous scaffold (Osteobiol Dual Block) (DB) and xenofree ex vivo culture of human Periodontal Ligament Stem Cells (hPDLSCs) induced to osteogenic differentiation. hPDLSCs placed in xeno-free media formulation mantained the stem cells features, the expression of stemness and pluripotency markers, and the capacity to differentiate in different mesenchymal cell lines (1). Micrographs performed by transmission electron microscopy suggested that after one week of culture, both uninduced and osteogenic induced cells joined and grew on DB secreting extracellular matrix, hierarchically assembled in fibrils in osteogenic differentiation induced samples (2). Quantitative RT-PCR (qRT-PCR) of 92 osteogenesis-related assays of hPDLSCs seeded on the DB showed the upregulation of key genes involved in the osteogenic differentiation pathway such as RUNX2, collagens and SMAD. hPDLSCs induced to osteogenic differentiation in presence of DB expressed osteogenic- related transcripts such as BMP1-4-6, RUNX-2, collagens, MSX1-2, TGFβ3 and SMAD. Functional study revealed a significant increased response of calcium transients, in presence of the 3D-DB both in undifferentiated and differentiated cells stimulated with calcitonin and parathormone, suggesting that the biomaterial could drive the osteogenic differentiation process of hPDLSCs. These data were confirmed from the increase of gene expression of L-type voltage-dependent Ca2+ (VDCCL), subunits α1C and α2D1 in undifferentiated cells in presence of DB. Our results propose to consider DB a biocompatible, osteoinductive and osteoconductive biomaterial making it promising tools to regulate cell activities in biological environments and for a potential use for the development of new custom made tissue-engineering.

Intermittent Stretching and Osteogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells via the p38MAPK-Osterix Signaling Pathway

Aims: The relationship between the p38MAPK signaling pathway and osterix in osteogenic differentiation of BMMSCs subjected to intermittent stretching was investigated. Methods: BMMSCs derived from C57BL/6J mice were divided into the following groups: 1) control, 2) stretch, and 3) SB203580+stretch (SB203580 is a p38MAPK signal pathway inhibitor). BMMSCs were exposed to an intermittent mechanical strain of 0.8% (8000μ strain) at 0.5 Hz, twice a day for 30 min each application. BMMSCs were harvested on days 1, 3, and 5 post-treatment. The expression of ALP, COL I, OCN, and osterix mRNA was assessed utilizing RT-PCR while the expression of P-p38MAPK and osterix protein was assessed by Western blot analysis. The osterix gene in mouse BMMSCs was knocked down using RNAi technology and its protein expression was also assessed by Western blot. RT-PCR was used to detect ALP, COL I, and OCN mRNA expression. Results: Intermittent stretching was found to promote expression of ALP, COL I, OCN, and osterix mRNA. Silencing the osterix gene was found to reduce levels of ALP, COL I, and OCN mRNA. Western blot analysis demonstrated that the levels of osterix and P-p38MAPK proteins in the stretch group were significantly higher than in the control group (P<0.05). There was less expression of ALP, COL I, OCN, and osterix mRNA in the SB203580+stretch group than in the control and stretch groups. Conclusions: Data demonstrate that intermittent stretching promotes osteogenic differentiation of BMMSCs, and the p38MAPK-osterix pathway has an important role in the control of osteogenesis-related gene expression.

Nitric Oxide Activates Signaling by c-Raf, MEK, p-JNK, p38 MAPK and p53 in Human Mesenchymal Stromal Cells inhibits their Osteogenic Differentiation by Blocking Expression of Runx2

Introduction: Mesenchymal stromal cells (MSC) are a promising therapy for wound healing and regeneration of inflamed tissues. They are used clinically for different symptoms and diseases and are being investigated in clinical trials world wide at an increasing rate. However, depending on the application protocol and site of treatment, MSC may face an inflammatory environment. Objective: Nitric oxide (NO) is one of the soluble factors produced in acute and chronic inflammation and influences growth, apoptosis, proliferation and differentiation of cells. NO therefore my have an influence on MSC injected into inflamed sites. Thus we investigated the effects of NO radicals on human MSC. Methods: Human MSC were expanded and characterized. Expression of the mesenchymal linage markers was determined by flow cytometry and their tri-lineage differentiation was explored in vitro. MSC were incubated with the NO-donor sodium nitroprusside (SNP) at different concentrations (5 μM-5 mM) and over different periods of time (15 min–24 hrs), and analyzed for their respiratory activity, gene expression responses, cell signalling pathways, and differentiation potential. Results: Human MSC expressed the mesenchymal marker proteins CD73, CD90, CD105, CD146, but failed to express the hematopoietic markers CD11b, CD14, CD34, and CD45. Activation of the MSC in vitro by nitric oxide activated c-Raf-, p-38-MAPK, and p-JNK- mediated signalling in a dose dependent manner, and also significantly regulated genes involved in cellular proliferation (cyclin D1, GAS1), apoptotis (p53), and induced an intense nuclear factor E2-related factor (NRF2)-associated stress response. Moreover, NO inhibited the entry of MSC in the osteogenic differentiation pathway and NO-treated MSC expressed less of the transcription factor Runx2. In contrast, the expression of the adipogenic marker gene PPARγ2 remained unchanged. Conclusion: We conclude that NO modulates the metabolism of MSC and compromises their osteogenic differentiation potential, which may have detrimental consequences for bone remodelling or bone regeneration.