Expression Of Osteoblast Marker Genes Research Articles

Combined Effects of Cyclic Hypoxic and Mechanical Stimuli on Human Bone Marrow Mesenchymal Stem Cell Differentiation: A New Approach to the Treatment of Bone Loss.

Background: The prevention and treatment of bone loss and osteoporotic fractures is a public health challenge. Combined with normobaric hypoxia, whole-body vibration has a high clinic potential in bone health and body composition. The effect of this therapy may be mediated by its action on bone marrow mesenchymal stem cells (MSCs). Objectives: Evaluate the effects of cyclic low-vibration stimuli and/or hypoxia on bone marrow-derived human MSC differentiation. Methods: MSCs were exposed four days per week, two hours/day, to hypoxia (3% O2) and/or vibration before they were induced to differentiate or during differentiation into osteoblasts or adipocytes. Gene and protein expression of osteoblastic, adipogenic, and cytoskeletal markers were studied, as well as extracellular matrix mineralization and lipid accumulation. Results: early osteoblastic markers increased in undifferentiated MSCs, pretreated in hypoxia and vibration. This pretreatment also increased mRNA levels of osteoblastic genes and beta-catenin protein in the early stages of differentiation into osteoblasts without increasing mineralization. When MSCs were exposed to vibration under hypoxia or normoxia during osteoblastic differentiation, mineralization increased with respect to cultures without vibrational stimuli. In MSCs differentiated into adipocytes, both in those pretreated as well as exposed to different conditions during differentiation, lipid formation decreased. Changes in adipogenic gene expression and increased beta-catenin protein were observed in cultures treated during differentiation. Conclusions: Exposure to cyclic hypoxia in combination with low-intensity vibratory stimuli had positive effects on osteoblastic differentiation and negative ones on adipogenesis of bone marrow-derived MSCs. These results suggest that in elderly or frail people with difficulty performing physical activity, exposure to normobaric cyclic hypoxia and low-density vibratory stimuli could improve bone metabolism and health.

The effects of all-trans retinoic acid on prednisolone-induced osteoporosis in zebrafish larvae

Glucocorticoids (GCs) are extensively used as anti-inflammatory and immunosuppressive medications in the long-term treatment of rheumatic disorders, respiratory diseases, renal diseases, and organ transplantation. Prolonged use of GCs can reduce bone mineral density, leading to osteoporosis (Glucocorticoid Induced Osteoporosis, GIOP) and fracture. All-trans retinoic acid (ATRA) is an active vitamin A metabolite that regulates embryonic development and adult organ function. ATRA has been found in studies to enhance osteogenesis. To examine the interventional effects of ATRA on GIOP and the mechanisms of ATRA activities, we first performed bioinformatic analysis to identify potential gene targets of ATRA. Zebrafish larvae were recruited as experimental animals, and the frequently used GC, prednisolone, was administered to larvae to construct a GIOP model. We evaluated the influence of exogenous ATRA on the activities of bone metabolic enzymes, the expression of genes linked to osteoblasts and osteoclasts, and the restoration of bone mineral density and bone mass in GIOP zebrafish larvae. Furthermore, we studied the influence of RBM14, a transcriptional coactivator and negative reciprocal factor of ATRA, on the regulation of osteoblastic gene expression during the anti-GIOP process of ATRA using the morpholino knockdown approach. The findings of bone metabolic enzyme activity (alkaline phosphatase, ALP and tartrate-resistant acid phosphatase, TRAP) and expression assays of osteoblastic marker genes (Runx2a, Runx2b, SP7, Csf1a, RANKL, and CTSK) indicated that ATRA had bidirectional effects on osteogenesis. However, in the GIOP model, ATRA reversed the GIOP-induced osteoporosis phenotype by inhibiting the GIOP-induced suppression of osteoblastic metabolic enzyme (ALP) activities and osteoblastic marker gene expression (Runx2a, Runx2b, and SP7), and this antagonism was concentration-dependent. We also observed that ATRA inhibited RBM14 expression in zebrafish larvae, while ATRA alone and RBM14 knockdown showed a consistent induction of osteoblast marker gene expression, implying that ATRA's inhibitory effect on RBM14 expression may underlie ATRA's osteogenic effects. Based on these data, we postulated that ATRA may ameliorate GIOP by decreasing RBM14 expression, thereby enhancing osteoblastic marker gene expression.

Crebanine mitigates glucocorticoid-induced osteonecrosis of the femoral head by restoring bone remodelling homeostasis via attenuating oxidative stress.

The onset of osteonecrosis of the femoral head (ONFH) is intimately associated with the extensive administration of glucocorticoids (GCs). Long-term stimulation of GCs can induce oxidative stress in both osteoclasts (OCs) and osteoblasts (OBs), resulting in the disturbance of bone remodelling. An alkaloid named crebanine (CN) demonstrates pharmacological properties including anti-inflammation and reactive oxygen species (ROS) modulation. Our objective is to assess the therapeutic potential of CN in treating ONFH and elucidate the associated underlying mechanisms. The network pharmacology analysis uncovered that CN played a role in regulating ROS metabolism. Invitro, CN demonstrated its ability to reduce the dexamethasone (DEX)-stimulated generation of OCs and suppress their resorptive function by downregulating the level of osteoclast marker genes. Concurrently, CN also mitigated DEX-induced damage to OBs, facilitating the restoration of osteoblast marker gene expression, cellular differentiation and function. These effects were achieved by CN augmenting the antioxidant system to reduce intracellular ROS levels. Furthermore, invitro results were corroborated by micro-CT and histological data, which also showed that CN attenuated MPS-induced ONFH in mice. This study highlights the therapeutic potential of CN in counteracting GCs-induced ONFH.

Vasorin-deficient mice display disturbed vitamin D and mineral homeostasis in combination with a low bone mass phenotype

Vasorin (Vasn) is a pleiotropic molecule involved in various physiological and pathological conditions, including cancer. Vasn has also been detected in bone cells of developing skeletal tissues but no function for Vasn in bone metabolism has been implicated yet. Therefore, this study aimed to investigate if Vasn plays a significant role in bone biology. First, we investigated tissue distribution of Vasn expression, using lacZ knock-in reporter mice. We detected clear Vasn expression in skeletal elements of postnatal mice. In particular, osteocytes and bone forming osteoblasts showed high expression of Vasn, while the bone marrow was devoid of signal. Vasn knockout mice (Vasn−/−) displayed postnatal growth retardation and died after four weeks. MicroCT analysis of femurs from 22- to 25-day-old Vasn−/−mice demonstrated reduced trabecular and cortical bone volume corresponding to a low bone mass phenotype. Ex vivo bone marrow cultures demonstrated that osteoclast differentiation and activity were not affected by Vasn deficiency. However, osteogenesis of Vasn−/− bone marrow cultures was disturbed, resulting in lower numbers of alkaline phosphate positive colonies, impaired mineralization and lower expression of osteoblast marker genes. In addition to the bone phenotype, these mice developed a vitamin D3-related phenotype with a strongly reduced circulating 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 and urinary loss of vitamin D binding protein. In conclusion, Vasn-deficient mice suffer from severe disturbances in bone metabolism and mineral homeostasis.

Extracellular Histones Promote Calcium Phosphate-Dependent Calcification in Mouse Vascular Smooth Muscle Cells.

Vascular calcification, a major risk factor for cardiovascular events, is associated with a poor prognosis in chronic kidney disease (CKD) patients. This process is often associated with the transformation of vascular smooth muscle cells (VSMCs) into cells with osteoblast-like characteristics. Damage-associated molecular patterns (DAMPs), such as extracellular histones released from damaged or dying cells, are suspected to accumulate at calcification sites. To investigate the potential involvement of DAMPs in vascular calcification, we assessed the impact of externally added histones (extracellular histones) on calcium and inorganic phosphate-induced calcification in mouse VSMCs. Our study found that extracellular histones intensified calcification. We also observed that the histones decreased the expression of VSMC marker genes, while simultaneously increasing the expression of osteoblast marker genes. Additionally, histones treated with DNase I, which degrades dsDNA, attenuated this calcification, compared with the non-treated histones, suggesting a potential involvement of dsDNA in this process. Elevated levels of dsDNA were also detected in the serum of CKD model mice, underlining its potential role in vascular calcification in CKD. Our findings suggest that extracellular histones could play a pivotal role in the vascular calcification observed in CKD.

Titanium micro-nano textured surface with strontium incorporation improves osseointegration: an in vivo and in vitro study.

This study aimed to investigate the osseointegration of titanium (Ti) implants with micro-nano textured surfaces functionalized with strontium additions (Sr) in a pre-clinical rat tibia model. Ti commercially pure (cp-Ti) implants were installed bilaterally in the tibia of 64 Holtzman rats, divided into four experimental groups (n=16/group): (1) Machined surface - control (C); (2) Micro-nano textured surface treatment (MN); (3) Micro-nano textured surface with Sr2+ addition (MNSr); and (4) Micro-nano textured surface with a higher complementary addition of Sr2+ (MNSr+). In total, two experimental euthanasia periods were assessed at 15 and 45 days (n=8/period). The tibia was subjected to micro-computed tomography (μ-CT), histomorphometry with the EXAKT system, removal torque (TR) testing, and gene expression analysis by PCR-Array of 84 osteogenic markers. Gene expression and protein production of bone markers were performed in an in vitro model with MC3T3-E1 cells. The surface characteristics of the implants were evaluated by scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and laser scanning confocal microscopy. SEM, confocal, and EDS analyses demonstrated the formation of uniform micro-nano textured surfaces in the MN group and Sr addition in the MNSr and MNSr+ groups. TR test indicated greater osseointegration in the 45-day period for treated surfaces. Histological analysis highlighted the benefits of the treatments, especially in cortical bone, in which an increase in bone-implant contact was found in groups MN (15 days) and MNSr (45 days) compared to the control group. Gene expression analysis of osteogenic activity markers showed modulation of various osteogenesis-related genes. According to the in vitro model, RT-qPCR and ELISA demonstrated that the treatments favored gene expression and production of osteoblastic differentiation markers. Micro-nano textured surface and Sr addition can effectively improve and accelerate implant osseointegration and is, therefore, an attractive approach to modifying titanium implant surfaces with significant potential in clinical practice.

Bioenergetic profile and redox tone modulate in vitro osteogenesis of human dental pulp stem cells: new perspectives for bone regeneration and repair

BackgroundRedox signaling and energy metabolism are known to be involved in controlling the balance between self-renewal and proliferation/differentiation of stem cells. In this study we investigated metabolic and redox changes occurring during in vitro human dental pulp stem cells (hDPSCs) osteoblastic (OB) differentiation and tested on them the impact of the reactive oxygen species (ROS) signaling.MethodshDPSCs were isolated from dental pulp and subjected to alkaline phosphatase and alizarin red staining, q-RT-PCR, and western blotting analysis of differentiation markers to assess achievement of osteogenic/odontogenic differentiation. Moreover, a combination of metabolic flux analysis and confocal cyto-imaging was used to profile the metabolic phenotype and to evaluate the redox tone of hDPSCs.ResultsIn differentiating hDPSCs we observed the down-regulation of the mitochondrial respiratory chain complexes expression since the early phase of the process, confirmed by metabolic flux analysis, and a reduction of the basal intracellular peroxide level in its later phase. In addition, dampened glycolysis was observed, thereby indicating a lower energy-generating phenotype in differentiating hDPSCs. Treatment with the ROS scavenger Trolox, applied in the early-middle phases of the process, markedly delayed OB differentiation of hDPSCs assessed as ALP activity, Runx2 expression, mineralization capacity, expression of stemness and osteoblast marker genes (Nanog, Lin28, Dspp, Ocn) and activation of ERK1/2. In addition, the antioxidant partly prevented the inhibitory effect on cell metabolism observed following osteogenic induction.ConclusionsAltogether these results provided evidence that redox signaling, likely mediated by peroxide species, influenced the stepwise osteogenic expansion/differentiation of hDPSCs and contributed to shape its accompanying metabolic phenotype changes thus improving their efficiency in bone regeneration and repair.

Sustained release of a highly specific GSK3β inhibitor SB216763 in the PCL scaffold creates an osteogenic niche for osteogenesis, anti-adipogenesis, and potential angiogenesis.

The safe and effective use of Wnt signaling is a hot topic in developing osteogenic drugs. SB216763 (S33) is a widely used highly specific GSK3β inhibitor. Here, we show that S33 initiates canonical Wnt signaling by inhibiting GSK3β activity in the bone marrow stromal cell line ST2 and increases osteoblast marker alkaline phosphatase activity, osteoblast marker gene expression including Alpl, Col1α1, and Runx2, promoting osteogenic differentiation and mineralization of ST2 cells. In addition, S33 suppressed the expression of adipogenic transcription factors Pparg and Cebpa in ST2 cells to suppress adipogenesis. ICRT-14, a specific transcriptional inhibitor of Wnt signaling, reversed the effects of S33 on the differentiation of ST2 cells. S33 also increased the expression of osteoclast cytokines RANKL and Opg but decreased the RANKL/Opg ratio and had the potential to inhibit osteoclast differentiation. In addition, we printed the PSCI3D (polycaprolactone, S33, cell-integrated 3D) scaffolds using a newly established integrated 3D printing system for hard materials and cells. S33 sustained release in the hydrogel of the scaffold with 25.4% release on day 1% and 81.7% release over 7days. Cells in the scaffolds had good cell viability. The ratio of live/dead cells remained above 94% for 7days, while the cells in the scaffolds proliferated linearly, and the proliferative activity of the PSCI3D scaffold group increased 1.4-fold and 1.7-fold on days 4 and 7, respectively. Similarly, in PSCI3D scaffolds, osteogenic differentiation of st2 cells was increased. The alkaline phosphatase activity increased 1.4- and 4.0-fold on days 7 and 14, respectively, and mineralization increased 1.7-fold at 21days. In addition, PSCI3D conditioned medium promoted migration and tubulogenesis of HUVECs, and S33 upregulated the expression of Vegfa, a key factor in angiogenesis. In conclusion, our study suggests that S33 functions in osteogenesis, anti-adipogenesis, and potential inhibition of osteoclast differentiation. And the sustained release of S33 in PSCI3D scaffolds creates a safe osteogenic niche, which promotes cell proliferation, osteogenesis, and angiogenesis and has application prospects.

Absence of TRIC-B from type XIV Osteogenesis Imperfecta osteoblasts alters cell adhesion and mitochondrial function – A multi-omics study

Osteogenesis Imperfecta (OI) is a heritable collagen-related bone dysplasia characterized by bone fractures, growth deficiency and skeletal deformity. Type XIV OI is a recessive OI form caused by null mutations in TMEM38B, which encodes the ER membrane intracellular cation channel TRIC-B. Previously, we showed that absence of TMEM38B alters calcium flux in the ER of OI patient osteoblasts and fibroblasts, which further disrupts collagen synthesis and secretion. How the absence of TMEM38B affects osteoblast function is still poorly understood. Here we further investigated the role of TMEM38B in human osteoblast differentiation and mineralization. TMEM38B-null osteoblasts showed altered expression of osteoblast marker genes and decreased mineralization. RNA-Seq analysis revealed that cell-cell adhesion was one of the most downregulated pathways in TMEM38B-null osteoblasts, with further validation by real-time PCR and Western blot. Gap and tight junction proteins were also decreased by TRIC-B absence, both in patient osteoblasts and in calvarial osteoblasts of Tmem38b-null mice. Disrupted cell adhesion decreased mutant cell proliferation and cell cycle progression. An important novel finding was that TMEM38B-null osteoblasts had elongated mitochondria with altered fusion and fission markers, MFN2 and DRP1. In addition, TMEM38B-null osteoblasts exhibited a significant increase in superoxide production in mitochondria, further supporting mitochondrial dysfunction. Together these results emphasize the novel role of TMEM38B/TRIC-B in osteoblast differentiation, affecting cell-cell adhesion processes, gap and tight junction, proliferation, cell cycle, and mitochondrial function.

Mir-4699 promotes the osteogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are drawing considerable attention in the field of regenerative medicine due to their differentiation capabilities. The miRNAs are among the most important epigenetic regulators of MSC differentiation. Our previous study identified miR-4699 as a direct suppressor of the DKK1 and TNSF11 gene expression. However, the precise osteogenic-related phenotype or mechanism caused by miR-4699 change has yet to be dealt with in depth. In the present study, miR-4699 mimics were transfected into human Adipose tissue-derived mesenchymal stem cells (hAd-MSCs) and osteoblast marker gene expression (RUNX2, ALP, and OCN), was analyzed to investigate whether miR-4699 promotes osteoblast differentiation of hAd-MSCs through targeting the DKK-1 and TNFSF11. We further examined and compared the effects of recombinant human BMP2 with miR-4699 on cell differentiation. In addition to quantitative PCR, analysis of alkaline phosphatase activity, calcium content assay, and Alizarin red staining were used to explore osteogenic differentiation. To evaluate the effect of miR-4699 on its target gene (on protein level) we utilized the western blotting technique. The overexpression of miR-4699 in hAd-MSCs resulted in the stimulation of alkaline phosphatase activity, osteoblast mineralization, and the expression of RUNX2, ALP, and OCN osteoblast marker genes. Our findings indicated that miR-4699 supported and synergized the BMP2-induced osteoblast differentiation of mesenchymal stem cells. We suggest, thereof, the utilization of hsa-miR-4699 for further in vivo experimental investigation to reveal the potential therapeutic impact of regenerative medicine for different types of bone defects.

DKK-1 Is Underexpressed in Mesenchymal Stem Cells from Patients with Ankylosing Spondylitis and Further Downregulated by IL-17

Dickkopf-1 (Dkk-1) is a key regulator of bone remodeling in spondyloarthropathies. Nevertheless, data regarding its expression in cells of pathophysiologic relevance, such as mesenchymal stem cells (MSCs), are lacking. Herein, we aimed to address DKK1 gene expression and Wnt pathway activation in MSCs from patients with ankylosing spondylitis (AS) and explore the effect of IL-17 on MSCs with respect to DKK-1 expression and Wnt pathway activation. Primary MSCs were isolated from the bone marrow of the femoral head of two patients with AS and two healthy controls undergoing orthopedic surgery. MSCs were cultured for 7 days in expansion medium and for 21 days in osteogenic medium in the presence or absence of IL-17A. Gene expression of DKK-1 and osteoblastic markers was determined by RT-PCR. Alkaline phosphatase activity, alizarin red and Van Kossa staining were used to assess osteoblastic function and mineralization capacity. DKK-1 was significantly downregulated in MSCs and osteoblasts from patients with AS compared to controls. Moreover, MSCs and osteoblasts from AS patients displayed increased Wnt pathway activation and enhanced osteoblastic activity, as indicated by increased expression of osteoblast marker genes and alkaline phosphatase activity. IL-17 downregulated DKK-1 expression and increased osteoblastic activity and mineralization capacity. DKK-1 is underexpressed in MSCs from AS patients compared to controls, whereas IL-17 has an inhibitory effect on DKK-1 expression and stimulates osteoblastic function. These data may have pathogenetic and clinical implications in AS.

Network Pharmacology Deciphers the Action of Bioactive Polypeptide in Attenuating Inflammatory Osteolysis via the Suppression of Oxidative Stress and Restoration of Bone Remodeling Balance.

Oxidative stress involves enormously in the development of chronic inflammatory bone disease, wherein the overproduction of reactive oxygen species (ROS) negatively impacts the bone remodeling via promoting osteoclastogenesis and inhibiting osteogenesis. Lacking effective therapies highlights the importance of finding novel treatments. Our previous study screened a novel bioactive peptide D7 and demonstrated it could enhance the cell behaviors and protect bone marrow mesenchymal stem cells (BMSCs). Since BMSCs are progenitor cells of osteoblast (OB), we therefore ask whether D7 could also protect against the progress of inflammatory osteolysis. To validate our hypothesis and elucidate the underlying mechanisms, we first performed network pharmacology-based analysis according to the molecule structure of D7, and then followed by pharmacological evaluation on D7 by in vitro lipopolysaccharide(LPS)-induced models. The result from network pharmacology identified 20 candidate targets of D7 for inflammatory osteolysis intervention. The further analysis of Gene Ontology (GO)/KEGG pathway enrichment suggested the therapeutic effect of D7 may primarily affect osteoclast (OC) differentiation and function during the inflammatory osteolysis. Through validating the real effects of D7 on OC and OB as postulated, results demonstrated suppressive effects of D7 on LPS-stimulated OC differentiation and resorption, via the inhibition on OC marker genes. Contrarily, by improving the expression of OB marker genes, D7 displayed promotive effects on OB differentiation and alleviated LPS-induced osteogenic damage. Further mechanism study revealed that D7 could reduce LPS-induced ROS formation and strengthen antioxidants expressions in both OC and OB precursors, ameliorating LPS-triggered redox imbalance in bone remodeling. Taken together, our findings unveiled therapeutic effects of D7 against LPS-induced inflammatory osteolysis through the suppression of oxidative stress and the restoration of the bone remodeling process, providing a new therapeutic candidate for chronic inflammatory bone diseases.

PCL nanofibrous incorporating unique matrix fusion protein adsorbed mesoporous bioactive glass for bone tissue engineering

Mesoporous bioactive glass (MBG) is a potential biomedical material in bone defect repairment because of its bioactivity, biocompatibility, and osteoinduction properties. Here we report that Mg-doped MBG scaffold with 3:1 Ca/Mg ratio (MBG-Ca/Mg-3) is good for MC3T3-E1 osteoblast differentiation and mineralization. Mimicking bone extracellular matrix structure by electrospinning, we used MBG-Ca/Mg-3 adsorbed with Osteocalcin-Osteopontin-Biglycan (OOB), a new unique matrix fusion protein, to form OOB@MBG-Ca/Mg-3 scaffold, which has multifunctional ability in calvarial bone defect repairment in vivo. Intriguingly, we found that OOB@MBG-Ca/Mg-3 scaffold increases the expression of osteoblastic marker genes, including bone morphogenetic protein (Bmp2), osteopontin (Opn), Osterix, Runx2 through activation of ERK1/2. We concluded that OOB@MBG-Ca/Mg-3 scaffold promotes osteoblast differentiation and mineralization through ERK1/2 pathway and it can also enhance bone formation in vivo, which provides a new biomaterial in bone tissue engineering.

Titanium with nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by regulating histone methylation

The bone remodeling process is crucial for titanium (Ti) osseointegration and involves the crosstalk between osteoclasts and osteoblasts. Considering the high osteogenic potential of Ti with nanotopography (Ti Nano) and that osteoclasts inhibit osteoblast differentiation, we hypothesized that nanotopography attenuate the osteoclast-induced disruption of osteoblast differentiation. Osteoblasts were co-cultured with osteoclasts on Ti Nano and Ti Control and non-co-cultured osteoblasts were used as control. Gene expression analysis using RNAseq showed that osteoclasts downregulated the expression of osteoblast marker genes and upregulated genes related to histone modification and chromatin organization in osteoblasts grown on both Ti surfaces. Osteoclasts also inhibited the mRNA and protein expression of osteoblast markers, and such effect was attenuated by Ti Nano. Also, osteoclasts increased the protein expression of H3K9me2, H3K27me3 and EZH2 in osteoblasts grown on both Ti surfaces. ChIP assay revealed that osteoclasts increased accumulation of H3K27me3 that represses the promoter regions of Runx2 and Alpl in osteoblasts grown on Ti Control, which was reduced by Ti Nano. In conclusion, these data show that despite osteoclast inhibition of osteoblasts grown on both Ti Control and Ti Nano, the nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by preventing the increase of H3K27me3 accumulation that represses the promoter regions of some key osteoblast marker genes. These findings highlight the epigenetic mechanisms triggered by nanotopography to protect osteoblasts from the deleterious effects of osteoclasts, which modulate the process of bone remodeling and may benefit the osseointegration of Ti implants.

Mapping Bone Marrow Cell Response from Senile Female Rats on Ca-P-Doped Titanium Coating.

Chemical and topographical surface modifications on dental implants aim to increase the bone surface contact area of the implant and improve osseointegration. This study analyzed the cellular response of undifferentiated mesenchymal stem cells (MSC), derived from senile rats’ femoral bone marrow, when cultured on a bioactive coating (by plasma electrolytic oxidation, PEO, with Ca2+ and P5+ ions), a sandblasting followed by acid-etching (SLA) surface, and a machined surface (MSU). A total of 102 Ti-6Al-4V discs were divided into three groups (n = 34). The surface chemistry was analyzed by energy dispersive spectroscopy (EDS). Cell viability assay, gene expression of osteoblastic markers, and mineralized matrix formation were investigated. The cell growth and viability results were higher for PEO vs. MSU surface (p = 0.001). An increase in cell proliferation from 3 to 7 days (p < 0.05) and from 7 to 10 days (p < 0.05) was noted for PEO and SLA surfaces. Gene expression for OSX, ALP, BSP, and OPN showed a statistical significance (p = 0.001) among groups. In addition, the PEO surface showed a higher mineralized matrix bone formation (p = 0.003). In conclusion, MSC from senile female rats cultured on SLA and PEO surfaces showed similar cellular responses and should be considered for future clinical investigations.

Inhibition of TET-mediated DNA demethylation suppresses osteoblast differentiation.

DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten-eleven translocation (TET) enzyme family catalyzes the hydroxymethylation and subsequent demethylation of DNA by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Little is known about TET protein function due to a lack of pharmacological tools to manipulate DNA hydroxymethylation levels. In this study, we examined the role of TET-mediated DNA hydroxymethylation during BMP-induced C2C12 osteoblast differentiation using a novel cytosine-based selective TET enzyme inhibitor, Bobcat339 (BC339). Treatment of C2C12 cells with BC339 increased global 5mC and decreased global 5hmC without adversely affecting cell viability, proliferation, or apoptosis. Furthermore, BC339 treatment inhibited osteoblast marker gene expression and decreased alkaline phosphatase activity during differentiation. Methylated DNA immunoprecipitation and bisulfite sequencing showed that inhibition of TET with BC339led to increased 5mC at specific CpG-rich regions at the promoter of Sp7, a key osteoblast transcription factor. Consistent with promoter 5mC marks being associated with transcriptional repression, luciferase activity of an Sp7-promoter-reporter construct was repressed by in vitro DNA methylation or BC339. Chromatin immunoprecipitation analysis confirmed that TET2 does indeed occupy the promoter region of Sp7. Accordingly, forced overexpression of SP7 rescued the inhibition of osteogenic differentiation by BC339. In conclusion, our data suggest that TET-mediated DNA demethylation of genomic regions, including the Sp7 promoter, plays a role in the initiation of osteoblast differentiation. Furthermore, BC339 is a novel pharmacological tool for the modulation of DNA methylation dynamics for research and therapeutic applications.

UPLC-ESI-Q-TOF-MSE-based metabolomics analysis of Acer mono sap and evaluation of osteogenic activity in mouse osteoblast cells.

Investigation of phytochemicals and bioactive molecules is tremendously vital for the applications of new plant resources in chemistry, food, and medicine. In this study, the chemical profiling of sap of Acer mono (SAM), a Korean syrup known for its anti-osteoporosis effect, was performed using UPLC-ESI-Q-TOF-MSE analysis. A total of 23 compounds were identified based on the mass and fragmentation characteristics and most of the compounds have significant biomedical applications. The in vitro antioxidant assessment of SAM indicated excellent activity by scavenging DPPH and ABTS-free radicals and were found to be 23.35 mg mL-1 and 29.33 mg mL-1, respectively, as IC50 concentrations. As well, the in vitro proliferation effect of the SAM was assessed against mouse MC3T3-E1 cells, and the results showed that the SAM enhanced the proliferation of the cells, and 12.5 mg mL-1 and 25 mg mL-1 of SAM were selected for osteogenic differentiation. The morphological analysis clearly evidenced the SAM enhanced the osteogenic activity in MC3T3-E1 cells by the increased deposition of extracellular calcium and nodule formation. Moreover, the qRT-PCR analysis confirmed the increased expression of osteoblast marker gene expression including ALP, osteocalcin, osteopontin, collagen1α1, Runx2, and osterix in SAM-treated MC3T3-E1 cells. Together, these results suggest that SAM possesses osteogenic effects and can be used for bone regeneration and bone loss-associated diseases such as osteoporosis.

Effects of the Lysine Methyltransferase Inhibitor AZ505 on Bone Metabolism.

BackgroundProtein methylation has important role in regulating diverse cellular responses, including differentiation, by affecting protein activity, stability, and interactions. AZ505 is an inhibitor of the SET and MYND domain-containing protein 2 lysine methylase. In this study, we investigated the effect of AZ505 on osteoblast and osteoclast differentiation in vitro and evaluated the effect of AZ505 in vivo on the long bones in mice.MethodsOsteoblast differentiation was assessed by alkaline phosphatase (ALP) and Alizarin red staining after culturing calvarial preosteoblasts in an osteogenic medium. Osteoclast differentiation was analyzed by tartrate-resistant acid phosphatase (TRAP) staining in bone marrow-derived macrophages cultured with macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). For in vivo experiments, mice were intraperitoneally injected with AZ505 and femurs were examined by micro-computed tomography.ResultsAZ505 increased ALP and Alizarin red staining in cultured osteoblasts and the expression of osteoblast marker genes, including Runx2 and osteocalcin. AZ505 resulted in decreased TRAP-staining of osteoclasts and expression of c-Fos and nuclear factor of activated T cells transcription factors and osteoclast marker genes, including cathepsin K and dendritic cell-specific transmembrane protein. Unexpectedly, in vivo administration of AZ505 markedly decreased the trabecular bone mass of femurs. In support of this catabolic result, AZ505 strongly upregulated RANKL expression in osteoblasts.ConclusionsThe results indicate that AZ505 has a catabolic effect on bone metabolism in vivo despite its anabolic effect in bone cell cultures. The findings indicate that cell culture data should be extrapolated cautiously to in vivo outcomes for studying bone metabolism.

Milk peptide induced miRNA-369 facilitates bone functions in the rat osteoblasts

The animal’s health and productivity is chiefly influenced by its anatomy and physiology. Moreover, bone growth in animals is also associated to overall health and production. Besides, bone fractures accounts to constrained food intake, reduced immune function and general productivity losses. In poultry animals, bone deformities, renders severe costs in the meat production and egg quality. The treatment for various bone related issues is both time and capital seeking. Besides, currently available therapies, i.e. bisphosphonates, raloxifene are associated with various side effects like jaw osteonecrosis, vaginal bleeding etc. The present study reports the bone promoting effect in milk peptide induced miRNA, miR369. Findings represents an enhanced bone proliferation and differentiation under the influence of miR369 in in vitro rat osteoblasts culture. The bone marker enzyme alkaline phosphatase and osteoblast specific protein expression were tested using ELISA. miR369 significantly stimulated the marker protein levels. Additionally, miR369 when injected to neonatal rat pups, showed improved osteoblast marker gene(s) expression. The study demonstrates the positive role of miR369 in the bone functions and hence, advocates its functional utility for the management of various bone ailments prevailing in animals and/or humans. Besides miR369, a putative molecular gene therapeutic agent can prove revolutionary in controlling the animal health losses ascribed to deteriorating bone health.

Four and a Half LIM Domains Protein 2 Mediates Bortezomib-Induced Osteogenic Differentiation of Mesenchymal Stem Cells in Multiple Myeloma Through p53 Signaling and β-Catenin Nuclear Enrichment.

Myeloma bone disease (MBD), caused by the inhibition of osteoblast activity and the activation of osteoclast in the bone marrow environment, is the most frequent and life-threatening complication in multiple myeloma (MM) patients. Bortezomib (Bzb) was shown to promote MM-derived mesenchymal stem cells (MM-MSCs) differentiation to osteoblast in vitro and in animal models, promoting the bone formation and regeneration, may be mediated via β-catenin/T-cell factor (TCF) pathway. Further defining molecular mechanism of Bzb-enhanced bone formation in MM will be beneficial for the treatment of myeloma patients. The present study has identified for the first time four and a half LIM domains protein 2 (FHL2), a tissue-specific coregulator that interacts with many osteogenic marker molecules, as a therapeutic target to ameliorate MM bone disease. First, increased messenger RNA (mRNA) and protein levels of FHL2, and the mRNA level of main osteoblast markers (including Runx2, ALP, and Col1A1), were found in MM-patients-derived MSCs after Bzb treatment. FHL2 KD with short hairpin RNA (shRNA) reduced the expression of osteoblast marker genes and blocked the osteogenic differentiation of MM-MSCs regardless of the presence or absence of Bzb, implying that FHL2 is an important activator of the osteogenic differentiation of human MSCs under a proteasome inhibition condition. Molecular analysis showed that the enhanced expression of FHL2 was associated with the Bzb-induced upregulation of p53. No significant change at protein level of total β-catenin was observed with or without Bzb treatment. However, it was mostly enriched to nuclei in MSCs after Bzb treatment. Moreover, β-catenin was restricted to the perinuclear region in FHL2 KD cells. These data provide evidence that FHL2 is essential for promoting β-catenin nuclear enrichment in MM-MSCs. In conclusion, FHL2 is critical for Bzb-induced osteoblast differentiation of MM-MSCs and promotes the osteogenesis, through p53 signaling and β-catenin activation. Targeting FHL2 in MM may provide a new therapeutic strategy for treating MBD.