Osteoblastic Cell Line Research Articles

SLC38A2 provides proline and alanine to regulate postnatal bone mass accrual in mice.

Amino acids have recently emerged as important regulators of osteoblast differentiation and bone formation. Osteoblasts require a continuous supply of amino acids to sustain biomass production to fuel cell proliferation, osteoblast differentiation and bone matrix production. We recently identified proline as an essential amino acid for bone development by fulfilling unique synthetic demands that are associated with osteoblast differentiation. Osteoblasts rely on the amino acid transporter SLC38A2 to provide proline to fuel endochondral ossification. Despite this, very little is known about the function or substrates of SLC38A2 during bone homeostasis. Here we demonstrate that the neutral amino acid transporter SLC38A2 is expressed in osteoblast lineage cells and provides proline and alanine to osteoblast lineage cells. Genetic ablation of SLC38A2 using Prrx1Cre results in decreased bone mass in both male and female mice due to a reduction in osteoblast numbers and bone forming activity. Decreased osteoblast numbers are attributed to impaired proliferation and osteogenic differentiation of skeletal stem and progenitor cells. Collectively, these data highlight the necessity of SLC38A2-mediated proline and alanine uptake during postnatal bone formation and bone homeostasis.

Efficacy of Trevesia palmata (Roxb. ex Lindl.) Vis. Extract on MG 63 cell lines and arthritis-induced animal models

Ethnopharmacological relevanceDespite widespread use of herbal remedies for treating arthritis and osteosarcoma, many plants are still not pharmacologically evaluated for their efficacy. Contrary to many non-steroidal, immunosuppressants, antibiotics, and antineoplastic drugs that have adverse effects, phytotherapeutic compounds have promising benefits with fewer complications. In this study the unexplored Northeastern India indigenous plant Trevesia palmata (Roxb. ex Lindl.) Vis. used in traditional medicine to cure bone fractures is chosen for studying anti-proliferative and anti-rheumatic properties. Aim of the studyThis study designed to explore the polyphenolic composition, antioxidant, anti-inflammatory and anti-arthritic potential of T. palmata leaf extracts. Further, the cellular activity was studied using MG 63 osteoblast cell lines and pharmacologically evaluated using Complete Freund's Adjuvant (CFA) induced arthritic rat model. Materials and methodsIn vitro free radical scavenging activity, anti-inflammatory and anti-arthritic activities of extracts were analyzed using standardized methods. The polyphenolic profiling and apoptosis inducing ability of T. palmata ethyl acetate (TPEA) extract on MG 63 osteoblast cell lines were analyzed. The in vivo pharmacological studies were carried out with low dose 250 mg/kg and high dose of 500 mg/kg of T. palmata. The biochemical and haematological parameters and in vivo antioxidant activity were evaluated for the control and treated groups. Radiological and histological study were done to understand the impact and penetration of inflammatory arthritis from tissues to joint bones. ResultsTPEA showed highest free radical scavenging activity (DPPH - 4.72 IC50, ABTS - 242.33 ± 6.81 mM TE/g extract), anti-inflammatory (40.04% inhibition of RBC lysis) and anti-arthritic activity (32.4% inhibition of protein denaturation) with the presence of gallic acid, catechin, caffeic acid, rutin, quercetin and naringenin. The TPEA extract inhibited cell proliferation of MG 63 osteoblast cells and induced apoptosis by arresting cell cycle at different phases. After acute toxicity studies the doses 250 mg/kg and 500 mg/kg were fixed and showed better results in CFA-induced arthritic animals. Thus, the extract phytoconstituents may have immense potential against chronic inflammation, joint ailments, bone cancer and arthritis which serves as a phytomedicine contrary to synthetic medications. ConclusionsThe potential treatment of polyphenolic compounds in the T. palmata extract on osteosarcoma and arthritis was demonstrated from this study. Thus, cellular inflammatory infiltrates are significantly reduced in bone and joint tissues as well.

Loss of Vhl alters trabecular bone loss during S. aureus osteomyelitis in a cell-specific manner.

Osteomyelitis, or bone infection, is a major complication of accidental trauma or surgical procedures involving the musculoskeletal system. Staphylococcus aureus is the most frequently isolated pathogen in osteomyelitis and triggers significant bone loss. Hypoxia-inducible factor (HIF) signaling has been implicated in antibacterial immune responses as well as bone development and repair. In this study, the impact of bone cell HIF signaling on antibacterial responses and pathologic changes in bone architecture was explored using genetic models with knockout of either Hif1a or a negative regulator of HIF-1α, Vhl. Deletion of Hif1a in osteoblast-lineage cells via Osx-Cre (Hif1aΔOB ) had no impact on bacterial clearance or pathologic changes in bone architecture in a model of post-traumatic osteomyelitis. Knockout of Vhl in osteoblast-lineage cells via Osx-Cre (VhlΔOB ) caused expected increases in trabecular bone volume per total volume (BV/TV) at baseline and, intriguingly, did not exhibit an infection-mediated decline in trabecular BV/TV, unlike control mice. Despite this phenotype, bacterial burdens were not affected by loss of Vhl. In vitro studies demonstrated that transcriptional regulation of the osteoclastogenic cytokine receptor activator of NF-κB ligand (RANKL) and its inhibitor osteoprotegerin (OPG) is altered in osteoblast-lineage cells with knockout of Vhl. After observing no impact on bacterial clearance with osteoblast-lineage conditional knockouts, a LysM-Cre model was used to generate Hif1aΔMyeloid and VhlΔMyeloid mouse models to explore the impact of myeloid cell HIF signaling. In both Hif1aΔMyeloid and VhlΔMyeloid models, bacterial clearance was not impacted. Moreover, minimal impacts on bone architecture were observed. Thus, skeletal HIF signaling was not found to impact bacterial clearance in our mouse model of post-traumatic osteomyelitis, but Vhl deletion in the osteoblast lineage was found to limit infection-mediated trabecular bone loss, possibly via altered regulation of RANKL-OPG gene transcription.

Investigation of the influence of SrO addition on the bioactivity of CaO[sbnd]Na2O-P2O5-B2O3 glass system

In this work, the role of strontium oxide in the bioactive glasses calcium-sodium-borate was studied to evaluate the bioactivity of the system and the rate of the ion release in Simulated Body Fluid (SBF). Five samples were prepared, with concentration of strontium oxide (SrO) from 0 to 20 wt%, in order to understand how SrO change the glass structure and the ionic leaching process in SBF. Results of density and molar volume, XRD, FTIR, weight loss, pH and cytotoxicity assay were presented and discussed as a function of SrO. The measured pH values of SBF, presented lower values for samples with greater amounts of SrO, suggesting that the increase of SrO in the glasses can reduce the release of the ions in SBF. The fraction of tetracoordinate boron, N4, calculated by FTIR results, increases with the addition of SrO, which shows an increase in the glass connectivity and a decrease of the chemical reactivity. The FTIR spectra and X-ray diffraction results, after immersion in SBF, confirmed the materials bioactivity with the formation of carbonated hydroxyapatite or crystalline calcium phosphate. The samples did not show in vitro cytotoxicity against osteoblast cell line. Also, we highlight that 10SrO sample has induced proliferation of osteoblast-like cells in vitro to all concentrations of glass. These results show that SrO doped calcium-sodium-borate glasses are materials with high rate of bioactivity, low cytotoxicity and reduced dissolution rate of the glasses in an aqueous medium, besides induce cells proliferation and bone tissue growth and their biocompatibility with living tissues.

Anti-inflammatory properties of S53P4 bioactive glass implant material

ObjectivesTo assess whether the dissolution products of S53P4 bioactive glass (BG) affect cellular response of macrophages and clinically relevant peri‑implant cell populations to dental implant particles in vitro. Cells chosen were human gingival fibroblasts (HGFs), osteoblasts and bone marrow derived stromal cells (HBMSCs). MethodsMelt-derived S53P4 bioactive glass were prepared. HGFs, Saos-2 human osteoblastic cell line, HBMSCs and macrophages, derived from THP-1 human monocytic cell line, were cultured in the presence of particles from commercially pure titanium (Ti-CP4), grade 5 titanium alloy (Ti-6Al-4V), titanium-zirconium alloy (Ti-15Zr) or zirconia (Zr) (with respective diameters of 34.1 ± 3.8, 33.3 ± 4.4, 97.8 ± 8.2 and 71.3 ± 6.1 µm) with or without S53P4 dissolution products (conditioned media contained 327.30 ± 2.01 ppm Ca, 51.34 ± 0.41 ppm P and 61.48 ± 1.17 ppm Si, pH 8.01 ± 0.21). Inflammatory and macrophage polarisation markers including TNF-ɑ, IL-1, IL-6 and CD206 were quantified using enzyme-linked immunosorbent assay (ELISA). ResultsThe presence of Ti-6Al-4V implant particles significantly induced the expression of pro-inflammatory markers in all tested cell types. S53P4 BG dissolution products regressed the particle induced up-regulation of pro-inflammatory markers and, appeared to suppress M1 macrophage polarisation. ConclusionsImplant particles, Ti-6Al-4V in particular, resulted in significant inflammatory responses from cells. S53P4 BG may possess anti-inflammatory properties and potentially mediate macrophage polarisation behaviour. Clinical SignificanceThe findings highlight that the use and benefits of BG is a promising field of study. Authors believe more collective efforts are required to fully understand the reliability, efficiency and exact mechanisms of action of BG in the search for new generation of treatment modalities in dentistry.

Effects of rmBMP-7 on Osteoblastic Cells Grown on a Nanostructured Titanium Surface

This study evaluates the effects of the availability of exogenous BMP-7 on osteoblastic cells’ differentiation on a nanotextured Ti surface obtained by chemical etching (Nano-Ti). The MC3T3-E1 and UMR-106 osteoblastic cell lines were cultured for 5 and 7 days, respectively, on a Nano-Ti surface and on a control surface (Control-Ti) in an osteogenic medium supplemented with either 40 or 200 ng/mL recombinant mouse (rm) BMP-7. The results showed that MC3T3-E1 cells exhibited distinct responsiveness when exposed to each of the two rmBMP-7 concentrations, irrespective of the surface. Even with 40 ng/mL rmBMP-7, important osteogenic effects were noticed for Control-Ti in terms of cell proliferation potential; Runx2, Osx, Alp, Bsp, Opn, and Smad1 mRNA expression; and in situ ALP activity. For Nano-Ti, the effects were limited to higher Alp, Bsp, and Opn mRNA expression and in situ ALP activity. On both surfaces, the osteogenic potential of UMR-106 cultures remained unaltered with 40 ng/mL rmBMP-7, but it was significantly reduced when the cultures were exposed to the 200 ng/mL concentration. The availability of rmBMP-7 to pre-osteoblastic cells at the concentrations used alters the expression profile of osteoblast markers, indicative of the acquisition of a more advanced stage of osteoblastic differentiation. This occurs less pronouncedly on the nanotextured Ti and without reflecting in higher mineralized matrix production by differentiated osteoblasts on both surfaces.

Farklı Tiplerdeki Kök Kanal Yıkama Solüsyonlarının İnsan Osteoblastik Hücre Canlılığı Üzerine Etkileri

ABSTRACT
 Objective: Root canal irrigation is very important for successful root canal treatment. During this procedure, root dentin and periapical tissues are in contact with irrigation solutions. These solutions should ideally remove pulpal tissues without damaging the oral tissues. The aim of this study was to evaluate the effects of 7 different irrigation solutions (Rocanal, BioPure MTAD, SmearClear, chlorhexidine gluconate, EDTA, hydrogen peroxide, NaOCl) on the viability of human osteoblastic cell lines (SaOS-2).
 Methods: The tested solutions were diluted with culture medium and prepared in 6 different concentrations (1:2, 1:4, 1:8, 1:16, and 1:32). SaOS-2 cells were exposed to all concentrations of irrigation solutions, respectively (n = 12). Cell culture medium without test material was used as a control group. Cell viability was evaluated with the MTT cytotoxicity test. The cell viability of the control group was equal to 100%, and the data were statistically evaluated by One-way ANOVA and post-hoc Tukey's HSD tests.
 Results: All tested solutions had cytotoxic effects on SaOS-2 cells at all concentrations (P < 0.05). There was difference in percentage between the cell viability values of the materials (P

Functionalization with Polyphenols of a Nano-Textured Ti Surface through a High-Amino Acid Medium: A Chemical-Physical and Biological Characterization.

The study aimed to identify an effective mechanism of adsorption of polyphenols on a nano-textured Ti surface and to evaluate the osteogenic differentiation on it. The source of polyphenols was a natural extract from red grape pomace. A chemical etching was used to form an oxide layer with a nanoscale texture on Ti; this layer is hydrophilic, but without hydroxyl groups with high acidic–basic chemical reactivity. The samples were characterized by electron and fluorescence microscopies, UV–Vis spectroscopy, contact angle measurements, zeta potential titration curves, and Folin–Ciocâlteu test. The presence of an adsorbed layer of polyphenols on the functionalized surface, maintaining redox ability, was confirmed by several tests. Consistent with the surface features, the adsorption was maximized by dissolving the extract in a high–amino acid medium, with respect to an inorganic solution, exploiting the high affinity of amino acids for polyphenols and for porous titanium surfaces. The osteogenic differentiation was assessed on an osteoblastic cell line by immunofluorescence, cell viability, expression of key osteoblast markers, and extracellular matrix mineralization. The surfaces functionalized with the extract diluted in the range 1 × 10−5–1 mg/mL resulted in having a greater osteogenic activity for the highest concentration, with lower values of cell viability; higher expression of alkaline phosphatase, bone sialoprotein, and collagen; and lower levels of osteopontin. In conclusion, the functionalization of a nano-textured Ti surface with polyphenols can potentially favor the osteogenic activity of osseointegrated implants.

Synthesis of nano hydroxyapatite from Hypopthalmichthys molitrix (silver carp) bone waste by two different methods: a comparative biophysical and in vitro evaluation on osteoblast MG63 cell lines.

More than a thousand tonnes of fish bone wastes can be transformed into biomedical products annually. Alkaline hydrolysis and thermal calcification were used to create nanosized hydroxyapatite (HAp) crystals from Silver carp bone wastes. Biophysical tests were used to determine the nano size and chemical composition of synthesised hydroxyapatite. Alkaline hydrolysis hydroxyapatite (AH-HAp) was 58.3nm, while Thermal calcination hydroxyapatite (TC-HAp) was 64.3nm in size, confirmed by Atomic Force Microscopy. Energy Dispersive X-ray Analysis studies showed Ca/P (Calcium phosphate) ratio of AH-HAp to be 1.65, whereas TC-HAp as 1.45, confirming AH-HAp to be organically rich along with a similar Ca/P ratio as natural HAp. Fourier Transform Infrared Spectroscopy spectra indicated HAp formation from both procedures, however AH-HAp had superior crystallinity than TC-HAp confirmed from X-Ray Diffraction spectra. MG63 osteoblast cell lines showed 91% cell viability in cytotoxicity studies and 70.1% proliferation efficiency in Alkaline Phosphatase assay, which was higher than TC-HAp. The present study shows that HAp produced via alkaline hydrolysis has better biocompatibility which enhances its applicability as a biomaterial, than HAp synthesized through thermal calcination, which tends to incinerate organic moieties.

Maresin1 Suppresses High-Glucose-Induced Ferroptosis in Osteoblasts via NRF2 Activation in Type 2 Diabetic Osteoporosis.

Maresin1 (MaR1) is an endogenous pro-resolving lipid mediator produced from polyunsaturated fatty acids and is believed to have antioxidant and anti-inflammatory properties. The objective of this study was to estimate MaR1′s impact on type 2 diabetic osteoporosis (T2DOP) and its pharmacological mode of action. An in vitro high-glucose model of the osteoblast cell line MC3T3-E1 was constructed and stimulated with MaR1. Type 2 diabetic rats were used to establish in vivo models of calvarial defects and were treated in situ with MaR1. The results revealed that, aside from preventing mortality and promoting the osteogenic capacity of MC3T3-E1 cells, MaR1 increased nuclear factor erythroid-2 related factor 2 (NRF2) signaling as well as the activity of glutathione peroxidase 4 (GPX4) and cystine-glutamate antiporter (SLC7A11) and caused the restraint of ferroptosis under hyperglycemic stimulation. However, the therapeutic impact of MaR1 was significantly diminished due to NRF2-siRNA interference and the ferroptosis activator Erastin. Meanwhile, these results were validated through in vivo experiments. These findings imply that MaR1 activated the NRF2 pathway in vivo and in vitro to alleviate high-glucose-induced ferroptosis greatly. More crucially, MaR1 might effectively reduce the risk of T2DOP.

Loading-induced bone formation is mediated by Wnt1 induction in osteoblast-lineage cells.

Mechanical loading on the skeleton stimulates bone formation. Although the exact mechanism underlying this process remains unknown, a growing body of evidence indicates that the Wnt signaling pathway is necessary for the skeletal response to loading. Recently, we showed that Wnts produced by osteoblast lineage cells mediate the osteo-anabolic response to tibial loading in adult mice. Here, we report that Wnt1 specifically plays a crucial role in mediating the mechano-adaptive response to loading. Independent of loading, short-term loss of Wnt1 in the Osx-lineage resulted in a decreased cortical bone area in the tibias of 5-month-old mice. In females, strain-matched loading enhanced periosteal bone formation in Wnt1F/F controls, but not in Wnt1F/F; OsxCreERT2 knockouts. In males, strain-matched loading increased periosteal bone formation in both control and knockout mice; however, the periosteal relative bone formation rate was 65% lower in Wnt1 knockouts versus controls. Together, these findings show that Wnt1 supports adult bone homeostasis and mediates the bone anabolic response to mechanical loading.

New PCL/PEC Blends: In Vitro Cell Response of Preosteoblasts and Human Mesenchymal Stem Cells.

Simple SummaryA topic of great relevance is the design of new biocompatible materials for their application in tissue engineering. The preparation of new polycaprolactone (PCL)/poly (ethylene carbonate) (PEC) blends has resulted in a series of new biomaterials obtained in a simple manner, which favor cell proliferation and subsequent differentiation using two different cell lines, the osteoblastic cell line (MC3T3-E1) and human mesenchymal cells (hMSCs), whose results indicate that these materials could be used in future to carry out 3D forming with the purpose of testing their potential application in tissue engineering for bone regeneration through “in vivo” assays.In this study, new blends of PCL/PEC have been prepared in an easy manner by casting with the objective of obtaining new biomaterials to apply to tissue engineering and bone regeneration. The PCL/PEC blends obtained, together with neat polymer blends, were characterized by infrared spectroscopy (FTIR), atomic force microscopy (AFM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). This full characterization is the key to disentangle the miscibility, which means good compatibility, of the polymer blends used in this work. The addition of increasing amounts of PEC, has shown in the new biomaterials obtained, a remarkable improvement in relation with the mechanical properties (manageable materials) and above all, in terms of an increase in their hydrophilic character with respect to the PCL neat polymer. The improvement of all these properties is reflected in their biological properties. With these thoughts in mind, the blends obtained were tested through the assessment of several biological parameters such as cell viability, proliferation, and differentiation of both the MC3T3-E1 osteoblastic cell line and hMSCs to evaluate their cell response to different polymer membranes aimed at bone tissue regeneration. “In vitro” biocompatibility methods have been chosen rather than in vivo studies due to their lower cost, faster procedure time, and minimum ethical concerns, and because it was the first time that the biological effects of these blends were studied. The results show that the PCL/PEC blends obtained, with tunable properties in terms of hydrophilic character and hydrolytic degradation, may be regarded as good candidates to perform “in vivo” tests and check their real-life applicability for bone regeneration. The polymer acronym (the weight percentage in the sub index) is PCLx/PECy as noted in table one with the summary of compositions.

Osteoblast Lineage Cell-derived Sema3A Regulates Bone Homeostasis Independently of Androgens.

Semaphorin 3A (Sema3A) coordinates bone resorption and formation under the control of estrogen signaling. However, the contribution of osteoblast lineage cell-derived Sema3A to vertebral homeostasis has remained unclear. Moreover, it is unknown whether androgen signaling is involved in Sema3A expression in osteoblast lineage cells. In this study, we show that osteoblast lineage cell-derived Sema3A plays a key role in bone homeostasis independent of androgen signaling. Sema3a deletion with Sp7-Cre did not alter the trabecular bone mass in lumbar vertebrae, along with there being no significant difference in Sema3a mRNA expression. In contrast, osteoblast lineage cell-specific deletion of Sema3A with BGLAP-Cre led to decreased bone volume in both long bones and lumbar vertebrae. In addition, osteoblast lineage cell-derived Sema3A was not involved in orchidectomy-induced bone loss because androgen deficiency did not affect Sema3A protein expression. Thus, these results indicate that Sema3A derived from osteoblast lineage cells acts as an osteoprotective factor, even in vertebrae, and its expression is controlled in an androgen-independent manner.

Enhancing physicochemical, mechanical, and bioactive performances of monetite nanoparticles reinforced chitosan‐PEO electrospun scaffold for bone tissue engineering

AbstractIn the current work, monetite (DCPA) nanoparticles incorporated chitosan (CH)‐based composite was prepared using by electrospinning technique. A smooth and defect‐free fibrous matrix with an average fiber diameter in the range between 311 ± 13 to 380 ± 28 nm was obtained for 5 wt% (CH‐DCPA5) and 7 wt% DCPA (CH‐DCPA7) containing electrospun scaffolds. The results indicated that the addition of 7 wt% DCPA nanoparticles to chitosan caused an increase in the tensile strength of scaffolds from 6.2 to 12.34 MPa. A slow degradation rate (~37% in 28 days) indicates its suitability for bone tissue regeneration. A study on the biomineralization capability of the electrospun scaffolds after their immersion in simulated body fluid in vitro revealed that the addition of DCPA strongly promoted the deposition of the apatite layer onto electrospun nanofibrous scaffolds. The culture of MG‐63 osteoblast cells on the scaffolds demonstrated that the incorporation of DCPA nanoparticles into the chitosan polymeric matrix helped in the adhesion, spreading, and migration of cells. CH‐DCPA5 and CH‐DCPA7 scaffolds exhibited a higher capacity to proliferate osteoblast cell lines as compared to pure CH scaffold as confirmed by MTT assay and qualitative cell viability analysis from fluorescence microscopy. Immunocytochemistry analysis for osteocalcin expression revealed a higher capacity of DCPA‐containing scaffolds to differentiate MG‐63 cells into bone lineage as compared to pure chitosan scaffolds. DCPA incorporation into chitosan also resulted in MG‐63 cultured on to it to elicit higher alkaline phosphatase activity suggesting the highest capacity of CH‐DCPA7 to differentiate MG 63 cell line among all the scaffolds studied here. The results indicated superior osteogenic properties of DCPA nanoparticle reinforced chitosan matrix for use in BTE in the form of nanofibrous scaffolds.

Immunomodulation Effect of Biomaterials on Bone Formation.

Traditional bone replacement materials have been developed with the goal of directing the osteogenesis of osteoblastic cell lines toward differentiation and therefore achieving biomaterial-mediated osteogenesis, but the osteogenic effect has been disappointing. With advances in bone biology, it has been revealed that the local immune microenvironment has an important role in regulating the bone formation process. According to the bone immunology hypothesis, the immune system and the skeletal system are inextricably linked, with many cytokines and regulatory factors in common, and immune cells play an essential role in bone-related physiopathological processes. This review combines advances in bone immunology with biomaterial immunomodulatory properties to provide an overview of biomaterials-mediated immune responses to regulate bone regeneration, as well as methods to assess the bone immunomodulatory properties of bone biomaterials and how these strategies can be used for future bone tissue engineering applications.

Hydroxyapatite from Natural Sources for Medical Applications.

The aim of this work is to study the physical-chemical, mechanical, and biocompatible properties of hydroxyapatite obtained by hydrothermal synthesis, at relatively low temperatures and high pressures, starting from natural sources (Rapana whelk shells), knowing that these properties influence the behavior of nanostructured materials in cells or tissues. Thus, hydroxyapatite nanopowders were characterized by chemical analysis, Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), scanning electron microscopy (SEM), and X-ray diffraction (XRD). In vitro studies on osteoblast cell lines (cytotoxicity and cell proliferation), as well as preliminary mechanical tests, have been performed. The results showed that the obtained powders have a crystallite size below 50 nm and particle size less than 100 nm, demonstrating that hydrothermal synthesis led to hydroxyapatite nanocrystalline powders, with a Ca:P ratio close to the stoichiometric ratio and a controlled morphology (spherical particle aggregates). The tensile strength of HAp samples sintered at 1100 °C/90 min varies between 37.6–39.1 N/mm2. HAp samples sintered at 1300 °C/120 min provide better results for the investigated mechanical properties. The coefficient of friction has an appropriate value for biomechanical applications. The results of cell viability showed that the cytotoxic effect is low for all tested samples. Better cell proliferation is observed for osteoblasts grown on square samples.

Circulating Monocytes Act as a Common Trigger for the Calcification Paradox of Osteoporosis and Carotid Atherosclerosis via TGFB1-SP1 and TNFSF10-NFKB1 Axis.

BackgroundOsteoporosis often occurs with carotid atherosclerosis and causes contradictory calcification across tissue in the same patient, which is called the “calcification paradox”. Circulating monocytes may be responsible for this unbalanced ectopic calcification. Here, we aimed to show how CD14+ monocytes contribute to the pathophysiology of coexisting postmenopausal osteoporosis and carotid atherosclerosis.MethodsWe comprehensively analyzed osteoporosis data from the mRNA array dataset GSE56814 and the scRNA-seq dataset GSM4423510. Carotid atherosclerosis data were obtained from the GSE23746 mRNA dataset and GSM4705591 scRNA-seq dataset. First, osteoblast and vascular SMC lineages were annotated based on their functional expression using gene set enrichment analysis and AUCell scoring. Next, pseudotime analysis was applied to draw their differentiated trajectory and identify the key gene expression changes in crossroads. Then, ligand–receptor interactions between CD14+ monocytes and osteoblast and vascular smooth muscle cell (SMC) lineages were annotated with iTALK. Finally, we selected calcification paradox-related expression in circulating monocytes with LASSO analysis.ResultsFirst, we found a large proportion of delayed premature osteoblasts in osteoporosis and osteogenic SMCs in atherosclerosis. Second, CD14+ monocytes interacted with the intermediate cells of the premature osteoblast and osteogenic SMC lineage by delivering TGFB1 and TNFSF10. This interaction served as a trigger activating the transcription factors (TF) SP1 and NFKB1 to upregulate the inflammatory response and cell senescence and led to a retarded premature state in the osteoblast lineage and osteogenic transition in the SMC lineage. Then, 76.49% of common monocyte markers were upregulated in the circulating monocytes between the two diseases, which were related to chemotaxis and inflammatory responses. Finally, we identified 7 calcification paradox-related genes on circulating monocytes, which were upregulated in aging cells and downregulated in DNA repair cells, indicating that the aging monocytes contributed to the development of the two diseases.ConclusionsOur work provides a perspective for understanding the triggering roles of CD14+ monocytes in the development of the calcification paradox in osteoporosis- and atherosclerosis-related cells based on combined scRNA and mRNA data. This study provided us with an elucidation of the mechanisms underlying the calcification paradox and could help in developing preventive and therapeutic strategies.

Effect of Menin Deletion in Early Osteoblast Lineage on the Mineralization of an In Vitro 3D Osteoid-like Dense Collagen Gel Matrix.

Bone has a complex microenvironment formed by an extracellular matrix (ECM) composed mainly of mineralized type I collagen fibres. Bone ECM regulates signaling pathways important in the differentiation of osteoblast-lineage cells, necessary for bone mineralization and in preserving tissue architecture. Compared to conventional 2D cell cultures, 3D in vitro models may better mimic bone ECM and provide an environment to support osteoblastic differentiation. In this study, a biomimetic 3D osteoid-like dense collagen gel model was used to investigate the role of the nuclear protein menin plays in osteoblastic differentiation and matrix mineralization. Previous in vitro and in vivo studies have shown that when expressed at later stages of osteoblastic differentiation, menin modulates osteoblastogenesis and regulates bone mass in adult mice. To investigate the role of menin when expressed at earlier stages of the osteoblastic lineage, conditional knockout mice in which the Men1 gene is specifically deleted early (i.e., at the level of the pluripotent mesenchymal stem cell lineage), where generated and primary calvarial osteoblasts were cultured in plastically compressed dense collagen gels for 21 days. The proliferation, morphology and differentiation of isolated seeded primary calvarial osteoblasts from knockout (Prx1-Cre; Men1f/f) mice were compared to those isolated from wild-type (Men1f/f) mice. Primary calvarial osteoblasts from knockout and wild-type mice did not show differences in terms of proliferation. However, in comparison to wild-type cells, primary osteoblast cells derived from knockout mice demonstrated deficient mineralization capabilities and an altered gene expression profile when cultured in 3D dense collagen gels. In summary, these findings indicate that when expressed at earlier stages of osteoblast differentiation, menin is important in maintaining matrix mineralization in 3D dense collagen gel matrices, in vitro.

TBX3 regulates the transcription of VEGFA to promote osteoblasts proliferation and microvascular regeneration.

ObjectiveOsteochondral decellularization can promote local vascular regeneration, but the exact mechanism is unknown. The aim of this study is to study osteogenic microvascular regeneration in single cells.MethodsThe scRNA-seq dataset of human periosteal-derived cells (hPDCs) were analyzed by pySCENIC. To examine the role of TBX3 in osteogenesis and vascularization, cell transfection, qRT-PCR, western blot, and CCK-8 cell proliferation assays were performed.ResultsTCF7L2, TBX3, FLI1, NFKB2, and EZH2 were found to be transcription factors (TFs) most closely associated with corresponding cells. The regulatory network of these TFs was then visualized. Our study knocked down the expression of TBX3 in human osteoblast cell lines. In the TBX3 knockdown group, we observed decreased expression of VEGFA, VEGFB, and VEGFC. Moreover, Western blot analysis showed that downregulating TBX3 resulted in a reduction of VEGFA expression. And TBX3 stimulated osteoblast proliferation in CCK-8 assays.ConclusionTBX3 regulates VEGFA expression and promotes osteoblast proliferation in skeletal microvasculature formation. The findings provide a theoretical basis for investigating the role of TBX3 in promoting local vascular regeneration.

Development of ternary nanocomposites based on hydroxyapatite/silver oxide for biomedical applications: morphology, structure and antibacterial activity

Nanocomposites based on hydroxyapatite (HAP) are fabricated with/without combining graphene oxide (GO) and silver oxide (Ag2O). All compositions are structurally investigated using x-ray diffraction (XRD). In addition, the lattice parameters were estimated. Lattice constants a and c of hexagonal HAP symmetry are around 9.42 and 6.88 Å, respectively. Further, the lattice constant of Ag2O is around 4.736 Å. Moreover, Fourier-transform infrared (FTIR) spectroscopy is used besides X-ray photoelectron (XPS) to investigate the composition. The morphological study was done with the scanning electron microscope (SEM) and transmission electron microscope (TEM). In this regard, Ag2O is exhibited as nano-spherical grains, while HAP was shown in nanorod grains. The dimensions of HAP decreased from 39 to 16 to 34 and 7 nm for length and diameter. The average roughness parameter (Ra) increased from 23.3 to 67.5 nm from HAP to TNC. The parameters of average maximum height (Rtm), maximum peak height (Rp), and maximum valley depth (Rv) reached 439.3, 261.3, and 251.8 nm respectively. Further, the Ca/P ratio is detected with a value of 1.5. Besides, cell viability of osteoblast cell lines increased from 95.9 ± 3 to 101.2 ± 5%. In addition, the antibacterial behavior increased to 21.6 ± 2.3 and 21.1 ± 1.4 mm for TNC against Escherichia coli and Staphylococcus aureus, respectively.