Induced Osteoblast Proliferation Research Articles

METTL3-mediated m6A modification of SOX4 regulates osteoblast proliferation and differentiation via YTHDF3 recognition

N6-methyladenosine (m6A), the most prevalent internal modification in mRNA, is related to the pathogenesis of osteoporosis (OP). Although methyltransferase Like-3 (METTL3), an m6A transferase, has been shown to mitigate OP progression, the mechanisms of METTL3-mediated m6A modification in osteoblast function remain unclear. Here, fluid shear stress (FSS) induced osteoblast proliferation and differentiation, resulting in elevated levels of METTL3 expression and m6A modification. Through Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and Transcriptomic RNA Sequencing (RNA-seq), SRY (Sex Determining Region Y)-box 4 (SOX4) was screened as a target of METTL3, whose m6A-modified coding sequence (CDS) regions exhibited binding affinity towards METTL3. Further functional experiments demonstrated that knockdown of METTL3 and SOX4 hampered osteogenesis, and METTL3 knockdown compromised SOX4 mRNA stability. Via RNA immunoprecipitation (RIP) assays, we further confirmed the direct interaction between METTL3 and SOX4. YTH N6-Methyladenosine RNA Binding Protein 3 (YTHDF3) was identified as the m6A reader responsible for modulating SOX4 mRNA and protein levels by affecting its degradation. Furthermore, in vivo experiments demonstrated that bone loss in an ovariectomized (OVX) mouse model was reversed through the overexpression of SOX4 mediated by adeno-associated virus serotype 2 (AAV2). In conclusion, our research demonstrates that METTL3-mediated m6A modification of SOX4 plays a crucial role in regulating osteoblast proliferation and differentiation through its recognition by YTHDF3. Our research confirms METTL3-m6A-SOX4-YTHDF3 as an essential axis and potential mechanism in OP.

The Use of Lactide Polymers in Bone Tissue Regeneration in Dentistry-A Systematic Review.

(1) Background: Different compositions of biodegradable materials are being investigated to successfully replace non-resorbable ones in bone tissue regeneration in dental surgery. The systematic review tried to address the question, "Can biodegradable polymers act as a replacement for conventional materials in dental surgery procedures?" (2) Methods: An electronic search of the PubMed and Scopus databases was conducted in October 2022. The following keywords were used: (lactide polymers) and (hydroxyapatite or fluorapatite) and (dentistry) and (regeneration). Initially, 59 studies were found. Forty-one studies met the inclusion criteria and were included in the review. (3) Results: These usually improved the properties and induced osteogenesis, tissue mineralisation and bone regeneration by inducing osteoblast proliferation. Five studies showed higher induction of osteogenesis in the case of biomaterials, UV-HAp/PLLA, ALBO-OS, bioresorbable raw particulate hydroxyapatite/poly-L-lactide and PLGA/Hap, compared to conventional materials such as titanium. Four studies confirmed improvement in tissue mineralisation with the usage of biomaterials: hydroxyapatite/polylactic acid (HA/PLA) loaded with dog's dental pulp stem cells (DPSCs), Coll/HAp/PLCL, PDLLA/VACNT-O:nHAp, incorporation of hydroxyapatite and simvastatin. Three studies showed an acceleration in proliferation of osteoblasts for the use of biomaterials with additional factors such as collagen and UV light. (4) Conclusions: Lactide polymers present higher osteointegration and cell proliferation rate than the materials compared. They are superior to non-biodegradable materials in terms of the biocompability, bone remodelling and healing time tests. Moreover, because there is no need of reoperation, as the material automatically degrades, the chance of scars and skin sclerosis is lower. However, more studies involving greater numbers of biomaterial types and mixes need to be performed in order to find a perfect biodegradable material.

Extracellular vesicles derived from bone marrow mesenchymal stem cells loaded on magnetic nanoparticles delay the progression of diabetic osteoporosis via delivery of miR-150-5p

Extracellular vesicles derived from bone marrow mesenchymal stem cells (BMSC-EVs) are emerged as carriers of therapeutic targets against bone disorders, yet its isolation and purification are limited with recent techniques. Magnetic nanoparticles (MNPs) can load EVs with a unique targeted drug delivery system. We constructed gold-coated magnetic nanoparticles (GMNPs) by decorating the surface of the Fe3O4@SiO2 core and a silica shell with poly(ethylene glycol) (PEG)-aldehyde (CHO) and examined the role of BMSC-EVs loaded on GMNPs in diabetic osteoporosis (DO). The osteoporosis-related differentially expressed miR-150-5p was singled out by microarray analysis. DO models were then established in Sprague–Dawley rats by streptozotocin injection, where poor expression of miR-150-5p was validated in the bone tissues. Next, GMNPE was prepared by combining GMNPs with anti-CD63, after which osteoblasts were co-cultured with the GMNPE-BMSC-EVs. The re-expression of miR-150-5p facilitated osteogenesis in osteoblasts. GMNPE could promote the enrichment of EVs in the bone tissues of DO rats. BMSC-EVs delivered miR-150-5p to osteoblasts, where miR-150-5p targeted MMP14 and consequently activated Wnt/β-catenin pathway. This effect contributed to the enhancement of osteoblast proliferation and maturation. Furthermore, GMNPE enhanced the EV-based delivery of miR-150-5p to regulate the MMP14/Wnt/β-catenin axis, resulting in promotion of osteogenesis. Overall, our findings suggest the potential of GMNP-BMSC-EVs to strengthen osteoblast proliferation and maturation in DO, showing promise as an appealing drug delivery strategy against DO.Graphical abstract1. GMNPs-BMSCs-EVs-miR-150-5p promotes the osteogenesis of DO rats.2. miR-150-5p induces osteoblast proliferation and maturation by targeting MMP14.3. Inhibition of MMP14 activates Wnt/β-catenin and increases osteogenesis.4. miR-150-5p activates the Wnt/β-catenin pathway by downregulating MMP14.

Melatonin Mediates Osteoblast Proliferation Through the STIM1/ORAI1 Pathway.

Based on the positive correlation between bone mineral density and melatonin levels in blood, this study confirmed that melatonin supplementation prevents postmenopausal osteoporosis. We further confirmed that melatonin promotes an increase in intracellular calcium concentrations through the STIM1/ORAI1 pathway, thereby inducing the proliferation of osteoblasts. Introduction: Osteoporosis (OP) is a progressive, systemic bone disease that is one of the main causes of disability and death in elderly female patients. As an amine hormone produced by the human pineal gland, melatonin plays an important role in regulating bone metabolism. This study intends to investigate the relationship between melatonin levels in human blood and bone density and to suggest the efficacy of melatonin in treating osteoporosis by performing in vivo and in vitro experiments. Methods: We used liquid chromatography-tandem mass spectrometry to determine the serum melatonin levels in postmenopausal women with osteoporosis and young women with a normal bone mass. The bone density, BV/TV, Tb.Th, Tb.Sp and other indicators of postmenopausal osteoporosis and mice with a normal bone mass were detected by measuring bone density and micro-CT. The intracellular calcium ion concentration was detected using fluorescence microscopy and a full-wavelength multifunctional microplate reader, and the expression of SOCE-related genes and STIM1/ORAI1 proteins was detected using PCR and WB. Results: This study confirmed that bone density positively correlates with the melatonin level in human blood. In the animal model, melatonin supplementation reverses postmenopausal osteoporosis. We explored the internal mechanism of melatonin treatment of osteoporosis. Melatonin promotes an increase in intracellular calcium ion concentrations through the STIM1/ORAI1 pathway to induce osteoblast proliferation. Conclusions: This study provides an important theoretical basis for the clinical application of melatonin in patients with osteoporosis and helps to optimize the diagnosis and treatment of postmenopausal osteoporosis.

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.

JAGGED1 stimulates cranial neural crest cell osteoblast commitment pathways and bone regeneration independent of canonical NOTCH signaling.

Craniofacial bone loss is a complex clinical problem with limited regenerative solutions. Currently, BMP2 is used as a bone-regenerative therapy in adults, but in pediatric cases of bone loss, it is not FDA-approved due to concerns of life-threatening inflammation and cancer. Development of a bone-regenerative therapy for children will transform our ability to reduce the morbidity associated with current autologous bone grafting techniques. We discovered that JAGGED1 (JAG1) induces cranial neural crest (CNC) cell osteoblast commitment during craniofacial intramembranous ossification, suggesting that exogenous JAG1 delivery is a potential craniofacial bone-regenerative approach. In this study, we found that JAG1 delivery using synthetic hydrogels containing O9-1 cells, a CNC cell line, into critical-sized calvarial defects in C57BL/6 mice provided robust bone-regeneration. Since JAG1 signals through canonical (Hes1/Hey1) and non-canonical (JAK2) NOTCH pathways in CNC cells, we used RNAseq to analyze transcriptional pathways activated in CNC cells treated with JAG1±DAPT, a NOTCH-canonical pathway inhibitor. JAG1 upregulated expression of multiple NOTCH canonical pathway genes (Hes1), which were downregulated in the presence of DAPT. JAG1 also induced bone chemokines (Cxcl1), regulators of cytoskeletal organization and cell migration (Rhou), signaling targets (STAT5), promoters of early osteoblast cell proliferation (Prl2c2, Smurf1 and Esrra), and, inhibitors of osteoclasts (Id1). In the presence of DAPT, expression levels of Hes1 and Cxcl1 were decreased, whereas, Prl2c2, Smurf1, Esrra, Rhou and Id1 remain elevated, suggesting that JAG1 induces osteoblast proliferation through these non-canonical genes. Pathway analysis of JAG1+DAPT-treated CNC cells revealed significant upregulation of multiple non-canonical pathways, including the cell cycle, tubulin pathway, regulators of Runx2 initiation and phosphorylation of STAT5 pathway. In total, our data show that JAG1 upregulates multiple pathways involved in osteogenesis, independent of the NOTCH canonical pathway. Moreover, our findings suggest that JAG1 delivery using a synthetic hydrogel, is a bone-regenerative approach with powerful translational potential.

Ovotransferrin Exhibits Osteogenic Activity Partially via Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Activation in MC3T3-E1 Cells.

Ovotransferrin, a major protein in egg white, induces osteoblast proliferation and survival in vitro. However, it is unclear which receptor(s) drive the beneficial activities of this bioactive glycoprotein. We examined the role of the low-density lipoprotein receptor-related protein 1 (LRP1) in the actions of ovotransferrin on osteoblasts. Here, we showed that LRP1 in part regulates osteogenic action of ovotransferrin. Mouse osteoblasts, MC3T3-E1, with LRP1 deletion displayed diminished osteogenic activity. Our findings indicate that the bone-stimulatory impact of ovotransferrin on RUNX2, COL1A2, and Ca2+ signaling is LRP1-dependent. This shows that LRP1 not only acts as a scavenger receptor but also participates in ovotransferrin-mediated gene transcription. However, some of the key bone formatting factors such as ALP synthesis and serine residue phosphorylation of Akt by ovotransferrin remained independent of LRP1. Overall, this study shows that LRP1-ovotransferrin interaction might underline in part the ability of ovotransferrin to promote bone formation.

Does 2D correlation Raman spectroscopy distinguish polymer nanomaterials due to the nanoaddition?

Polymer nanocomposites obtain many new functional properties by adding appropriate nanoparticles, making them more suitable for applications in tissue engineering. The subjects of the presented research are nanocomposites composed of poly(ε-caprolactone) (PCL) matrix and carbon nanofibers as nanoadditions. Each of the tested materials contained a different type of fibrous nanoparticles: first, carbon nanofibers in the initial state (CNFs), and the second, carbon nanofibers after oxidative functionalization (CNFs-f). Both materials, PCL/CNFs and PCL/CNFs-f, were confirmed to be characterized by high bioactivity, and at the same time induced osteoblast proliferation. The aim of this work was to resolve the details of the complex relations between osteoblast-like U-2 OS cells and material, that denote the nanoaddition specificity. The 2D Raman correlation spectroscopy technique revealed that in both of the composites the key component of their interactions with the cells was the carbonous domains of the materials. In the PCL/CNFs the G-mode of the CNFs correlate asynchronously with Amide I of the U-2 OS cells, whereas, the D1-band is mostly engaged into interaction with the side chain amino acids in the case of the PCL/CNFs-f nanocomposite.

Does Vitamin D3 Prevent the Inhibitory Effect of Vancomycin on Osteoblasts?

BackgroundThe utility of vancomycin powder to prevent surgical site infection, mainly in spinal surgery, has been widely examined, and the local administration of vancomycin powder to wounds has been reported to be effective in preventing surgical site infections after spine surgery. However, in vitro studies have shown that high local concentrations of vancomycin may inhibit osteogenesis, although it remains unclear how these high concentrations influence osteoblasts. No candidate drug has been reported to recover cytotoxicity with high concentrations of vancomycin, but we suggest that vitamin D3, which induces osteoblast proliferation, may be administrated concomitantly with vancomycin in these situations.Questions/purposes(1) Does a high concentration of vancomycin reduce viable osteoblast numbers in cell culture compared with controls? (2) Does vitamin D3 administration confer a protective effect on osteoblasts when administered with continuous vancomycin? (3) Does vitamin D3 administration confer a protective effect on osteoblasts when administered with pulsed vancomycin (24 hours of administration)? (4) Does vitamin D3 administration confer alkaline phosphatase, mineralization, and gene expression when administered with pulsed vancomycin?MethodsMC3T3-E1 cells were cultured at 37° C in an α-minimum essential medium supplemented with 10% fetal bovine serum in a humidified incubator containing 5% CO2. The experimental concentrations of vancomycin (2500 μg/mL, 5000 μg/mL, and 7500 μg/mL) were determined based on previous reports and preliminary experiments. We concomitantly administered vitamin D3 (0.01 nM) to prevent cytotoxicity in osteoblasts, using two different treatments: continuous vancomycin administration (measured at 6 hours, 12 hours, 24 hours, and 72 hours) and pulsed vancomycin for 24 hours (measured at 1 days, 3 days, and 7 days). We analyzed cell numbers and morphologic changes in cells treated with vancomycin or vancomycin plus 0.01 nM vitamin D3. Osteoblast differentiation was assessed with alkaline phosphatase staining, alkaline phosphatase activity, and Alizarin red S staining.ResultsThe number of cells was reduced at 6 hours, 24 hours, 48 hours, and 72 hours in response to continuous vancomycin administration at 7500 µg/mL (at 72 hours, control 14.6 × 104 cells/mL ± 0.260 × 104 cells/mL, vancomycin at 0.917 × 104 cells/mL ± 0.288 × 104 cells/mL, mean difference -13.7 × 104 cells/mL ± 0.388 × 104 cells/mL [95% CI -14.5 to -12.9]; p < 0.001). Vitamin D3 did not have a protective effect when vancomycin was administered continuously at 7500 µg/mL (at 72 hours, vancomycin alone 0.917 × 104 cells/mL ± 0.288 × 104 cells/mL, vancomycin + vitamin D3 1.67 × 104 cells/mL ± 0.310 × 104 cells/mL, mean difference 0.75 × 104 cells/mL ± 0.423 × 104 cells/mL [95% CI -0.127 to 1.63]; p = 0.09).With pulsed administration for only the first 24 hours, the number of cells was reduced at 1 day, 3 days, and 7 days at 7500 μg/mL (at 7 days, control 18.6 × 104 cells/mL ± 1.29 × 104 cells/mL, vancomycin at 3.46 × 104 cells/mL ± 0.292 × 104 cells/mL, mean difference -15.1 × 104 cells/mL ±1.33 × 104 cells/mL [95% CI -17.9 to -12.4]; p < 0.001 for all). However, vitamin D3 had a recovery effect when vancomycin was administered only for 24 hours (cell number with 7500 μg/mL, day 7: vancomycin alone 3.46 × 104 cells/mL ± 0.292 × 104 cells/mL, vancomycin +vitamin D3 10.6 × 104 cells/mL ± 0.900 × 104 cells/mL, mean difference 7.13 × 104 cells/mL ± 0.946 × 104 cells/mL [95% CI 5.16 to 9.09]; p < 0.001).With the addition of vitamin D3, we observed recovery of alkaline phosphatase staining and Alizarin red staining (evidence of calcification) but no difference in the gene expression of Type I collagen (vancomycin alone 0.319 ± 0.0730, vancomycin + vitamin D3 0.511 ± 0.139, mean difference 0.192 ± 0.157 [95% CI -0.483 to 0.867]; p = 0.345), alkaline phosphatase (vancomycin alone 0.532 ± 0.0210, vancomycin + vitamin D3 0.785 ± 0.0590, mean difference 0.253 ± 0.0620 [95% CI -0.0150 to 0.521]; p = 0.0550), and cathelicidin antimicrobial peptide (vancomycin alone 0.885 ± 0.0520, vancomycin + vitamin D3 1.24 ± 0.125, mean difference 0.355 ± 0.135 [95% CI -0.0200 to 0.730]; p = 0.0580).ConclusionWe found that 7500 µg/mL of vancomycin is cytotoxic to osteoblasts. Cytotoxicity could be prevented by administering vitamin D3 in combination with vancomycin.Clinical RelevanceThe high concentrations of vancomycin routinely used clinically raises concerns related to osteoblast cytotoxicity, which may contribute to pseudoarthrosis after spinal surgery. Thus, vitamin D3, which is frequently used to treat osteoporosis, may have efficacy as a concomitantly administered drug by inducing the proliferation of osteoblasts. These results indicate that a combination therapy of vancomycin and vitamin D3 may prevent adverse events such as osteoblast cytotoxicity.

Freeze-Dried Platelet-Rich Plasma Induces Osteoblast Proliferation via Platelet-Derived Growth Factor Receptor-Mediated Signal Transduction.

Study DesignControlled laboratory study.Purpose This study aimed to evaluate the in vitro pharmacological activity of growth factors (GFs) in freeze-dried platelet-rich plasma (FD-PRP) after storage for 4 weeks.Overview of LiteratureFreshly prepared PRP is a rich source of many GFs. We reported that FD-PRP stored for 8 weeks accelerated bone union in a rat posterolateral fusion model equally well as fresh-PRP. However, the pharmacological activity of FD-PRP after long-term storage has not been shown in vitro.MethodsImmediately after preparation, as well as 4 weeks after freeze-dried storage, the platelet count was measured. Human osteoblasts were treated with fresh-PRP and FD-PRP, respectively. Western blotting was used to assess the phosphorylation of the platelet-derived growth factor (PDGF) receptor (PDGFR) and its downstream target, extracellular signal-regulated kinase (ERK). The proliferation rates of osteoblasts were investigated by immunocytochemistry and MTT cell viability assays. Furthermore, we used western blotting to evaluate the effect of PDGFR knockdown on the phosphorylation of ERK stimulated with fresh-PRP and FD-PRP.Results Platelet counts in both the fresh-PRP and FD-PRP samples were approximately 10-fold higher than in peripheral blood samples. The phosphorylation and activation of the PDGFR and ERK were evenly induced by fresh-PRP and FD-PRP stimulation. Both freshPRP and FD-PRP significantly induced osteoblast proliferation in MTT cell viability assays. Furthermore, osteoblast PDGFR knockdown attenuated the downstream ERK activation by fresh PRP and FD-PRP.ConclusionsWe demonstrated the pharmacological activity of PDGF in FD-PRP in vitro after 4 weeks of storage.

Loading Psoralen into liposomes to enhance its stimulatory effect on the proliferation and differentiation of mouse calvarias osteoblasts

Psoralen (PSR), a well-known traditional Chinese medicine has been claimed for the treatment of osteoporosis. However, its hydrophobicity and the first-pass metabolism restrict the potential application of PSR. Thus, the development of PSR-loaded liposome was done to improve the solubility and bioavailability of PSR. The PSR/liposomes exhibited a particle size of approximately 110 nm and were quite stable during 30 days of storage. The entrapment efficiency (EE), drug loading (DL) and zeta potentials of PSR/liposome were 85.0 ± 1.6%, 5.0 ± 1.6% and -36 mV, respectively. Small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) measurements suggested that PSR/liposomes are the mixture of unilamellar and multilamellar vesicles. The in vitro drug release profile of PSR/liposome exhibited a gradual behavior. Both pure PSR and PSR/liposome promoted osteoblast proliferation in a dose-dependent manner. The proliferation effect was firstly enhanced with drug concentration increased, and then decreased when the concentration was higher than 20 µM. But PSR/liposome could induce osteoblast proliferation in more gentle way through the sustained release of PSR. For the level of ALP activity, PSR/liposome was 1.2 times higher than pure PSR. Above all, it is expected that PSR/liposome could be used in osteoporosis treatment in the future.

Tooth transformer®: A new method to prepare autologous tooth grafts – Histologic and histomorphometric analyses of 11 consecutive clinical cases

Introduction: Human dentin matrix could be successfully used for bone grafting procedures. It was well accepted that dentin grafts can induce osteoblast proliferation. An innovative preparation method, using the dedicated automated device Tooth Transformer®, which can transform autologous teeth in suitable grafting material, has been recently introduced. The aim of the present article is to analyze the histologic outcomes in 11 consecutive human cases, in which autologous tooth graft materials, starting from the whole tooth of the patient, were used for bone regeneration. Results: The bone defects were completely filled by newly formed tissue after 4 months of healing. Histologic analysis revealed no inflammatory or infective reactions against the tooth graft. Tooth granules were surrounded by newly formed bone. Some tooth granules were incorporated in the bony trabeculae, and they appeared partially resorbed. This fact testified that tooth grafts underwent remodeling processes just like the native bone. Discussion: Results from the present histologic case series analysis revealed that tooth graft appeared well integrated in the regenerative tissue without any inflammatory or infective reaction. The tooth of the patient may be used as an autologous regenerative material, avoiding any foreign graft material.

Abstract B050: The role of activated beta-catenin/Wnt signaling in the progression of castration resistance of prostate cancer in bone

Abstract Bone metastases typically develop in patients with advanced prostate cancer (PCa). These metastases are osteoblastic (bone-forming) and constitute the main cause of morbidity and mortality. Androgen deprivation is commonly used to treat bone metastases of PCa, but responses to such therapy are short, and eventually the disease progresses to a castration-resistant form. Bone is the primary site of castration-resistant PCa (CRPC) progression. Further development of therapies for bone metastases of PCa requires an understanding of the mechanisms underlying the growth of CRPC in bone. Clinical and laboratory-based investigations have implicated a role for PCa cell-bone cell interaction in the growth of PCa and have demonstrated that osteoblasts produce factors that favor the growth and survival of PCa cells. Collectively, these observations strongly suggest that PCa cells interact with osteoblasts and that this interaction contributes to the growth of PCa in bone. Recently, a study showed 18% of patients with metastatic CRPC harbored alterations in the Wnt canonical pathway, including hotspot mutations of β-catenin (β-cat), the molecular node of the Wnt canonical pathway, that leads to pathway activation. This report is in agreement with previous publications of β-cat nuclear accumulation, an indirect measure of Wnt-canonical pathway activation, in 20 to 40% of advanced PCas. We previously reported that a patient-derived xenograft (PDX) MDA-PCa-118b (118b) developed in our lab harbors a β-cat mutation (D32G) and has an activated Wnt canonical pathway. 118b induces an osteoblastic reaction when it is implanted in the bone of mice in vivo. In vitro, 118b induces osteoblast proliferation in coculture. In contrast, the PCa cell line, PC3, induces osteolytic lesions in vivo and in vitro inhibits osteoblast proliferation in coculture. Strikingly, expression of β-cat mutated at D32G in PC3 cells reverses the osteoblast inhibitory effect of PC3 cells, resulting in increased proliferation of osteoblasts when grown in coculture. We therefore hypothesize that β-cat/Wnt signaling in PCa cells mediates the PCa-bone interaction that favors PCa growth in bone. Our future studies are focusing on studying the importance of β-cat mutations recently identified in patients with metastatic CRPC and determining whether these mutations also result in an osteoblastic phenotype, which in turn supports growth of prostate cancer in bone. Additionally, we are screening our collection of PCa PDX lines for increased β-cat expression through immunohistochemistry and will sequence DNA from PDX lines that demonstrate increased nuclear β-cat expression to test for activating mutations in β-cat. Our aim is to gain insight into the mechanism of the paracrine interaction between PCa cells and bone that supports the growth of PCa bone lesions, with the ultimate goal of developing therapies to block these interactions and reducing the morbidity and mortality associated with metastatic disease. Citation Format: Justin M. Roberts, Estefania Labanca, Peter Shepherd, Jun Yang, Michael W. Starbuck, Nora M. Navone. The role of activated beta-catenin/Wnt signaling in the progression of castration resistance of prostate cancer in bone [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B050.

Salvianolic acid B alleviate the osteoblast activity decreasing under simulated microgravity by Keap1/Nrf2/ARE signaling pathway

Astronauts suffer bone loss during long-term space flight, and the bone loss is hard to recover after returning to the ground. Salvianolic acid B (Sal B) is proved to have stimulation effect on osteoblasts. In order to study the mechanisms on the protect effect of Sal B on simulated microgravity (SMG)-induced osteoblast proliferation inhibition, cell proliferation, cell apoptosis, antioxidant activity and related osteogenic gene expression of osteoblasts were studied. Results show that osteoblasts proliferation was increased by Sal B. And it can also reduce apoptosis under SMG. In case of anti-oxidation, Sal B decreases the whole content of MDA, stimulates the activity of SOD, CAT and LDH. Meanwhile, the expression of Nrf2, HO-I, Runx2, Osx, OPN, ALP and Col-I was up-regulated by Sal B. In conclusion, by stimulating the osteoblasts’s activity and the capacity of anti-oxidation, salvianolic acid B has a protection effect on osteoblasts under SMG.

99Tc-MDP-induced human osteoblast proliferation, differentiation and expression of osteoprotegerin.

The aim of the present study was to examine the influence of technetium methylenediphosphonate (99Tc-MDP) on the proliferation and differentiation of human osteoblasts. Human iliac cancellous bone was isolated and cultured with either 99Tc-MDP, β fibroblast growth factor (as a positive control) or medium only (as a negative control). Proliferation was assessed by direct cell counting, CCK-8 assay and bromodeoxyuridine staining. The cell cycle and rate of apoptosis was assessed by propidium iodide staining and flow cytometry. Alkaline phosphatase (ALP) activity was assessed by the p-nitrophenyl phosphate method and mineralized nodules were stained with Alizarin Red. Expression of osteocalcin (OCN) and bone morphogenetic protein-2 (BMP-2) was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and expression levels of osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) were assessed by RT-qPCR and ELISA. Isolated human osteoblasts stained positively for ALP and developed mineralized nodules. Treatment with 10−5-10−10 M 99Tc-MDP enhanced proliferation and 48 h incubation with 10−8 M 99Tc-MDP increased the proportion of cells in S-phase, decreased the proportion in G0/G1 phase, and increased the cell proliferation index. The rate of apoptosis also increased, but the increase was not significant. Cells incubated with 10−6-10−9 M 99Tc-MDP for 3–9 days exhibited increased ALP activity and mineralized nodule development. 10−8 M 99Tc-MDP increased BMP-2 and OPG expression levels and OPG secretion, but OCN mRNA expression levels and RANKL secretion were not significantly altered at 72 h. 99Tc-MDP treatment induced osteoblast proliferation and differentiation without affecting apoptosis. These findings provide proof of concept for the future use of 99Tc-MDP in the treatment of bone-destructive diseases.

Effects of nano tantalum implants on inducing osteoblast proliferation and differentiation.

In the present study, we examined the effects of nano tantalum (Ta) dental implants on inducing osteoblast proliferation and differentiation. The MG-63 osteoblasts were divided into 3 groups after recovery, passage and storage: i) Osteoblast culturing group (control group); ii) osteoblast and titanium (Ti) implant co-culturing group (Ti group); and iii) osteoblast and Ta implant co-culturing group (Ta group). After 7 days, a scanning electron microscope was used to observe the growth status, number and morphological changes of the cells on the surfaces of the materials. An MTT assay was used to detect cell proliferation after culturing for 1, 3 and 7 days. ELISA assay was used to detect the levels of alkaline phosphatase (ALP) after 1, 3 and 7 days. Western blot analysis was used to detect the expression levels of collagen type I (Col-1) and osteocalcin after 1, 3 and 7 days. There was significant cell spreading on the surfaces of Ti and of Ta after 7 days, flat and with many pseudopodia. Additionally, there were more cell components in the Ta group. Concurrently, cell proliferation in the Ti and Ta groups increased. There was also an increase in the level of ALP and the expression level of Col-1 over time. The indexes of the Ta group were more apparent than those of the Ti group at each time-point, and the differences were statistically significant (p<0.05). In conclusion, compared with Ti implants, Ta implants induced more osteoblast proliferation and differentiation.

Identification of buffalo casein-derived bioactive peptides with osteoblast proliferation activity

Osteoblast cells are the building blocks of bone architecture and most suitable cells for screening osteoanabolic agents. As bone formation begins with osteoblast proliferation, it is the essential stage for screening osteoanbolics. LC–MS/MS analysis of the 11th RP–HPLC fraction of pepsin–trypsin (PT) hydrolysates of buffalo casein showed 15 peptides. Out of these 15 peptides, four peptides of less than 1 kDa were custom-synthesized and evaluated for osteoblast proliferating ability. Proliferation effect of the peptides was studied by formazan crystal formation (MTT method) and relative expression of proliferation marker genes (clay and cdk2). PT hydrolysates and all the four peptides significantly induced osteoblast proliferation indicated by both MTT and relative gene expression studies. This study showed the osteoblast-promoting property of peptides derived from milk proteins, thereby further substantiating the earlier epidemiological data regarding bone health-promoting effect of milk.

Effects of vascular endothelial growth factor on osteoblasts and osteoclasts.

Bone remodeling is crucial to the success of many dental procedures and is tightly regulated. Vascular endothelial growth factor (VEGF), a key cytokine for angiogenesis, is also an important regulator of bone remodeling. We aimed to examine the mechanisms by which VEGF induces bone remodeling by studying its effects on cultured osteoblasts and osteoclasts. Preosteoblastic MC3T3-E1 cells were treated with vehicle or VEGF-A165. Cell proliferation, migration, and invasion potentials were assessed. Preosteoclastic RAW264.7cells were treated with vehicle or VEGF with or without the receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoclast formation was measured with tartrate-resistant acid phosphatase staining. Conditioned media from vehicle-treated or VEGF-treated MC3T3-E1 cells were tested for the levels of RANKL and osteoprotegerin (OPG) and were used to treat RAW264.7cells to observe osteoclast formation. VEGF significantly induced MC3T3-E1 cell proliferation, migration, and invasion. VEGF did not directly induce osteoclastogenesis but significantly increased the RANKL/OPG ratio in the conditioned media from the MC3T3-E1 cultures; this significantly increased osteoclast formation. VEGF stimulates osteoclast differentiation by increasing the osteoblastic RANKL/OPG ratio but has no direct effect on osteoclast precursor cells, and it induces osteoblast proliferation, migration, and invasion potentials invitro.

Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism

Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 μg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity.

A comparative study of mechanical strain, icariin and combination stimulations on improving osteoinductive potential via NF-kappaB activation in osteoblast-like cells

BackgroundThe combination of drugs and exercise was the effective treatment in bone injure and rebuilding in clinic. As mechanical strain has potential in inducing the differentiation of osteoblasts in our previous study, the further research to investigate the combination of mechanical strain and icariin stimulation on inducing osteoblast proliferation, differentiation and the possible mechanism in MC3T3-E1 cell line.MethodsA whole cell enzyme-linked immunosorbent assay that detects the bromodeoxyuridine incorporation during DNA synthesis was applied to evaluate the proliferation. The mRNA expression of alkaline phosphatase (ALP), osteocalcin (OCN), type I collagen (Col I), bone morphogenetic protein-2 (BMP-2) and BMP-4 was detected by real-time reverse-transcription polymerase chain reaction. The activity of ALP was analyzed by ELISA and the protein expression of OCN, Col I and BMP-2 was assessed by western blot. Moreover, the activity of nuclear transcription factor kappa-B (NF-κB) signaling pathway was investigated with the expression of inhibitor of κB (IκB) α, phosphorylation of IκB-α (P-IκB-α), p65, P-p65 by western blot.ResultsWe observed that compared to single mechanical strain or icariin stimulation, the mRNA and protein expressions of ALP (P < 0.05 or P < 0.01), OCN (P < 0.01) and Col I (P < 0.05 or P < 0.01) were increased significantly by the combination of mechanical strain and icariin stimulation. Moreover, the combination of mechanical strain and icariin stimulation could up-regulate the expression of BMP-2 (P < 0.01) and BMP-4 compared to single mechanical strain or icariin stimulation. The combination of mechanical strain and icariin stimulation could activate NF-κB signaling pathway by increasing the expression of IκB α, P-IκB-α, p65, P-p65 (P < 0.01).ConclusionThe combination of mechanical strain and icariin stimulation could activate the NF-κB pathway to improve the proliferation, differentiation of osteoblast-like cells.