Osteoblast Mineralization Research Articles

Ferric ammonium citrate (FAC)-induced inhibition of osteoblast proliferation/differentiation and its reversal by soybean-derived peptides (SDP)

Ferric citrate has been used to treat hyperphosphatemia, a prevalent symptom in patients with chronic kidney disease while ferric ammonium citrate (FAC), a more dissolvable format, is widely used as food additive. However, excess iron is associated with osteoporosis. Dietary soybean products have been shown to prevent the progression of osteoporosis. In this study, a group of peptides, referred as P3, was identified from the enzymolysis of soybean protein isolates, and its biological functions were investigated. The results showed that MC3T3-E1 cell cycle progression from G0/G1 to S phase was accelerated by P3 treatment. MC3T3-E1 cell proliferation was enhanced by P3 via ERK1/2 activation. Importantly, P3 treatment abolished the antiproliferative effect of FAC on MC3T3-E1 cell. In addition, P3 treatment increased the expression of ALP, COL-1, OCN, consequently promoting the differentiation and mineralization of MC3T3-E1 cells via activation of p38 MAPK pathway. Consequently, P3 treatment was able to reverse the inhibitory effect of FAC on osteoblasts differentiation and mineralization. Our findings suggest P3, as a dietary supplement, has a potential therapeutic function to attenuate the adverse effects of FAC on bone metabolism and to prevent osteoporosis progression.

Chicago sky blue 6B (CSB6B), an allosteric inhibitor of macrophage migration inhibitory factor (MIF), suppresses osteoclastogenesis and promotes osteogenesis through the inhibition of the NF-κB signaling pathway

Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory mediator involved in various pathophysiological and inflammatory states. Accumulating line of evidence suggests a role for MIF in regulating bone metabolism and therefore a prime candidate for therapeutic targeting. In this study, we showed that Chicago sky blue 6B (CSB6B) suppresses RANKL-induced osteoclast and bone resorption in vitro via the inhibition of NF-κB signaling activation and promoting proteasome-mediated degradation of MIF. Consequently, the induction of NFATc1 was impaired resulting in downregulation of NFATc1-responsive osteoclast genes. We also demonstrated that CSB6B treatment enhanced primary calvarial osteoblast differentiation and bone mineralization in vitro via the suppression of NF-κB activation and upregulation of Runx expression. Using two murine models of osteolytic bone disorders, we further showed that administration of CSB6B protected mice against pathological inflammatoryc calvarial bone destruction induced by titanium particles mice as well as estrogen-deficiency induced bone loss as a result of ovariectomy. Together, as an MIF inhibitor, CSB6B can inhibit osteoclast differentiation and bone resorption function and enhance the mineralization of osteoblasts through the inhibition of NF-κB pathway. MIF is a prime target for therapeutic targeting for the treatment of osteolytic bone disorders and the MIF inhibitor CSB6B could be potential anti-osteoporosis drug.

Effect of TiO2 nanotubes size, heat treatment, and UV irradiation on osteoblast behavior

Titanium implants have been extensively used in orthopedic and dental applications due to their high performance at the biomechanical level and their excellent biocompatibility. Currently, in bone implants, the design of surfaces is used to address a biological behavior that includes the improvement of osseointegration or the immobilization of active molecules. This paper aimed to describe the effect of the nanotube characteristics on the adhesion, proliferation, and mineralization of osteoblasts. To the best of our knowledge, the effect of reduced internal diameters (≈10 nm) with and without UV irradiation and with and without heat treatment on osteoblast adhesion, proliferation, and mineralization has not been reported. All the anodic coatings were biocompatible; however, surface characteristics such as topography, wettability, charge, and phase composition could affect osteoblast adhesion, proliferation, and mineralization. Nanotubular coatings with higher roughness and UV irradiation improved early cell adhesion; however, the nanotubes with a diameter of about 10 nm and composed entirely by rutile or anatase phases negatively affected early cell adhesion. The number of filopodia rises with the reduction in the internal diameter of the porous anodic coating, and the mineralization had the same behavior, namely: a decrease of the internal diameter induced a superior rate of matrix mineralization.

Proteolytic processing of secretory pathway kinase Fam20C by site-1 protease promotes biomineralization

Family with sequence similarity 20C (Fam20C), the major protein kinase in the secretory pathway, generates the vast majority of the secreted phosphoproteome. However, the regulatory mechanisms of Fam20C transport, secretion, and function remain largely unexplored. Here, we show that Fam20C exists as a type II transmembrane protein within the secretory compartments, with its N-terminal signal peptide-like region serving as a membrane anchor for Golgi retention. The secretion and kinase activity of Fam20C are governed by site-1 protease (S1P), a key regulator of cholesterol homeostasis. We find that only mature Fam20C processed by S1P functions in osteoblast differentiation and mineralization. Together, our findings reveal a unique mechanism for Fam20C secretion and activation via proteolytic regulation, providing a molecular link between biomineralization and lipid metabolism.

IL-17F promotes osteoblastic osteogenesis via the MAPK/ERK1/2 signaling pathway.

Osteoimmunology is a field that focuses on the interactions between the skeletal and immune systems, and has become a focus of research over the years. The role of interleukin (IL)-17F, a proinflammatory cytokine, in bone regeneration and its signal transduction are not completely understood. The aim of the present study was to evaluate the function of IL-17F and the possible mechanisms underlying IL-17F in osteoblasts in vitro. Osteoblasts derived from newborn rats were treated with various concentrations of IL-17F. The pro-osteogenic effects of IL-17F were assessed at the cellular and molecular level. The results demonstrated that IL-17F promoted osteoblast proliferation, differentiation and mineralization. Reverse transcription-quantitative PCR and western blotting indicated that IL-17F treatment upregulated osteogenesis-related factors, including bone morphogenetic protein-2, Runt-related transcription factor-2 (Runx2) and Osterix, and downregulated Noggin compared with the control group. Subsequently, whether the IL-17F receptors, IL-17 receptor (IL-17R) A and IL-17RC, served a role in the effects of IL-17F on osteoblasts was investigated. The mRNA expression levels of IL-17RA and IL-17RC were upregulated in IL-17F-treated osteoblasts compared with control osteoblasts. Furthermore, U0126, a MAPK/ERK1/2 inhibitor, was utilized to investigate the mechanisms underlying IL-17F. The results indicated that compared with the control group, IL-17F increased the protein expression of phosphorylated-ERK1/2, Runx2 and Osterix, whereas U0126 reversed IL-17F-mediated effects. Collectively, the results of the present study suggested that IL-17F promoted osteoblastic osteogenesis via the MAPK/ERK1/2-mediated signaling pathway. IL-17F promoted osteogenesis, including proliferation, differentiation and mineralization activity, indicating that IL-17F may serve as a potential therapeutic target for osteoblast-mediated bone loss disease.

Multi-element processed pyritum mixed to β-tricalcium phosphate to obtain a 3D-printed porous scaffold: An option for treatment of bone defects

Bone defects remain a challenging problem for doctors and patients in clinical practice. Processed pyritum is a traditional Chinese medicine that is often used to clinically treat bone fractures. It contains mainly Fe, Zn, Cu, Mn, and other elements. In this study, we added the extract of processed pyritum to β-tricalcium phosphate and produced a porous composite TPP (TCP/processed pyritum) scaffold using digital light processing (DLP) 3D printing technology. Scanning electron microscopy (SEM) analysis revealed that TPP scaffolds contained interconnected pore structures. When compared with TCP scaffolds (1.35 ± 0.15 MPa), TPP scaffolds (5.50 ± 0.24 MPa) have stronger mechanical strength and can effectively induce osteoblast proliferation, differentiation, and mineralization in vitro. Meanwhile, the in vivo study showed that the TPP scaffold had better osteogenic capacity than the TCP scaffold. Furthermore, the TPP scaffold had good biosafety after implantation. In summary, the TPP scaffold is a promising biomaterial for the clinical treatment of bone defects.

The Role of BMP Signaling in Osteoclast Regulation.

The osteogenic effects of Bone Morphogenetic Proteins (BMPs) were delineated in 1965 when Urist et al. showed that BMPs could induce ectopic bone formation. In subsequent decades, the effects of BMPs on bone formation and maintenance were established. BMPs induce proliferation in osteoprogenitor cells and increase mineralization activity in osteoblasts. The role of BMPs in bone homeostasis and repair led to the approval of BMP2 by the Federal Drug Administration (FDA) for anterior lumbar interbody fusion (ALIF) to increase the bone formation in the treated area. However, the use of BMP2 for treatment of degenerative bone diseases such as osteoporosis is still uncertain as patients treated with BMP2 results in the stimulation of not only osteoblast mineralization, but also osteoclast absorption, leading to early bone graft subsidence. The increase in absorption activity is the result of direct stimulation of osteoclasts by BMP2 working synergistically with the RANK signaling pathway. The dual effect of BMPs on bone resorption and mineralization highlights the essential role of BMP-signaling in bone homeostasis, making it a putative therapeutic target for diseases like osteoporosis. Before the BMP pathway can be utilized in the treatment of osteoporosis a better understanding of how BMP-signaling regulates osteoclasts must be established.

Factor VIII Plays a Direct Role in Osteoblast Development

Our laboratory has previously reported that Factor VIII (FVIII)-deficient mice have decreased skeletal health compared to their wild-type littermates. We also demonstrated a defect in growth and differentiation of osteoblasts derived from primary mesenchymal cells (MSCs) from FVIII-deficient mice. Osteoblasts, cells that build bone, are critical in bone remodeling along with osteoclasts, cells that resorb bone. Decreased osteoblast activity can explain our observations, but not the mechanism of how FVIII is involved. To further understand how FVIII affects osteoblasts in vivo, FVIII-deficient mice were given factor replacement before cells were isolated and cultured.Methods:10-12 week old FVIII-deficient male mice were given 100 U/kg doses of FVIII replacement at 24 and 4 hours before being euthanized and marrow isolated for MSCs. In addition, MSCs were isolated from wild-type and FVIII-deficient male mice without treatment. Cells were then plated in 6-well plates at 2.0x106 cells/cm2 in αMEM +1% antibiotic/antimycotic +10% FBS + 50 ug/ml ascorbic acid and 10mM β-glycerophosphate added to differentiate pre-osteoblasts into osteoblasts. Cells were cultured for 15-20 days at 37˚C with 5.0% CO2 with media collected every 3-4 days and replaced with fresh differentiation media. Media is collected and stored at -80°C for analysis on alkaline phosphatase activity in the cultures. Alkaline phosphatase activity was determined by the release of para-nitrophenol from para-nitrophenyl-phosphate spectrophotometrically. Mineralized osteoblast cultures are fixed and stained with a 4mM Alizarin Sodium Sulfonate which binds to calcium incorporated in the mineralized bone matrix. Reverse transcription-polymerase chain reaction (RT-PCR) is run on cDNA made from osteoblast RNA to look at markers of osteoblast function and signaling.Results:Replacing FVIII in FVIII-deficient mice restores osteoblast function and/or mineralization as seen in Figure 1. As alkaline phosphatase activity correlates to maturing osteoblasts, we see no differences at 5 and 8 days in culture; however, after 12 and 15 days of culture, FVIII-deficient cultures have more than 60% reduced alkaline phosphatase activity when compared to wild-type as well as the FVIII-deficient with FVIII replacement (p<0.05). There is no significant difference between the wild-type and the FVIII replacement cell culture in terms of alkaline phosphatase activity indicating FVIII treatment is important to osteoblast maturation and bone mineralization. Several bone markers were analyzed using RT-PCR, with the most significant differences seen in receptor activator of nuclear factor kappa-B ligand (RANKL) which is reduced in the FVIII-deficient osteoblast cultures that received FVIII replacement by 50% when compared to wild-type (p<0.03) and by 25% compared to FVIII with no treatment (p<0.003). RANKL is part of the signaling pathway between osteoblasts and osteoclast to activate osteoclast activity and is integral to bone remodeling and resorption.Conclusion:These results suggest that FVIII plays a direct role in osteoblast devlopment and help explain the mechanism of bone disease associated with hemophilia A.Figure 1: Alizarin red stains for calcium deposits in cultured wild-type osteoblasts (Left), FVIII-deficient osteoblasts (Middle), FVIII-deficient osteoblasts with in vivo FVIII replacement (Right). [Display omitted] DisclosuresTaylor:Bioverativ: Consultancy, Research Funding; Janssen: Research Funding; Novo Nordisk: Research Funding; CSL Behring: Consultancy, Research Funding; Shire: Research Funding.

Electrically facilitated mineralization of osteoblasts and polypyrrole micro-bowl coatings for promotion of the osteogenic activity

To improve the osseointegration of metallic implants, micro-bowl polypyrrole (PPY) coatings are fabricated electrochemically on substrates in emulsion electrolyte. Phytic acid is adopted as dopants which plays a dominate role for the morphology, wettability and conductivity of PPY. By virtue of the micro-bowl structures, PPY coatings possess promoted adhesion of MC3T3-E1 cells. Besides, the PPY coating, together with external electrical stimulation (ES) facilitate osteogenetic differentiation of cells in a synergistic way. After 12 days incubation, alkaline phosphatase (ALP) activity and extracellular calcium contents of cells show 3 and 2-fold increases compared with bare substrates, respectively. In addition, in-vitro mineralization of calcium phosphates (CaP) on PPY coating is also accelerated in physiological conditions through ES. This work not only utilizes conducting polymer and ES to promote intracellular mineralization but also promotes CaP deposition on polymer for the first time, which might have potential applications in bone regeneration and repair.

Epigallocatechin gallate regulates inflammatory responses and new bone formation through Wnt/β-Catenin/COX-2 pathway in spondyloarthritis

ObjectiveSpondyloarthritis (SpA) is mainly characterized by bone erosion, new bone formation, inflammation and potential disability. Epigallocatechin gallate (EGCG) has been proved to be closely related with the regulation of inflammation and bone metabolism. However, whether EGCG could improve SpA remains unclear. MethodsSpA animal model was established using proteoglycan. Cell proliferation were measured by CCK-8 assay. The mRNA expression levels of genes were detected using qRT-PCR, protein levels were assessed via western blotting and immunohistochemistry. ELISA assay was performed to examined the inflammatory cytokine release. Lesions in spine cartilage tissues were observed using hematoxylin-eosin (HE) and Safranin O staining. Alkaline phosphatase (ALP) assay and Alizarin Red S staining was used to investigate osteoblast mineralization. ResultsWe found that EGCG could inhibit inflammation and new bone formation in SpA mice. Besides, inflammatory factor expression and osteogenic differentiation in osteoblasts isolated from SpA mice were also decreased by EGCG. Further, EGCG treatment suppressed the activation of Wnt/β-Catenin/COX-2 pathway and the activator of this pathway partially reversed the effects of EGCG on inflammation and osteoblast differentiation. ConclusionsEGCG repressed inflammatory responses and new bone formation, and further improved SpA through Wnt/β-Catenin/COX-2 pathway. Our findings may provide a new thought for the prevention and treatment of SpA.

The Effect of Wi-Fi Radiation on the Mineralization and Oxidative Stress of Osteoblasts

Some articles reported that Wi-Fi radiation induced oxidative stress (OS) in cells and vital organs. However, the possible effects of Wi-Fi electromagnetic fields (EMFs) on bone cells have not yet been investigated. MC3T3-E1 cells were cultured in cell incubators during induced differentiation and placed 3 cm from Wi-Fi antenna. A 2.45-GHz Wi-Fi signal, transmitted between a Wi-Fi router and a laptop Wi-Fi antenna, radiated on cells for 30 min/day over a 7-day period. The two modes of the Wi-Fi irradiation were 100 mW and 500 mW. The specific absorption rates (SARs) in the cell layer by 100 mW and 500 mW Wi-Fi were 0.1671 W/kg and 0.8356 W/kg, respectively, represented as SARa and SARb, and the cell layer temperature increased by 0.065°C and 0.32°C, respectively, after 30 min of irradiation by finite difference-time domain (FDTD) simulation. The cell oxidative stress indexes were measured by a microplate reader, and the calcified nodules were examined by alizarin red S staining. At a 3-cm close range, 2.45-GHz Wi-Fi radiation increased Reactive oxygen species (ROS) and glutathione (GSH) levels in osteoblasts with the increase of irradiation time, and the quantity of mineralization slightly depended on the radiation intensity.

Aesculetin Promotes Osteoblastogenic Bone Formation Through Enhancing Osteoblast Differentiation and Mineralization

Abstract Objectives Osteoporosis is a common chronic disease elicited by imbalance between osteoblastic bone formation and osteoclastic bone resorption. Marked increase in bone resorption leads to the aberrant fall in bone mineral density. With increasing age, there is also a significant reduction in bone formation. Aesculetin, a derivative of coumarin, possesses anti-inflammatory and antioxidant effects. The purpose of this study was to identify that aesculetin accelerated bone formation through increased osteoblastic differentiation and mineralization. Methods MC3T3-E1 cells were cultured with 1–10 μM aesculetin in α-MEM supplemented with 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid and 10 μM dexamethasone for up to 21 days. Alkaline phosphatase (ALP) activity and staining, Alizarin red S staining, and Western blotting for induction of target proteins were conducted for the measurement of osteoblastic differentiation and mineralization. Results Aesculetin further enhanced the ALP activity of differentiated MC3T3-E1 cells, showing that aesculetin stimulated the osteoblast differentiation. Alizarin red S staining revealed that calcium deposits highly increased in 1–10 μM aesculetin-treated osteoblasts. In addition, aesculetin further increased cellular expression of the bone-forming markers of bone morphogenetic protein-2, osteopontin and collagen type I in osteoblasts. Conclusions Aesculetin was effective in enhancing osteoblast differentiation and bone mineralization for bone formation, indicating that this compound may be a potential agent for the treatment of osteoporosis. Funding Sources This work was supported by the BK21 FOUR(Fostering Outstanding Universities for Research, 4220200913807) funded by the National Research Foundation of Korea (NRF).

Plant-derived soybean peroxidase stimulates osteoblast collagen biosynthesis, matrix mineralization, and accelerates bone regeneration in a sheep model

Bone defects arising from fractures or disease represent a significant problem for surgeons to manage and are a substantial economic burden on the healthcare economy. Recent advances in the development of biomaterial substitutes provides an attractive alternative to the current “gold standard” autologous bone grafting. Despite on-going research, we are yet to identify cost effective biocompatible, osteo-inductive factors that stimulate controlled, accelerated bone regeneration.We have recently reported that enzymes with peroxidase activity possess previously unrecognised roles in extracellular matrix biosynthesis, angiogenesis and osteoclastogenesis, which are essential processes in bone remodelling and repair. Here, we report for the first time, that plant-derived soybean peroxidase (SBP) possesses pro-osteogenic ability by promoting collagen I biosynthesis and matrix mineralization of human osteoblasts in vitro. Mechanistically, SBP regulates osteogenic genes responsible for inflammation, extracellular matrix remodelling and ossification, which are necessary for normal bone healing. Furthermore, SBP was shown to have osteo-inductive properties, that when combined with commercially available biphasic calcium phosphate (BCP) granules can accelerate bone repair in a critical size long bone defect ovine model. Micro-CT analysis showed that SBP when combined with commercially available biphasic calcium phosphate (BCP) granules significantly increased bone formation within the defects as early as 4 weeks compared to BCP alone. Histomorphometric assessment demonstrated accelerated bone formation prominent at the defect margins and surrounding individual BCP granules, with evidence of intramembranous ossification. These results highlight the capacity of SBP to be an effective regulator of osteoblastic function and may be beneficial as a new and cost effective osteo-inductive agent to accelerate repair of large bone defects.

Comparative evaluation of surface-modified zirconia for the growth of bone cells and early osseointegration

Statement of problemRapid osseointegration between implant and bone tissue for early loading of a prosthesis with sufficient primary stability depends on the surface characteristics of the implant. The development and characterization of suitable surface coatings on dental implants is a major challenge. PurposeThe purpose of this in vitro study was to evaluate and compare the osteogenic potential and cytotoxicity of unmodified zirconia, acid-etched zirconia, bioactive glass–coated zirconia, and tamarind kernel polysaccharide with hydrophilic acrylic acid (TKP-AA) hydrogel-coated zirconia. Material and methodsThirty-six disks each of unmodified zirconia, acid-etched, 45S5 bioactive glass–coated, and TKP-AA hydrogel-coated zirconia were evaluated for osteogenic potential and cytotoxic effect by using human osteoblast Saos-2 cells. The surface topography of the disks and the morphology of the cells grown on these surfaces were examined by scanning electron microscopy (n=3). The cell attachment was evaluated by confocal imaging (n=3). The cytotoxic effect was evaluated by cell viability assay (n=9). Osteoblast maturation was assessed by alkaline phosphatase assay (n=9) and cell mineralization by alizarin red staining (n=9). ANOVA and Bonferroni multiple comparison post hoc tests were used to evaluate the statistical significance of the intergroup differences in these characteristics (α=.05). ResultsThe surface modifications resulted in distinct changes in the surface morphology of zirconia disks and the growth of Saos-2 cells. Zirconia disks coated with TKP-AA promoted higher proliferation of osteoblasts compared with unmodified disks (P<.001). Similarly, the surface modifications significantly increased the differentiation of mesenchymal stem cells to osteoblasts as compared with uncoated zirconia (P<.001). However, the rate of differentiation to osteoblasts was similar among the surface modifications. Acid-etched and TKP-AA-coated disks promoted mineralization of osteoblasts to the same extent, except bioactive glass coating, which significantly increased the rate of mineralization (P<.001). ConclusionsSurface modification of zirconia by acid etching and coating with Bioglass or TKP-AA hydrogel resulted in the improved growth and differentiation of osteoblasts. TKP-AA hydrogel coating promoted the proliferation of osteoblasts, whereas Bioglass coating showed better mineralization. TKP-AA hydrogel coating is a promising candidate for improving the osseointegration of dental implants that warrants further investigation.

The Clinical Application of Porous Tantalum and Its New Development for Bone Tissue Engineering.

Porous tantalum (Ta) is a promising biomaterial and has been applied in orthopedics and dentistry for nearly two decades. The high porosity and interconnected pore structure of porous Ta promise fine bone ingrowth and new bone formation within the inner space, which further guarantee rapid osteointegration and bone–implant stability in the long term. Porous Ta has high wettability and surface energy that can facilitate adherence, proliferation and mineralization of osteoblasts. Meanwhile, the low elastic modulus and high friction coefficient of porous Ta allow it to effectively avoid the stress shield effect, minimize marginal bone loss and ensure primary stability. Accordingly, the satisfactory clinical application of porous Ta-based implants or prostheses is mainly derived from its excellent biological and mechanical properties. With the advent of additive manufacturing, personalized porous Ta-based implants or prostheses have shown their clinical value in the treatment of individual patients who need specially designed implants or prosthesis. In addition, many modification methods have been introduced to enhance the bioactivity and antibacterial property of porous Ta with promising in vitro and in vivo research results. In any case, choosing suitable patients is of great importance to guarantee surgical success after porous Ta insertion.

Core-binding factor beta is required for osteoblast differentiation during fibula fracture healing

BackgroundGrowing evidence has implicated core-binding factor beta (Cbfb) as a contributor to osteoblast differentiation, which plays a key role in fracture healing. Herein, we aimed to assess whether Cbfb affects osteoblast differentiation after fibula fracture.MethodsInitially, we established a Cbfb conditional knockout mouse model for subsequent studies. Immunohistochemical staining was conducted to detect the expression of proliferating cell nuclear antigen (PCNA) and collagen II in the fracture end. Next, we isolated and cultured osteoblasts from specific Cbfb conditional knockout mice for BrdU analysis, alkaline phosphatase (ALP) staining, and von Kossa staining to detect osteoblast viability, differentiation, and mineralization, respectively. Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect the expression of osteoblast differentiation-related genes.ResultsThe Cbfb conditional knockout mice exhibited downregulated expression of PCNA and collagen II, reduced ALP activity, and mineralization, as well as diminished expression of osteoblast differentiation-related genes. Further, Cbfb knockout exerted no obvious effects on osteoblast proliferation.ConclusionsOverall, these results substantiated that Cbfb could promote fibula fracture healing and osteoblast differentiation and thus provided a promising therapeutic target for clinical treatment of fibula fracture.

Effects of fluid flow shear stress to mouse muscle cells on the bone actions of muscle cell-derived extracellular vesicless.

The interactions between skeletal muscle and bone have been recently noted, and muscle-derived humoral factors related to bone metabolism play crucial roles in the muscle/bone relationships. We previously reported that extracellular vesicles from mouse muscle C2C12 cells (Myo-EVs) suppress osteoclast formation in mice. Although mechanical stress is included in extrinsic factors which are important for both muscle and bone, the detailed roles of mechanical stress in the muscle/bone interactions have still remained unknown. In present study, we examined the effects of fluid flow shear stress (FFSS) to C2C12 cells on the physiological actions of muscle cell-derived EV. Applying FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in mouse bone marrow cells in the presence of receptor activator nuclear factor κB ligand (RANKL). Moreover, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed mitochondria biogenesis genes during osteoclast formation with RANKL treatment. In addition, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in Raw264.7 cells in the presence of RANKL. Small RNA-seq-analysis showed that FFSS elevated the expression of miR196a-5p and miR155-5p with the suppressive actions of osteoclast formation and low expression in mouse bone cells. On the other hand, muscle cell-derived EVs with or without FFSS to C2C12 cells did not affect the expression of osteogenic genes, alkaline phosphatase activity and mineralization in mouse osteoblasts. In conclusion, we first showed that FFSS to C2C12 cells enhances the suppressive effects of muscle cell-derived EVs on osteoclast formation in mouse cells. Muscle cell-derived EVs might be partly involved in the effects of mechanical stress on the muscle/bone relationships.

Mastocytosis-derived extracellular vesicles deliver miR-23a and miR-30a into pre-osteoblasts and prevent osteoblastogenesis and bone formation

Osteoporosis and other manifestations of bone disease are frequent in patients with systemic mastocytosis (SM) in association with the presence of mast cell infiltrates in bone marrow, although the mechanisms behind bone disease remain poorly understood. We find that extracellular vesicles (EVs) released by neoplastic mast cells and present in the serum of patients with SM (SM-EVs) block osteoblast differentiation and mineralization in culture, and when injected into mice diminish the expression of osteoblast markers, and trabecular bone volume and microarchitecture. We demonstrate that miRNA-30a and miRNA-23a, increased in SM-EVs and neoplastic mast cell-derived EVs, attenuate osteoblast maturation by suppressing expression of RUNX2 and SMAD1/5, essential drivers of osteogenesis. Thus, SM-EVs carry and deliver miRNAs that epigenetically interfere with bone formation and can contribute to bone mass reduction in SM. These findings also suggest possibilities for novel approaches to the management of bone disease in mast cell proliferative disorders.

Timing of geranylgeraniol addition increases osteoblast activities under alendronate condition

BackgroundAlendronate (ALN), a nitrogen-containing bisphosphonate, is prescribed to treat bone diseases. ALN acts as an inhibitor of enzymes in the mevalonate pathway, which results in reducing osteoblast viability and mineralization. Geranylgeraniol (GGOH) is a substrate in mevalonate pathway and mediates protein prenylation in the cells. ObjectiveTo investigate the effects of GGOH on ALN-treated osteoblast activities in order to improve the application of GGOH. MethodsMC3T3 cells were treated with ALN. GGOH were added at different time points. Cell activities were examined using alizarin red S, MTT assay, alkaline phosphatase (ALP) activity, and quantitative polymerase chain reaction. ResultsALN decreased mineralization. In the presence of ALN, GGOH addition at the first week of culture increased mineralization compared with the addition at other time points. ALN treatment for 7 days caused a reduction in osteoblast and pre-osteoblast viability compared with untreated cells. GGOH supplement partially rescued cell viability and increased total protein in cells treated with ALN. Furthermore, GGOH significantly upregulated gene expressions of Col I, OPN, VEGF, and VEGFR2. ConclusionGGOH could be best applied at the early stage of osteogenesis since GGOH helped increasing cell viability and differentiation at the first 7 day of treatment.

MiR-133a delivery to osteoblasts ameliorates mechanical unloading-triggered osteopenia progression in vitro and in vivo

Mechanical unloading-induced bone loss is a clinical challenge, and deep understanding for this disease is necessary for developing novel and effective therapies. MicroRNAs (miRNAs) are small non-coding RNAs, and involved in bone remodeling. In the study, we attempted to explore the potential of miR-133a in regulating osteoblast activation and its anti-osteopenia function both in vitro and in vivo. Our in vitro studies at first showed that miR-133a could significantly promote the expression of osteocalcin (OCN), Collagen I, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osterix (Osx), promoting the activation and mineralization of osteoblasts. Then, hindlimb unloading (HU)-challenged mice were established with or without intravenous injection of agomir-miR-133a using an osteoblast-targeting delivery system. We found that miR-133a in osteoblasts significantly alleviated the bone loss, microstructural, and biomechanical property in mice with mechanical unloading, contributing to osteopenia alleviation. Furthermore, both in vitro and in vivo experiments showed that miR-133a could restrain osteoclastogenesis via tartrate-resistant acid phosphatase (TRAP) staining. In conclusion, our results suggested that miR-133a may be a promising factor in mediating the occurrence and progression of osteopenia caused by mechanical unloading, and thus targeting miR-133a could be considered as an effective therapeutic strategy for the suppression of pathological osteopenia.