Alizarin Research Articles

Dehydrocostus lactone (DHC) promotes osteoblastic differentiation and mineralization through p38/RUNX-2 signaling.

Dysregulation of osteoblastic differentiation is an important risk factor of osteoporosis, the therapy of which is challenging. Dehydrocostus lactone (DHC), a sesquiterpene isolated from medicinal plants, has displayed anti-inflammatory and antitumor properties. In this study, we investigated the effects of DHC on osteoblastic differentiation and mineralization of MC3T3-E1 cells. Interestingly, we found that DHC increased the expression of marker genes of osteoblastic differentiation, such as alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Additionally, DHC increased the expressions of collagen type I alpha 1 (Col1a1) and collagen type I alpha 2 (Col1a2). We also demonstrate that DHC increased ALP activity. Importantly, the Alizarin Red S staining assay revealed that DHC enhanced osteoblastic differentiation of MC3T3-E1 cells. Mechanistically, it is shown that DHC increased the expression of Runx-2, a central regulator of osteoblastic differentiation. Treatment with DHC also increased the levels of phosphorylated p38, and its blockage using its specific inhibitor SB203580 abolished the effects of DHC on runt-related transcription factor 2 (Runx-2) expression and osteoblastic differentiation, suggesting the involvement of p38. Based on these findings, we concluded that DHC might possess a capacity for the treatment of osteoporosis by promoting osteoblastic differentiation.

Delonix regia biomass as an eco-friendly biosorbent for effective Alizarin Red S textile dye removal: Characterization, kinetics, and isotherm studies

Alizarin Red (ARS), a harmful dye known for its carcinogenic and mutagenic properties, is widely used in textile dyeing, posing a significant environmental concern due to its water-solubility and potential negative impact on aquatic life. This study explores the use of Delonix regia pods, obtained from the Gulmohar or Mayflower tree, as an eco-friendly adsorbent for ARS removal. The adsorption efficiency of Delonix regia pods was investigated by examining the effects of pH, dosage, initial concentration, and contact time. The optimal conditions for ARS removal were determined to be a pH of 2, a dosage of 2 g/L, and a contact time of 60 min, resulting in a significant 55 % removal of the dye. The experimental data exhibited a good fit with the Langmuir isotherm model (R2 = 0.9637), with the maximum adsorption capacity (qm) of the pods calculated as 4.4307 mg g−1 and the binding energy constant (Kl) as −1.0732. These findings highlight the potential of Delonix regia pods as a promising and environmentally friendly option for effective ARS removal. The study emphasizes the significance of utilizing biomass-based adsorbents in addressing the environmental challenges associated with dye removal.

Silica and barium: Comparison of microscopic appearance and characterization of effects of silica on cytomorphology.

Silica from plastic red top sample collection tubes and barium cause recognized artifacts in slide preparations for microscopic examination. The objectives of this study were to evaluate and directly compare the microscopic appearance of silica and barium particles and various slide preparation techniques (e.g., use of coverslips, oil immersion, and different stains). A secondary objective of this study was to evaluate the effects of silica particles on cellular morphology after mechanical trauma with cytocentrifugation. Fluid samples (deionized water, pleural effusion, peritoneal effusion, cerebrospinal fluid, and urine) were collected and evaluated in silica- and non-silica-containing tubes. Barium was added to silica and non-silica samples. Direct and cytocentrifuge preparations were compared to evaluate the effect of silica particles on cellular morphology. Preparations were stained with Wright-Giemsa, rhodizonic acid disodium salt, Alizarin Red, Grocott's methenamine silver, and Prussian blue. Silica and barium particles were identifiable via light microscopy with and without polarized light, although silica particles diminished with immersion oil. Barium particles retained their structure and diminished less under oil. Cytoseal mounting medium for coverslip placement resulted in diminished refractility of silica and some barium particles. Silica particles with mechanical interaction during cytocentrifugation resulted in disrupted cellular morphology with many lysed cells. Silica and barium particles were negative for all special stains tested. Silica from plastic red top tubes adversely affects cell morphology in cytocentrifuge preparations, potentially affecting manual differential cell counts and compromising diagnostic interpretation. Samples intended for microscopic evaluation should not be collected in silica-containing tubes.

Exploring the impact of oral bacteria remnants on stem cells from the Apical papilla: mineralization potential and inflammatory response.

Bacterial persistence is considered one of the main causal factors for regenerative endodontic treatment (RET) failure in immature permanent teeth. This interference is claimed to be caused by the interaction of bacteria that reside in the root canal with the stem cells that are one of the essentials for RET. The aim of the study was to investigate whether prolonged exposure of stem cells from the apical papilla (SCAP) to bacterial remnants of Fusobacterium nucleatum, Actinomyces gerensceriae, Slackia exigua, Enterococcus faecalis, Peptostreptococcaceae yurii, commonly found in infected traumatized root canals, and the probiotic bacteria Lactobacillus gasseri and Limosilactobacillus reuteri, can alter SCAP's inflammatory response and mineralization potential. To assess the effect of bacterial remnants on SCAP, we used UV-C-inactivated bacteria (as cell wall-associated virulence factors) and bacterial DNA. Histochemical staining using Osteoimage Mineralization Assay and Alizarin Red analysis was performed to study SCAP mineralization, while inflammatory and osteo/odontogenic-related responses of SCAPs were assessed with Multiplex ELISA. We showed that mineralization promotion was greater with UV C-inactivated bacteria compared to bacterial DNA. Immunofluorescence analysis detected that the early mineralization marker alkaline phosphatase (ALP) was increased by the level of E. coli lipopolysaccharide (LPS) positive control in the case of UV-C-inactivated bacteria; meanwhile, DNA treatment decreased the level of ALP compared to the positive control. SCAP's secretome assessed with Multiplex ELISA showed the upregulation of pro-inflammatory factors IL-6, IL-8, GM-CSF, IL-1b, neurotrophic factor BDNF, and angiogenic factor VEGF, induced by UV-C-killed bacteria. The results suggest that long term stimulation (for 21 days) of SCAP with UV-C-inactivated bacteria stimulate their mineralization and inflammatory response, while DNA influence has no such effect, which opens up new ideas about the nature of RET failure.

A Potential Intracanal Medicament, 2-Hydroxyisocaproic Acid (HICA): Cytotoxicity, Genotoxicity, and Its Effect on SCAP Differentiation.

Intracanal medicaments with maximal antimicrobial efficacy and minimal damage to resident stem cells are essential for successful regenerative endodontic procedures. 2-Hydroxyisocaproic acid (HICA) could have the attributes of a potential intracanal medicament. This study evaluates its cytotoxicity, genotoxicity, and effects on the odontogenic and osteogenic differentiation of the stem cells of the apical papilla (SCAP). Cytotoxicity and cell viability assays were performed on cells treated for 24, 48, and 72 h with varying concentrations of HICA and compared to the standard intracanal medicament, calcium hydroxide. The genotoxicity was assessed via immunofluorescence for two markers of DNA double-strand breaks: phosphorylated γH2AX and 53BP1. The SCAP differentiation was evaluated based on the alkaline phosphatase activity, Alizarin Red staining, and expression of odontogenic and osteogenic genes (DSPP1, BSP1, OCN, RUNX2) in the presence of selected HICA concentrations. HICA was not cytotoxic at concentrations up to 10 mg/mL, regardless of the exposure time, although it was cytostatic at all tested concentrations. HICA was not genotoxic at concentrations below 5 mg/mL. No difference in cytotoxicity or genotoxicity was found between HICA and calcium hydroxide at 1 mg/mL. HICA retained about 70% of the osteogenic differentiation potential at 1 mg/mL. Within the limitations of this in vitro study, we show that HICA at 1 mg/mL could be a potential intracanal medicament for REPs.

Colorimetric gel labels for the non-destructive freshness monitoring of fresh-cut papaya

Colorimetric gel labels (referred to as AKAX) were fabricated to monitor the freshness of fresh-cut papaya in real time using agarose (AR) and konjac glucomannan (KGM) composite gels containing alizarin (AL) and Substance X (X). The AKAX colorimetric gel label produced an ordered color variation from purple to pink-purple to orange-yellow, following the CO2 concentration in the package, to reflect the fresh-cut fruit freshness. Experimental characterization of the gel label structure revealed that AR, KGM, AL, and X were compatible, while the gel label texture, water-holding capacity, rheology, and thermal stability were improved with the addition of KGM. AKAX colorimetric gel labels maintained excellent color stability and CO2 sensitivity during storage at 4 °C. The fresh-cut papaya approached spoilage as the color varied from purple to pink-purple, and an orange-yellow color indicated that the fresh-cut papaya had spoiled. Therefore, the developed AKAX colorimetric gel labels serve as effective tools for monitoring the spoilage of fresh-cut papaya.

The Use of Rapid Precipitation to Synthesise Multivariate UiO-66 Metal–Organic Frameworks for Photocatalysis

A rapid synthesis method is used to form multivariate metal–organic frameworks (MTV-MOFs) with the UiO-66 structure, where precipitation occurs upon mixing solutions of ligands and metal salts at temperatures less than 60 °C. The materials include mixtures of metals and ligands, Ce/Zr-UiO-66(1,4-NDC/BDC), Ce/Zr-UiO-66(1,4-NDC/2,6-NDC), Ce/Zr-UiO-66(1,4-NDC), Ce/Ti-UiO-66(1,4-NDC), and Ce/Ti-UiO-66(BDC-NH2) (NDC = naphthalene dicarboxylate, BDC = benzene-1,4-dicarboxylate, BDC-NH2 = 2-amino-benzene-1,4-dicarboxylate). Phase purity was determined by powder X-ray diffraction (PXRD), with a broadening of the profile indicative of nanoscale crystallites, verified by scanning electron microscopy (SEM). The molar ratio of metals and organic ligands in Ce/Zr-UiO-66(1,4-NDC/2,6-NDC) was confirmed by X-ray fluorescence (XRF) and solution 1H nuclear magnetic resonance (NMR) after digestion, respectively. Analysis of the adsorption of dyes by the MTV-MOFs showed that a pseudo-first-order model accounts for the kinetics. The effectiveness of photocatalytic degradation of two cationic (methylene blue and rhodamine B) and two anionic (Congo red and Alizarin Red S (AR)) dyes was studied under UV and visible light. The most effective photocatalytic degradation was found between 1 and 15 min towards both cationic and anionic dyes by Ce/Zr-UiO-66(1,4-NDC/2,6-NDC). Measurements of recyclability and photostability showed retention of crystallinity after five cycles of use and exposure to light for 17 h, as confirmed by PXRD.

Phytic acid improves osteogenesis and inhibits the senescence of human bone marrow mesenchymal stem cells under high-glucose conditions via the ERK pathway

Hyperglycaemia causes impairment of osteogenic differentiation and accelerates stem cell senescence, resulting in weakened osteogenesis and disordered bone metabolism. Phytic acid (PA) is an antioxidant that is reportedly beneficial to bone homeostasis. The present study aims to clarify how PA affects the osteogenic capacity and cellular senescence of bone marrow mesenchymal stem cells (BMSCs) exposed to high-glucose environments, as well as the potential molecular mechanisms. Our results indicate that osteogenic differentiation in BMSCs cultivated in high-glucose conditions is enhanced by PA, as evidenced by increased alkaline phosphatase activity and staining, Alizarin Red S staining, osteogenic marker in in vitro studies, and increased osteogenesis in animal experiments. PA also prevented high-glucose-induced senescence of BMSCs, as evidenced by the repression of reactive oxygen species production, senescence-associated β-galactosidase staining, and P21 and P53 expression. Furthermore, it was found that PA rescued the high-glucose-inhibited expression of phosphorylated extracellular regulated protein kinases (p-ERK). The inhibition of ERK pathway by the specific inhibitor PD98059 blocked the PA-enhanced osteogenesis of BMSCs and promoted cell senescence. Our results revealed that PA enhances osteogenic differentiation and inhibits BMSC senescence in a high-glucose environment. In addition, the activation of the ERK pathway seems to mediate the beneficial effects of PA. The findings provide novel insights that could facilitate bone regeneration in patients with diabetes.

Efficacy of bone defect therapy involving various surface treatments of titanium alloy implants: an in vivo and in vitro study

Multiple surface treatment methods for titanium alloy prostheses, widely used in orthopedics, are available; however, these can affect bone integration and regeneration efficiency. In this study, through cell and animal experiments, we devised seven bone implant categories of Ti6Al4V based on surface preparation and post-processing technology (polishing, grit-blasting, fine titanium spraying, coarse titanium spraying, electron beam melting [EBM] printing, selective laser melting [SLM] printing, and post-processed SLM printing) and imaged each microscopic surface structure with a scanning electron microscope (SEM). Mechanical testing revealed excessive post-processing damaged the mechanical properties of the implants. In vitro, human bone marrow mesenchymal stem cells (hBMSCs) were cultured with implants, and the morphology of the cells adhering to the implant surface was observed using SEM and confocal laser scanning microscopy. Cell Counting Kit-8 (CCK-8) semi-quantitatively determined cell activity, indirectly reflecting the proliferation of hBMSCs. Alizarin red and alkaline phosphatase experiments assessed osteogenic differentiation. In vivo, experiments utilized the New Zealand rabbit femoral condyle bone defect model to assess bone regeneration and integration using micro-computed tomography, Van Giesen staining, and Masson staining. We found that 3D-printed implants with regular pore structures were more conducive to hBMSC osteogenic differentiation, while the presence of metal powder on NPT-SLM-printed implants hindered such differentiation. The post-treatment SLM scaffold surface may have some residual semi-melted powder; however, these powder residues have no significant effect on cell activity and differentiation. Surface treatment (grit-blasting and titanium spraying) of planar structures can enhance hBMSC adhesion but does not necessarily promote their differentiation. The framework structure of 3D printing may affect the osteogenic differentiation of hBMSCs, and for SLM-printed implants, excessive pursuit of a “powderless” state will damage the mechanical properties of the implant.

Gallic acid induces osteoblast differentiation and alleviates inflammatory response through GPR35/GSK3β/β-catenin signaling pathway in human periodontal ligament cells.

Gallic acid (GA) possesses various beneficial functions including antioxidant, anticancer, anti-inflammatory as well as inhibiting osteoclastogeneis. However, effects on osteogenic differentiation, especially in human ligament periodontal (hPDL) cells, remain unclear. Thus, the aim of this study was to evaluate the function of GA on osteogenesis and anti-inflammation in hPDL cells and to explore the involved underlying mechanism. Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) treatment was used as a model for periodontitis. ROS production was determined by H2DCFDA staining. Trans-well and wound healing assays were performed for checking the migration effect of GA. Alizarin red and alkaline phosphatase activity (ALP) assays were performed to evaluate osteogenic differentiation. Osteogenesis and inflammatory-related genes and proteins were measured by real-time PCR and western blot. Our results showed that GA-treated hPDL cells had higher proliferation and migration effect. GA inhibited ROS production-induced by Pg-LPS. Besides, GA abolished Pg-LPS-induced inflammation cytokines (il-6, il-1β) and inflammasome targets (Caspase-1, NLRP3). In addition, GA promoted ALP activity and mineralization in hPDL cells, lead to enhance osteoblast differentiation process. The effect of GA is related to G-protein-coupled receptor 35 (GPR35)/GSK3β/β-catenin signaling pathway. GA attenuated Pg-LPS-induced inflammatory responses and periodontitis in hPDL cells. Taken together, GA may be targeted for therapeutic interventions in periodontal diseases.

LncRNA JHDM1D-AS1 promotes osteogenic differentiation of periodontal ligament cells by targeting miR-532-5p to activate IGF1R signaling.

The aim of this study was to explore the mechanism underlying periodontal ligament cells (PDLCs) osteogenic differentiation. Periodontitis causes damage to tooth-supporting apparatus and eventually leads to tooth loss. PDLCs hold great promise in periodontal regeneration due to their osteogenic features. The expression of osteogenic markers, lncRNA JHDM1D-AS1, miR-532-5p and IGF1R was examined. For osteogenic differentiation, primary human PDLCs (hPDLCs) were cultured in an osteogenic medium, and it was assessed by ALP activity and Alizarin Red staining. The interaction between JHDM1D-AS1, miR-532-5p and IGF1R was analyzed via dual luciferase, RIP and RNA pull-down assays. JHDM1D-AS1 was up-regulated during osteogenic differentiation and its silencing inhibited hPDLC osteogenic differentiation. JHDM1D-AS1 worked as a miR-532-5p sponge in hPDLCs. miR-532-5p directly targeted IGF1R to suppress its expression, and miR-532-5p knockdown facilitated osteogenic differentiation of hPDLCs. Overexpression of IGF1R promoted osteogenic differentiation of hPDLCs via activating Notch/HES1 signaling in hPDLCs. JHDM1D-AS1 promotes osteogenic differentiation of hPDLCs via sponging miR-532-5p to facilitate IGF1R expression and activate Notch/HES1 signaling.

CeO2 Nanoparticle Bioactive Materials Promote MG-63 Osteogenic Differentiation and Antioxidant Activity Through NRF2 Signaling.

The incidence of bone-related diseases is higher in the elderly population, which greatly affects the patients' quality of life. Throughout this research, we synthesized a biocomposite nanomaterial of CeO2. The unique structural characteristics of CeO2 nanoparticles (CeO2 NPs) were studied by means of XRD, TEM, and SEM. Nanoparticles of an osteosarcoma cell line (MG-63) were assayed for ALP enzyme levels, key proteins in osteoblasts, and stained with Alizarin Red S to assess the physical properties, bioactivity, and calcium deposition of the osteosarcoma cell line. Moreover, we used H2O2 to construct an oxidative stress model to evaluate the antioxidant activity of CeO2 NPs. Experimental data showed that the CeO2 NPs increased the antioxidant capacity of MG-63 cells and significantly increased alkaline phosphatase activity, calcium deposition, and bone growth as manifested by increased expression of bone differentiation proteins BMP2, OCN, OPN, and type I collagen. Interestingly, RNA interference and functional recovery experiments confirmed that CeO2 NPs enhanced the antioxidant activity of MG-63 cells related to NRF2 signaling. In conclusion, the material is expected to be a potential treatment for bone-related diseases.

Introducing a novel ex-vivo calcification model for human aortic valves

Abstract Background Aortic stenosis (AS) is a narrowing of the aortic valve opening due to calcification and thickening of the leaflets. There are currently no effective pharmacological treatments for AS, and surgery or percutaneous interventions are the only therapeutic options. The cellular and molecular mechanisms underlying AS are complex and not fully understood due to the complexity and multi-factorial nature of AS and the absence of suitable test systems. In the current in-vitro models to study valvular calcification, isolated valvular interstitial cells (VICs) are cultured in 2D and in the absence of its native extracellular matrix. In addition, other cell types like valvular endothelial cells (VECs) and macrophages, which have been shown to impact the behavior of VICs, are absent. Purpose Our goal is to establish an ex-vivo calcification model for human aortic valves which maintains the original valve structure and cellular components. Methods Diseased aortic valves were harvested from patients undergoing aortic valve replacement. A 5mm x 7mm piece of this aortic valve tissue was dissected and embedded in agarose, then cut into 300 mm slices using a vibratome (Figure 1). In this way VICs remained in their valvular environment allowing the most native response of VICs to stimuli. The use of multiple slices from a single patient ensured proper internal controls. The slices were placed on a cell culture insert and cultured for 14 days. To induce calcification, the slices were cultured in the presence of 3 mM phosphate (n=23). As a control, valvular slices were cultured in the absence of 3mM phosphate (n=12). To validate if the calcification induced in the ex vivo culture system can be molecularly modulated the glucocorticoid dexamethasone was added to cultures containing 3mM phosphate (n=8). The slices were assessed for the presence of calcification using Alizarin Red staining and for the presence of osteogenic and inflammatory markers using (Immuno) histochemistry. Results In the presence of 3mM Phosphate, 74% of the slices demonstrated calcification, whereas no calcification was observed in all slices cultured without 3mM Phosphate (p<0.0001; Figure 2a). Furthermore, osteogenic genes such as Runx and alkaline phosphatase-1, as well as the inflammation marker NFkb were expressed in the calcified slices (Figure 2a). Dexamethason inhibited the calcification in the slices treated with 3 mM phosphate (Figure 2b). Conclusions We developed a novel ex-vivo calcification model for human aortic valves in which the initiation and progression of aortic valve calcification can be studied. In addition, the calcification can be modulated at a molecular level providing a unique opportunity to uncover the molecular signaling pathways involved in valvular calcification and to test compounds facilitating pre-clinical translational studies.Figure 1Figure 2

Boosting the performance of LaCoO3/MoS2 perovskite interface for sustainable decontaminants under visible light-driven photocatalysis

The novel composite LaCoO3/MoS2 hybrid nanostructure was synthesized via a combination of sol-gel, hydrothermal, and ultrasonication methods. Alizarin Red S (ARS) and Rhodamine B (RhB) were employed as a model pollutant, to assess the photodegradation efficiency of synthesized catalysts. The effect of MoS2 (2.5%, 5%, 7.5%, and 10%) on LaCoO3 (LCO) and its photocatalytic performance was studied. The properties of synthesized catalysts were assessed using various material characterization techniques. The photocatalytic dye degradation of ARS and RhB was investigated under visible light. Among the synthesized catalyst LM-5% composite (LaCoO3 with 5% MoS2) is determined to be the best photocatalyst as it degrades 96 % (ARS) and 90 % (RhB) in 40 min and 80 min, respectively. The photocatalyst is stable even after multiple runs and exhibits negligible loss in degradation efficiency during the cyclic test. Trapping experiments reveal the significance of superoxide anion and hydroxyl radicals against the photodegradation of ARS and RhB. The kinetics of photodegradation of ARS and RhB by LM-5% is found to be 5.70 × 10−2 and 2.25 × 10−2 min−1, respectively. Herein, we demonstrated a catalyst possessing excellent photodegradation activity which may ignite the possibilities of using efficient photocatalysts for environmental remediation.

Three-dimensional eco-friendly bacterial nanocellulose (BNC) scaffold for regenerative dentistry: Characterization, cytocompatibility and differentiation potential

ObjectiveRegenerative dentistry (RD) is an innovative strategy for treating necrotic teeth and regenerating damaged dental tissue. Biocompatible materials are pivotal for the advancement of RD, and the rising interest in environmental sustainability drives exploration of sustainable materials for dentistry. Bacterial nanocellulose (BNC) has emerged as a promising eco-friendly option and this study aims to assess BNC's suitability as scaffolds for regenerative dentistry applications. MethodsDifferent in vitro methods have been utilized to characterize the properties of BNC scaffolds in regenerative dentistry, such as scanning electron microscopy (SEM) to analyse surface property and porosity, as well as examining their absorption behaviour using phosphate-buffered saline and bovine serum. Dental pulp stem cell (DPSCs) attachment, viability, and proliferation were evaluated using SEM, live and dead, and tetrazolium reduction assays. The odontogenic potential of the scaffold was evaluated using Alizarin Red staining and qPCR (14 and 21 days). ResultsScanning electron microscopy (SEM) images and ethanol displacement method demonstrated the porous architecture of the BNC scaffold with an average porosity of 70.02 ± 4.74% and 50.26 ± 1.43% respectively. The scaffold absorbed 2846.54 ± 258.95 of BSA and 1648.63 ± 50.37% PBS after immersion in solution for 1 h, following pseudo first and second order kinetics. The biocompatibility assay indicated that cell density increased with time and that the scaffold was appropriate for cell adhesion and migration. Moreover, the BNC led to significantly higher mineralization and odontogenic expression compared to the control (BNC in conditioned media). SignificanceBNC showed fast adsorption of bovine serum, allowed DPSC attachment, migration, and odontogenic differentiation. This suggests its suitability as a biocompatible scaffold for triggering in situ mineralized tissue regeneration for regenerative dental applications.

Altered Osteogenic Differentiation in Mesenchymal Stem Cells Isolated from Compact Bone of Chicken Treated with Varying Doses of Lipopolysaccharides.

Persistent inflammation biologically alters signaling molecules and ultimately affects osteogenic differentiation, including in modern-day broilers with unique physiology. Lipopolysaccharides (LPS) are Gram-negative bacterial components that activate cells via transmembrane receptor activation and other molecules. Previous studies have shown several pathways associated with osteogenic inductive ability, but the pathway has yet to be deciphered, and data related to its dose-dependent effect are limited. Primary mesenchymal stem cells (MSCs) were isolated from the bones of day-old broiler chickens, and the current study focused on the dose-dependent variation (3.125 micrograms/mL to 50 micrograms/mL) in osteogenic differentiation and the associated biomarkers in primary MSCs. The doses in this study were determined using a cell viability (MTT) assay. The study revealed that osteogenic differentiation varied with dose, and the cells exposed to higher doses of LPS were viable but lacked differentiating ability. However, this effect became transient with lower doses, and this phenotypic character was observed with differential staining methods like Alizarin Red, Von Kossa, and alkaline phosphatase. The data from this study revealed that LPS at varying doses had a varying effect on osteogenic differentiation via several pathways acting simultaneously during bone development.

Ultra-thin electrospun nanocomposite scaffold of poly (3-hydroxybutyrate)-chitosan/magnetic mesoporous bioactive glasses for bone tissue engineering applications

Bioglass is widely used in skeletal tissue engineering due to its outstanding bioactive properties. In the present study, magnetic mesoporous bioglass (MMBG) synthesized through the sol-gel method was incorporated into poly(3-hydroxybutyrate)-chitosan (PHB-Cs) solution and the resulting electrospun nanocomposite scaffolds were investigated and compared with MMBG free scaffold. The addition of 10 wt% MMBG has an outstanding effect on producing ultra-thin electrospun nanocomposite fibers due to its magnetic content (diameter of ≃128 nm). This improvement led to better mechanical properties, including an increase in both tensile modulus (up to ≃229 MPa) and tensile strength (to ≃4.95 MPa). Although the inclusion of MMBG slightly decreased the surface roughness of the nanofibrous scaffold (RMS from ≃197 to 154 nm), it could improve the wettability (WCA from ≃54 to 44°). This achievement has the potential to bring an enhancement in biomineralization and biological response. These outputs, combined with the observed increase in human osteoblast MG-63 cell viability (≃53 % improvement) as measured by MTT assay, DAPI, and SEM indicate prefer cell behavior of this nanocomposite structure. Additionally, the qualitative improvement in Alizarin Red staining and the quantitative enhancement of ALP secretion, serve as further evidence of the PHB-Cs/MMBG ultrathin nanofibers potential in bone tissue engineering.

LncRNA SNHG14 activates autophagy via regulating miR-493-5p/Mef2c axis to alleviate osteoporosis progression

Osteoporosis is a progressive bone disease caused by impaired function of endogenous bone marrow-derived mesenchymal stem cells (BMSCs). Herein, we investigated the mechanism of lncRNA SNHG14 in osteoporosis progression. BMSCs were isolated from BALB/c mice. The osteogenic ability of BMSCs was assessed by Alkaline phosphatase (ALP) and Alizarin Red S Staining (ARS) staining. The interaction between miR-493-5p and SNHG14 or myocyte enhancer factor 2 C (Mef2c) was confirmed by dual-luciferase reporter assay. Bone histomorphometry changes were evaluated to analyze SNHG14’roles in osteoporosis in vivo. Our results illustrated SNHG14 and Mef2c levels were increased in a time-dependent manner in BMSCs, and miR-493-5p expression was decreased. SNHG14 knockdown inhibited osteogenic differentiation of BMSCs, and SNHG14 upregulation had the opposite effect. SNHG14 overexpression elevated bone mineral density and bone trabecular number, and alleviated osteoporosis progression in vivo. Mechanically, miR-493-5p was a target of SNHG14, and miR-493-5p targeted the Mef2c gene directly. SNHG14 overexpression reversed the inhibition of miR-493-5p on the osteogenic ability of BMSCs, and miR-493-5p silencing accelerated BMSCs osteogenesis by activating Mef2c-mediated autophagy to accelerate BMSCs osteogenesis. In short, SNHG14 activated autophagy via regulating miR-493-5p/Mef2c axis to alleviate osteoporosis progression, which might provide a new molecular target for osteoporosis treatment.

Superoxide dismutase 2 scavenges ROS to promote osteogenic differentiation of human periodontal ligament stem cells by regulating Smad3 in alveolar bone-defective rats.

Osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is an essential event in alveolar bone regeneration. Oxidative stress may be the main inhibiting factor of hPDLSC osteogenesis. Superoxide dismutase 2 (SOD2) is a key antioxidant enzyme, but its effect on hPDLSC osteogenic differentiation is unclear. Several surface markers were detected by flow cytometry, and the differentiation potential of hPDLSCs was validated by alkaline phosphatase (ALP), Alizarin Red S, and Oil Red O staining. Osteogenic indicators of hPDLSCs were detected by real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and ALP staining. Furthermore, alveolar bone defect rat models were analyzed through micro-CT, hematoxylin and eosin, and Masson staining. The intracellular reactive oxygen species (ROS) level was evaluated by a ROS assay kit. Finally, the expression of SOD2, Smad3, and p-Smad3 in hPDLSCs was detected by RT-qPCR and Western blotting (WB). SOD2 positively regulated the gene and protein expressions of ALP, BMP6, and RUNX2 in hPDLSCs (p<0.05). Ideal bone formation and continuous cortical bone were obtained by transplanting LV-SOD2 hPDLSCs (lentivirus vector for overexpressing SOD2 in hPDLSCs) in vivo. Exogenous H2O2 downregulated osteogenic indicators (ALP, BMP6, RUNX2) in hPDLSCs (p<0.05); this was reversed by overexpression of SOD2. WB results showed that the Smad3 and p-Smad3 signaling pathways participated in the osteogenic process of SOD2 in hPDLSCs. SOD2 positively regulated hPDLSC osteogenic differentiation in vitro and in vivo. Mechanistically, SOD2 promotes hPDLSC osteogenic differentiation by regulating the phosphorylation of Smad3 to scavenge ROS. This work provides a theoretical basis for the treatment of alveolar bone regeneration.

Cardioprotective function of sclerostin by reducing calcium deposition, proliferation, and apoptosis in human vascular smooth muscle cells

BackgroundSclerostin is an inhibitor of the Wnt/b-catenin pathway, which regulates bone formation, and can be expressed in vascular smooth muscle cells (VSMCs). Type 2 diabetes (T2D) is associated with an increased risk of cardiovascular disease (CVD) and increased serum and tissue expression of sclerostin. However, whether the role of sclerostin is detrimental or protective in the development of CVD is unknown. Therefore, our aims are to determine the level of sclerostin in T2D patients with/without CVD and in controls, both at serum and vascular tissue, and to analyze the role of sclerostin in VSMCs under calcified environments.MethodsCross-sectional study including 121 controls and 139 T2D patients with/without CVD (48/91). Sclerostin levels in serum were determined by ELISA, and sclerostin expression was analyzed by RT-qPCR and immunohistochemistry in calcified and non-calcified artery of lower limb from T2D patients (n = 7) and controls (n = 3). In vitro experiments were performed in VSMCs (mock and sclerostin overexpression) under calcifying conditions analyzing the sclerostin function by determination of calcium and phosphate concentrations, and quantification of calcium deposits by Alizarin Red. Proliferation and apoptosis were analyzed by MTT assay and flow cytometry, respectively. The regulation of the expression of genes involved in bone metabolism was determined by RT-qPCR.ResultsA significant increase in serum sclerostin levels in T2D patients with CVD compared to T2D patients without CVD and controls (p < 0.001) was observed. Moreover, higher circulating sclerostin levels were independently associated with CVD in T2D patients. Increased sclerostin expression was observed in calcified arteries of T2D patients compared to non-calcified arteries of controls (p = 0.003). In vitro experiments using VSMCs under calcified conditions, revealed that sclerostin overexpression reduced intracellular calcium (p = 0.001), calcium deposits (p < 0.001), cell proliferation (p < 0.001) and promoted cell survival (p = 0.015). Furthermore, sclerostin overexpression exhibited up-regulation of ALPL (p = 0.009), RUNX2 (p = 0.001) and COX2 (p = 0.003) and down-regulation of inflammatory genes, such as, IL1β (p = 0.005), IL6 (p = 0.001) and IL8 (p = 0.003).ConclusionsSclerostin could play a protective role in the development of atherosclerosis in T2D patients by reducing calcium deposits, decreasing proliferation and inflammation, and promoting cell survival in VSMCs under calcifying conditions. Therefore, considering the bone-vascular axis, treatment with anti-sclerostin for bone disease should be used with caution.Graphical