Osteoblastic Phenotype Research Articles

Involvement of Rab11 in osteoblastic differentiation: Its up-regulation during the differentiation and by tensile stress

Rab GTPases, the largest group of small monomeric GTPases, have been shown to participate in membrane trafficking involving many cellular processes. However, their roles during osteoblastic differentiation remain to be elucidated. In this study, we investigated Rab GTPase involvement in osteoblastic differentiation. Protein levels of a series of Rabs (Rab4, Rab5, Rab7, Rab9a, Rab11a/b, and Rab27) were increased during osteoblastic differentiation of MC3T3-E1 cells, and the Rab11a/b levels were particularly pronounced in the presence of Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, an activator of osteoblastogenesis. We subsequently investigated the functional contribution of Rab11a and Rab11b during osteoblastic differentiation. The alkaline phosphatase (ALP) levels were reduced by Rab11b depletion but not by Rab11a depletion. Because our result suggested that Rab11a and Rab11b could be regulated downstream of Runx2 (Runt-related transcription factor 2), a key transcription factor for osteoblastic differentiation, we investigated the effects of the double knockdown of Runx2 and Rab11a or Rab11b on osteoblastic phenotypes. The double knockdown significantly reduced ALP activity as well as collagen deposition compared with single Runx2 knockdown. Furthermore, the Rab11a and Rab11b response to mechanical stress in vivo was investigated using a mouse orthodontic tooth movement model. Rab11a and Rab11b expression was enhanced in the periodontal ligament, where bone formation is activated by tensile stress. This study shows that Rab11a and Rab11b are regulated downstream of Runx2 in osteoblastic differentiation, and their expressions are also controlled by tensile stress.

Optimization and Characterization of a Bone Culture Model to Study Prostate Cancer Bone Metastasis.

Nearly 90% of patients with advanced prostate cancer manifest bone metastases. Distinct from the osteolytic metastasis mostly observed in other cancer types, prostate cancer bone metastasis is typically more osteoblastic, which is relatively understudied due to the lack of reliable and efficient models to resemble the indolent cellular growth and complexity of metastatic progression. In our previous studies, we developed bone-in-culture array (BICA) to primarily model the osteoblast-involved, pre-osteolytic stage of breast cancer bone metastasis. Given that the progression of prostate cancer bone metastasis is largely osteoblastic, it is reasonable to speculate that the original BICA model can be adjusted to investigate prostate cancer bone metastases. In this study, we refined BICA by reducing the surgical labor and improving its reproducibility and capacity. The optimized BICA can successfully recapitulate important features of prostate cancer bone metastasis such as the osteoblastic phenotype, indolent growth, cancer-niche interactions, and response to hormones. Our efforts address the long-standing need for reliable and efficient models to study prostate cancer bone metastasis.

Implication of TRPM4 ionic channel in osteogenic differentiation of human valvular interstitial cells

The Ca2 + -activated non selective cationic channel, TRPM4, is involved in cardiac fibroblasts remodelling. In valvular interstitial cells (VIC), with similar properties than fibroblasts, its expression at the mRNA level was reported to be higher in conditions of valvular calcification leading to aortic stenosis. Endothelial dysfunction, followed by inflammation and fibrosis, promote the differentiation of VIC into osteoblastic phenotype cells which is, at term, responsible of valve ossification. The purpose of this study is to search for a potential implication of TRPM4 into osteogenic differentiation of human VIC. Human aortic valves are obtained from surgery for valve replacement. VIC are isolated by enzymatic digestion then maintained in culture. VIC are incubated during 14 days in a pro-calcific mixture in the presence or not of 9-phenanthrol, a pharmacological inhibitor of TRPM4. Expression and functionality of this channel were followed respectively by Western Blot and electrophysiological recordings in the inside-out configuration of the patch clamp technique. The implication of TRPM4 in VIC mineralization process was observed by measuring calcific crystals using alizarin red staining. A typical TRPM4 single channel current was detected in VIC in culture with a single channel conductance of 20 pS, a monovalent cationic permeability as well as voltage and calcium sensitivity. Protein expression of TRPM4 was confirmed by Western Blot. Pharmacological inhibition of the channel by 9-phenanthrol (3.10–6 M), during culture, reduced the mineralization by 30% without affecting cell viability. Thereby, TRPM4 would participate in osteogenic differentiation of human VIC in culture. Further studies are needed to understand which mecanisms link TRPM4 and ostegenic differentiation.

Electrospun fibers of poly (lactic acid) containing bioactive glass and magnesium oxide nanoparticles for bone tissue regeneration

Electrospun fibers of poly (lactic acid) (PLA) containing 10 and 20 wt% of bioactive glass (n-BG) and magnesium oxide (n-MgO) nanoparticles of ca. 27 and 23 nm respectively, were prepared toward to application in bone tissue engineering. The addition of both nanoparticles into the PLA will produce a synergic effect increasing its bioactivity and antimicrobial behavior. Neat PLA scaffold and the composites with MgO showed an average fiber diameter of 1.7 ± 0.6 μm, PLA/n-BG and PLA/n-BG/n-MgO fibers presented a significant diameter increase reaching values of ca. 3.1 ± 0.8 μm. Young's modulus of the electrospun scaffolds was affected by the direct presence of the particle and scaffold morphologies. All the composites having n-BG presented bioactivity through the precipitation of hydroxyapatite structures on the surface. Although n-MgO did not add bioactivity to the PLA fibers, they were able to render antimicrobial characteristics reducing the S. aureus viability around 30%, although an effect on E. coli strain was not observed. PLA/n-BG nanocomposites did not display any significant antimicrobial behavior. The different composites increased the alkaline phosphatase (ALP) expression as compared with pure PLA barely affecting the cell viability, meaning a good osteoblastic phenotype expression capacity, with PLA/n-BG presenting the highest osteoblastic expression.

Degradation Behavior and Mechanical Integrity of a Mg-0.7Zn-0.6Ca (wt.%) Alloy: Effect of Grain Sizes and Crystallographic Texture.

The microstructural characteristics of biodegradable Mg alloys determine their performance and appropriateness for orthopedic fixation applications. In this work, the effect of the annealing treatment of a Mg-0.7Zn-0.6Ca (ZX11) alloy on the mechanical integrity, corrosive behavior, and biocompatibility-osteoinduction was studied considering two annealing temperatures, 350 and 450 °C. The microstructure showed a recrystallized structure, with a lower number of precipitates, grain size, and stronger basal texture for the ZX11-350 condition than the ZX11-450. The characteristics mentioned above induce a higher long-term degradation rate for the ZX11-450 than the ZX11-350 on days 7th and 15th of immersion. In consequence, the mechanical integrity changes within this period. The increased degradation rate of the ZX11-450 condition reduces 40% the elongation at failure, in contrast with the 16% reduction for the ZX11-350 condition. After that period, the mechanical integrity remained unchanged. No cytotoxic effects were observed for both treatments and significant differentiation of mesenchymal stem cells into the osteoblast phenotype was observed.

The Biological Basis for Surface-dependent Regulation of Osteogenesis and Implant Osseointegration.

Bone marrow stromal cells are regulated by the chemical and physical features of a biomaterial surface. When grown on titanium (Ti) and Ti alloy surfaces, such as titanium-aluminum-vanadium, with specific topographies that mimic the microscale, mesoscale, and nanoscale features of an osteoclast resorption pit, they undergo a rapid change in cell shape to assume a columnar morphology typical of a secretory osteoblast. These cells exhibit markers associated with an osteoblast phenotype, including osteocalcin and osteopontin, and they secrete factors associated with osteogenesis, including bone morphogenetic protein 2, vascular endothelial growth factor, and neurotrophic semaphorins. The pathway involves a shift in integrin expression from α5β1 to α2β1 and signaling by Wnt5a rather than Wnt3a. Conditioned media from these cultures can stimulate vasculogenesis by human endothelial cells and osteoblastic differentiation of marrow stromal cells not grown on the biomimetic substrate, suggesting that the surface could promote osteogenesis in vivo through similar mechanisms. In vivo studies using a variety of animal models confirm that implants with biomimetic surfaces result in improved osseointegration compared with Ti implants with smooth surfaces, as do meta-analyses comparing clinical performance of implant surface topographies.

La tumeur à cellules géantes des os en 2022

Giant cell tumors of bone (GCTs) are rare mesenchymal tumors classified as intermediate in the WHO 2020 classification, i.e. neither completely benign nor definitely malignant, due to recurrence (frequent) and pulmonary metastases (rare). They involve the end of long bones as well as the axial bones of mature skeletons. They are made of mononuclear stromal tumor cells of (pre-) osteoblastic phenotype, mononuclear cells of the monocyte-macrophage lineage and osteoclast-like multinuclear giant cells responsible for tumor osteolysis. In 95% of cases, the stromal cells have a specific mutation in the H3F3A gene which encodes histone H3.3. The mutated H3.3 G34W protein (90% of cases) can be easily detected by immunohistochemistry, even on small samples. Many tumors or bone pseudotumors contain osteoclast-like giant cells, cells of the bone microenvironment, and should not be confused with GCT: mainly brown tumor of hyperparathyroidism, aneurysmal bone cyst, chondroblastoma, non-ossifying fibroma and central giant cell granuloma.

Additive Manufacturing of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/Poly(D,L-lactide-co-glycolide) Biphasic Scaffolds for Bone Tissue Regeneration.

Polyhydroxyalkanoates are biopolyesters whose biocompatibility, biodegradability, environmental sustainability, processing versatility, and mechanical properties make them unique scaffolding polymer candidates for tissue engineering. The development of innovative biomaterials suitable for advanced Additive Manufacturing (AM) offers new opportunities for the fabrication of customizable tissue engineering scaffolds. In particular, the blending of polymers represents a useful strategy to develop AM scaffolding materials tailored to bone tissue engineering. In this study, scaffolds from polymeric blends consisting of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(D,L-lactide-co-glycolide) (PLGA) were fabricated employing a solution-extrusion AM technique, referred to as Computer-Aided Wet-Spinning (CAWS). The scaffold fibers were constituted by a biphasic system composed of a continuous PHBV matrix and a dispersed PLGA phase which established a microfibrillar morphology. The influence of the blend composition on the scaffold morphological, physicochemical, and biological properties was demonstrated by means of different characterization techniques. In particular, increasing the content of PLGA in the starting solution resulted in an increase in the pore size, the wettability, and the thermal stability of the scaffolds. Overall, in vitro biological experiments indicated the suitability of the scaffolds to support murine preosteoblast cell colonization and differentiation towards an osteoblastic phenotype, highlighting higher proliferation for scaffolds richer in PLGA.

Parathyroid Hormone Induces Human Valvular Endothelial Cells Dysfunction That Impacts the Osteogenic Phenotype of Valvular Interstitial Cells.

Parathyroid hormone (PTH) is a key regulator of calcium, phosphate and vitamin D metabolism. Although it has been reported that aortic valve calcification was positively associated with PTH, the pathophysiological mechanisms and the direct effects of PTH on human valvular cells remain unclear. Here we investigated if PTH induces human valvular endothelial cells (VEC) dysfunction that in turn could impact the switch of valvular interstitial cells (VIC) to an osteoblastic phenotype. Human VEC exposed to PTH were analyzed by qPCR, western blot, Seahorse, ELISA and immunofluorescence. Our results showed that exposure of VEC to PTH affects VEC metabolism and functions, modifications that were accompanied by the activation of p38MAPK and ERK1/2 signaling pathways and by an increased expression of osteogenic molecules (BMP-2, BSP, osteocalcin and Runx2). The impact of dysfunctional VEC on VIC was investigated by exposure of VIC to VEC secretome, and the results showed that VIC upregulate molecules associated with osteogenesis (BMP-2/4, osteocalcin and TGF-β1) and downregulate collagen I and III. In summary, our data show that PTH induces VEC dysfunction, which further stimulates VIC to differentiate into a pro-osteogenic pathological phenotype related to the calcification process. These findings shed light on the mechanisms by which PTH participates in valve calcification pathology and suggests that PTH and the treatment of hyperparathyroidism represent a therapeutic strategy to reduce valvular calcification.

Osteogenic Commitment of Human Periodontal Ligament Cells Is Predetermined by Methylation, Chromatin Accessibility and Expression of Key Transcription Factors.

Periodontal ligament stem cells (PDLCs) can be used as a valuable source in cell therapies to regenerate bone tissue. However, the potential therapeutic outcomes are unpredictable due to PDLCs’ heterogeneity regarding the capacity for osteoblast differentiation and mineral nodules production. Here, we identify epigenetic (DNA (hydroxy)methylation), chromatin (ATAC-seq) and transcriptional (RNA-seq) differences between PDLCs presenting with low (l) and high (h) osteogenic potential. The primary cell populations were investigated at basal state (cultured in DMEM) and after 10 days of osteogenic stimulation (OM). At a basal state, the expression of transcription factors (TFs) and the presence of gene regulatory regions related to osteogenesis were detected in h-PDLCs in contrast to neuronal differentiation prevalent in l-PDLCs. These differences were also observed under stimulated conditions, with genes and biological processes associated with osteoblast phenotype activated more in h-PDLCs. Importantly, even after the induction, l-PDLCs showed hypermethylation and low expression of genes related to bone development. Furthermore, the analysis of TFs motifs combined with TFs expression suggested the relevance of SP1, SP7 and DLX4 regulation in h-PDLCs, while motifs for SIX and OLIG2 TFs were uniquely enriched in l-PDLCs. Additional analysis including a second l-PDLC population indicated that the high expression of OCT4, SIX3 and PPARG TFs could be predictive of low osteogenic commitment. In summary, several biological processes related to osteoblast commitment were activated in h-PDLCs from the onset, while l-PDLCs showed delay in the activation of the osteoblastic program, restricted by the persistent methylation of gene related to bone development. These processes are pre-determined by distinguishable epigenetic and transcriptional patterns, the recognition of which could help in selection of PDLCs with pre-osteoblastic phenotype.

Blocking the NLRP3 inflammasome reduces osteogenic calcification and M1 macrophage polarization in a mouse model of calcified aortic valve stenosis

Background and aimsActivated innate immune cells infiltrating the valve and their secreted cytokines drive the differentiation of valve interstitial cells into myofibroblastic and osteoblastic phenotypes in calcified aortic valve stenosis (CAVS). In this study, we investigated how NLRP3 inhibition with CY-09 reduces aortic valve stenosis and calcification. MethodsApoE−/− mice were fed a high-fat diet for 24 weeks with or without intraperitoneal injection of 2.5 mg/kg/day NLRP3 inhibitor CY-09 for 42 consecutive days, while the control group mice were fed a normal diet. The valve function was monitored by echocardiography; calcified nodules were assessed by Von Kossa staining; and calcification-related molecules, inflammatory factors, and white leucocyte influx into the valve were assessed by immunohistochemistry, TUNEL assay, and PCR. ResultsMice treated with CY-09 exhibited improved aortic valve function and reduced valve calcification deposition. CY-09 intervention significantly downregulated the elevated expression of the NLRP3 inflammasome pathway molecules NLRP3, caspase-1, and IL-1β and the osteogenic calcification markers RUNX2, SPARC, and BMP2 in stenotic valves, while the number of apoptotic cells and dystrophic calcification markers CDH11 and α- SMA did not change significantly. Inhibition of NLRP3 activity also reduced the ratio of M1/M2 macrophages, prevented the shift of macrophages towards the M1 phenotype, and downregulated the levels of the proinflammatory factors IL-6 and TNF-α. ConclusionsThis study provides a proof-of-concept that pharmacological inhibition of the NLRP3 inflammasome is a feasible strategy for alleviating aortic valve calcification and stenosis.

Uncoupling the Vicious Cycle of Mechanical Stress and Inflammation in Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD) is a common acquired valvulopathy, which carries a high burden of mortality. Chronic inflammation has been postulated as the predominant pathophysiological process underlying CAVD. So far, no effective medical therapies exist to halt the progression of CAVD. This review aims to outline the known pathways of inflammation and calcification in CAVD, focussing on the critical roles of mechanical stress and mechanosensing in the perpetuation of valvular inflammation. Following initiation of valvular inflammation, dysregulation of proinflammatory and osteoregulatory signalling pathways stimulates endothelial-mesenchymal transition of valvular endothelial cells (VECs) and differentiation of valvular interstitial cells (VICs) into active myofibroblastic and osteoblastic phenotypes, which in turn mediate valvular extracellular matrix remodelling and calcification. Mechanosensitive signalling pathways convert mechanical forces experienced by valve leaflets and circulating cells into biochemical signals and may provide the positive feedback loop that promotes acceleration of disease progression in the advanced stages of CAVD. Mechanosensing is implicated in multiple aspects of CAVD pathophysiology. The mechanosensitive RhoA/ROCK and YAP/TAZ systems are implicated in aortic valve leaflet mineralisation in response to increased substrate stiffness. Exposure of aortic valve leaflets, endothelial cells and platelets to high shear stress results in increased expression of mediators of VIC differentiation. Upregulation of the Piezo1 mechanoreceptor has been demonstrated to promote inflammation in CAVD, which normalises following transcatheter valve replacement. Genetic variants and inhibition of Notch signalling accentuate VIC responses to altered mechanical stresses. The study of mechanosensing pathways has revealed promising insights into the mechanisms that perpetuate inflammation and calcification in CAVD. Mechanotransduction of altered mechanical stresses may provide the sought-after coupling link that drives a vicious cycle of chronic inflammation in CAVD. Mechanosensing pathways may yield promising targets for therapeutic interventions and prognostic biomarkers with the potential to improve the management of CAVD.

Role of peripheral myelin protein 22 in chronic exercise-induced interactions of muscle and bone in mice.

Exercise is important for the prevention and treatment of sarcopenia and osteoporosis. Although the interactions between skeletal muscles and bone have recently been reported, the myokines linking muscle to bone during exercise remain unknown. We previously revealed that chronic exercise using treadmill running blunts ovariectomy-induced osteopenia in mice. We herein performed an RNA sequence analysis of the gastrocnemius and soleus muscles of male mice with or without chronic exercise to identify the myokines responsible for the effects of chronic exercise on the muscle/bone relationship. We extracted peripheral myelin protein 22 (PMP22) as a humoral factor that was putatively induced by chronic exercise in the soleus and gastrocnemius muscles of mice from the RNA sequence analysis. Chronic exercise significantly enhanced the expression of PMP22 in the gastrocnemius and soleus muscles of female mice. PMP22 suppressed macrophage-colony stimulating factor and receptor activator factor κB ligand-induced increases in the expression of osteoclast-related genes and osteoclast formation from mouse bone marrow cells. Moreover, PMP22 significantly inhibited osteoblast differentiation, alkaline phosphatase activity, and mineralization in mouse osteoblast cultures; however, the overexpression of PMP22 did not affect muscle phenotypes in mouse muscle C2C12 cells. A simple regression analysis revealed that PMP22 mRNA levels in the gastrocnemius and soleus muscles were positively related to cortical bone mineral density at the femurs of mice with or without chronic exercise. In conclusion, we identified PMP22 as a novel myokine induced by chronic exercise in mice. We first showed that PMP22 suppresses osteoclast formation and the osteoblast phenotype in vitro.

Suppressing phosphoinositide-specific phospholipases Cγ1 promotes mineralization of osteoarthritic subchondral bone osteoblasts via increasing autophagy, thereby ameliorating articular cartilage degeneration.

Phosphoinositide-specific phospholipases C-γ1 (PLC-γ1) signaling has been shown to modulate osteoarthritis (OA) chondrocyte metabolism. However, the role of PLC-γ1 in OA osteoblasts remains unclear. Herein, whether and how PLC-γ1 was involved in mineralization in OA subchondral bone osteoblasts were investigated. Primary non-OA and OA osteoblasts of human and rat isolated from the subchondral bone or the calvaria were cultured in vitro, as well as mouse pre-osteoblastic cell line MC3T3-E1 cells. Rat knee OA model was induced by anterior cruciate ligament transection (ACLT), in which bone canal was carried out from the surface of lateral epicondyle of femur using micro-electric drill. Morphological characteristics of subchondral bone structure and articular cartilage were assessed using CT, micro-CT, and Safranin O/Fast green staining, respectively. Mineralization was measured by alizarin red staining. The expression and production of genes involved in osteoblastic phenotype and mineralization were evaluated by qPCR, western blotting, and immunohistochemistry assays, respectively. The inhibitions were performed using inhibitors and ShRNAs. The decreased relative bone density and thickness in the early stage of OA and the increased one in the late stage of OA were observed in subchondral bone of ACLT-rat model. Decreased ALP and OCN levels and absorbance values of ARS content were observed in in vitro osteoblasts isolated from 2 w post-ACLT rat model, as well as IL-1β-treated (for maintaining and mimicking inflammatory status) human OA and rat osteoblasts. Decreased Atg7 level and LC3BII/I ratio in combination with an increase in the P62 level, was concomitant with decreased ALP and OCN mRNA levels and absorbance values of ARS content in OA or IL-1β-treated osteoblasts. Specific inhibition of PLC-γ1 by ShRNAs or inhibitor (U73122) elevated ALP and OCN mRNA levels and absorbance values of ARS content accompanied with increased Atg7 level and LC3BII/I ratio in combination with a decrease in the P62 level in OA osteoblasts. Furthermore, the promoting effect of PLC-γ1 inhibition on ALP and OCN mRNA levels and absorbance values of ARS content was reversed by endoplasmic reticulum (ER) stress activator HA15, as well as autophagic inhibitors CQ and 3MA. Injection with PLC-γ1 inhibitor U73122 from the surface of lateral epicondyle of femur reduced aberrant subchondral bone formation and attenuated articular cartilage degeneration in ACLT-rat. Aberrant changes of OA subchondral bone structure were concomitant with altered osteoblastic phenotype and mineralization. Impaired autophagy contributed to decreased osteoblastic mineralization in the early stage of OA. PLC-γ1 inhibition promoted osteoblastic mineralization through increasing autophagy in OA osteoblasts, which was partially attributed to suppression of ER stress. Targeting PLC-γ1 in subchondral bone osteoblasts could be more efficacious for OA therapy through treating the bone and cartilage at the same time. In summary, we hypothesize that suppressing PLCγ1 promotes mineralization of osteoarthritic subchondral bone osteoblasts via increasing autophagy, thereby ameliorating articular cartilage degeneration.

Influence of Medical Marijuana on Mesenchymal Stromal Cell Osteogenesis: An in Vitro Study

Category:Basic Sciences/Biologics; Basic Sciences/Biologics; Sports; OtherIntroduction/Purpose:Recently, purposes of medicinal marijuana including analgesia and reduction in inflammation. The active component activates the CB1 and CB2 receptors, thus mimicking the action of endogenous cannabinoids. CB1 and CB2 are important regulators of bone metabollism. In particular, endocannabinoid signaling has shown to regulate proliferation and differentiation of MSCs and the activities of osteoblasts and osteoclasts. Cannabinoids may be prescribed to patients suffering bone fractures involving prolonged immobilization and discomfort. Thus, there is an important need to understand more completely the role of cannanbinoid signaling in fracture healing. In this study, we examine the influence of a synthetic cannabinoid agonist, Win-55,212-2 (Win) on adult, human MSC-derived osteoblast activity. Cell viability and osteogenic phenotype were assessed by MTS assay, RT-PCR, and alizarin red staining.Methods:With an IRB approved protocol, MSCs were isolated from healthy human bone marrow and expanded to the passage five for experimentation. Cells were then plated at 5,200 cells/cm2 (6-well plate) and osteogenically stimulated for 21 days before treatment with increasing concentrations of Win (0.01, 0.1, or 1 µM) for an additional 48 hours. The MTS assay was employed to determine the half-maximal (50%) inhibitory concentration (IC50) at different time points. The cell phenotype was assessed by real-time PCR and alizarin red staining.Results:We first used the MTS assay (for metabolism and proliferation) to test 7 doses of Win-55, from 0.001µM up to 5 µM and determined that the human osteoblast IC50 of Win-55 over 1-7 days of exposure was ~ 1.5 µM (Figure 1), a dose slightly lower than concentrations found to affect chondrocytes in a previous study. At 1µM Win-55, RT-PCR revealed that the expression of inflammatory cytokines IL-1beta and TNF-alpha was significantly reduced, while that of IL-6 and COX2 was unchanged. The expression of most osteogenic markers was unchanged by Win, but we did observe significant increases in BMP2, RUNX2 and OPN (Figure 2). Alizarin Red staining revealed increased calcium deposition and mineralization in the presence of 1µM Win, corroborating the RT-PCR results (Figure 3).Conclusion:Win-55,212-2 may be beneficial to osteogenesis. 1 µM Win enhanced the osteoblastic phenotype over control, untreated MSC-derived osteoblasts, reduced inflammatory mediators and increased calcium deposition and mineralization. This suggests that cannabidiol treatment might lead to improvements in fracture healing and provide a novel therapeutic option for the bone regeneration. Future studies are aimed at characterizing the CB-1 and CB-2 signaling associated with the changes observed here and the effects of Win on MSC-derived osteoblasts in the context of inflammation.

8-Nitro-cGMP suppresses mineralization by mouse osteoblasts.

Nitric oxide and reactive oxygen species regulate bone remodeling, which occurs via bone formation and resorption by osteoblasts and osteoclasts, respectively. Recently, we found that 8-nitro-cGMP, a second messenger of nitric oxide and reactive oxygen species, promotes osteoclastogenesis. Here, we investigated the formation and function of 8-nitro-cGMP in osteoblasts. Mouse calvarial osteoblasts were found to produce 8-nitro-cGMP, which was augmented by tumor necrosis factor-α (10 ng/ml) and interleukin-1β (1 ng/ml). These cytokines suppressed osteoblastic differentiation in a NO synthase activity-dependent manner. Exogenous 8-nitro-cGMP (30 μmol/L) suppressed expression of osteoblastic phenotypes, including mineralization, in clear contrast to the enhancement of mineralization by osteoblasts induced by 8-bromo-cGMP, a cell membrane-permeable analog of cGMP. It is known that reactive sulfur species denitrates and degrades 8-nitro-cGMP. Mitochondrial cysteinyl-tRNA synthetase plays a crucial role in the endogenous production of RSS. The expression of osteoblastic phenotypes was suppressed by not only exogenous 8-nitro-cGMP but also by silencing of the Cars2 gene, indicating a role of endogenous 8-nitro-cGMP in suppressing the expression of osteoblastic phenotypes. These results suggest that 8-nitro-cGMP is a negative regulator of osteoblastic differentiation.

Dextran sulfate-amplified extracellular matrix deposition promotes osteogenic differentiation of mesenchymal stem cells

The development of bone-like tissues in vitro that exhibit key features similar to those in vivo is needed to produce tissue models for drug screening and the study of bone physiology and disease pathogenesis. Extracellular matrix (ECM) is a predominant component of bone in vivo; however, as ECM assembly is sub-optimal in vitro, current bone tissue engineering approaches are limited by an imbalance in ECM-to-cell ratio. We amplified the deposition of osteoblastic ECM by supplementing dextran sulfate (DxS) into osteogenically induced cultures of human mesenchymal stem cells (MSCs). DxS, previously implicated to act as a macromolecular crowder, was recently demonstrated to aggregate and co-precipitate major ECM components, including collagen type I, thereby amplifying its deposition. This effect was re-confirmed for MSC cultures undergoing osteogenic induction, where DxS supplementation augmented collagen type I deposition, accompanied by extracellular osteocalcin accumulation. The resulting differentiated osteoblasts exhibited a more mature osteogenic gene expression profile, indicated by a strong upregulation of the intermediate and late osteogenic markers ALP and OCN, respectively. The associated cellular microenvironment was also enriched in bone morphogenetic protein 2 (BMP-2). Interestingly, the resulting decellularized matrices exhibited the strongest osteo-inductive effects on re-seeded MSCs, promoted cell proliferation, osteogenic marker expression and ECM calcification. Taken together, these findings suggest that DxS-mediated enhancement of osteogenic differentiation by MSCs is mediated by the amplified ECM, which is enriched in osteo-inductive factors. We have thus established a simple and reproducible approach to generate ECM-rich bone-like tissue in vitro with sequestration of osteo-inductive factors. Statement of significanceAs extracellular matrix (ECM) assembly is significantly retarded in vitro, the imbalance in ECM-to-cell ratio hampers current in vitro bone tissue engineering approaches in their ability to faithfully resemble their in vivo counterpart. We addressed this limitation by leveraging a poly-electrolyte mediated co-assembly and amplified deposition of ECM during osteogenic differentiation of human mesenchymal stem cells (MSCs). The resulting pericelluar space in culture was enriched in organic and inorganic bone ECM components, as well as osteo-inductive factors, which promoted the differentiation of MSCs towards a more mature osteoblastic phenotype. These findings thus demonstrated a simple and reproducible approach to generate ECM-rich bone-like tissue in vitro with a closer recapitulation of the in vivo tissue niche.

Anti-osteoblastogenic, pro-inflammatory and pro-angiogenic effect of extracellular vesicles isolated from the human osteosarcoma cell line MNNG/HOS.

Extracellular Vesicles (EVs) are becoming increasingly recognized as integral signaling vehicles in several types of cancers, including bone malignancies. However, the specific mechanisms by which EVs influence osteosarcoma progression have not been fully determined. We evaluated the effects of EVs derived from the human osteosarcoma cell line MNNG/HOS (MNNG/HOS-EVs) on bone resident cells. We found that MNNG/HOS-EVs are internalized by osteoblasts and osteoclasts in vitro, with potent inhibitory effects on osteoblast metabolic activity, cell density and alkaline phosphatase activity. Consistently, MNNG/HOS-EVs reduced the expression of cell cycle and pro-osteoblastogenic genes, whilst increasing transcriptional expression and protein release of pro-osteoclastogenic/inflammatory cytokines (RankL, Il1b, Il6 and Lcn2), pro-tumoral cytokines (CCL2,5,6,12 and CXCL1,2,5) and the metalloproteinase MMP3. MNNG/HOS-EVs did not induce osteoclast differentiation, while promoting in vitro and in vivo angiogenesis. Intriguingly, EVs derived from another osteosarcoma cell line (U2OS) reduced ALP activity but had no other effect on osteoblast phenotype. MNNG/HOS-EVs were also found to dramatically increase Serpin b2 expression in osteoblasts. To evaluate the significance of this finding, osteoblasts were forced to overexpress Serpin b2, which however did not affect osteoblast differentiation, while Il6 and Lcn2 mRNAs were up regulated. Overall, we shed light on the interactions of osteosarcoma EVs with the cells of the bone microenvironment, identifying key anti-osteoblastogenic, pro-inflammatory and pro-angiogenic factors that could contribute to osteosarcoma expansion.

IL-18 Mediates Vascular Calcification Induced by High-Fat Diet in Rats With Chronic Renal Failure.

Objective: Vascular calcification (VC) is an important predictor of cardiovascular morbidity and mortality in patients with chronic renal failure (CRF). It is well-known that obesity and metabolic syndrome (OB/MS) predicts poor prognosis of CRF patients. However, the influence of OB/MS on VC in CRF patients isn't clear. IL-18 mediates OB/MS-related inflammation, but whether IL-18 is involved in OB/MS -mediated VC in CRF patients hasn't been studied. In this study, it was explored that whether OB/MS caused by high-fat diet (HFD) can affect the level of serum IL-18 and aggravate the degree of VC in CRF rats. Furthermore, it was studied that whether IL-18 induces rat vascular smooth muscle cells (VSMCs) calcification by activating the MAPK pathways.Approach: The rats were randomly assigned to the sham-operated, CRF and CRF + HFD groups. CRF was induced by 5/6 nephrectomy. Serum IL-18 levels and aortic calcification indicators were compared in each group. Primary rat VSMCs calcification were induced by β-glycerophosphate and exposed to IL-18. VSMCs were also treated with MAPK inhibitors.Results: The weight, serum levels of hsCRP, TG and LDL-C in CRF + HFD group were significantly higher than those in sham-operated and CRF groups (p < 0.05). Compared with the sham-operated group, the calcium content and the expression of BMP-2 of aorta in CRF and CRF + HFD groups were significantly increased (p < 0.05). Moreover, the calcium content and the expression of BMP-2 of aorta in CRF + HFD group was significantly higher than those in CRF group (p < 0.05). And the serum IL-18 level was positively correlated with aortic calcium content. It was also found that p38 inhibitor SB203580 can suppress the VSMCs calcification and osteoblast phenotype differentiation induced by IL-18. But the JNK inhibitor SP600125 can't suppress the VSMCs calcification and osteoblast phenotype differentiation induced by IL-18.Conclusions: These findings suggest that obesity-related inflammation induced by high-fat diet could exacerbate VC in CRF rats. Furthermore, serum IL-18 level had a positive correlation with the degree of VC. It is also found that IL-18 promoted osteogenic differentiation and calcification of rat VSMCs via p38 pathway activation.

Abstract 13243: Integrin Linked Kinase Expression in Human Valvular Endothelial Cells Plays a Protective Role in Calcific Aortic Valve Disease

Introduction: Calcific aortic valve disease (CAVD) is the most common valvular heart disease in the aging population. CAVD is a highly active ossification process originated in valvular interstitial cells which differentiate towards an osteoblastic phenotype. However, the role of valvular endothelial cells in CAVD has not been fully explored. We previously demonstrated that ILK expression in vascular endothelium plays an essential cardioprotective role. Hypothesis: Endothelial ILK plays a protective role in human valvular endothelial cells osteogenesis. Methods and Results: Human aortic valves were obtained from 52 patients with calcific aortic stenosis compared with 16 non-calcified aortic valves and ILK levels were analyzed. Our study showed a significant relationship between the degree of AV calcification and decreased ILK expression detected by IHQ and qPCR (p&lt; 0,013). Moreover, decreased ILK expression correlated with BMP2 and Runx2, two markers of valvular calcification (p&lt;0.01). Human valvular endothelial cells (hVECs) isolated from non-calcified controls display the ability to calcify when cultured in pro-calcifiying conditions and ILK levels were unaffected by the pro-calcifiying conditions. However, silencing ILK expression in hVECs resulted in decreased NO production, increased expression of calcific disease markers Runx2, BMP2, and osteopontin. ILK silencing in hVECs induced calcium deposition, determined by alizarin red staining, when cultured in pro-calcifiying conditions. Thus, our results suggest that decreased ILK expression could promote endothelial to osteogenic phenotype transition. Restoring nitric oxide levels by DETA-NONOate treatment in ILK silenced hVEC was able to reverse those effects partially. Conclusions: Collectively, our results point to a crucial role of ILK in maintaining human valve endothelial cell function preventing valvular calcification.