Expression Of Osteogenic Markers Research Articles

Three-dimensional bioprinting of mesenchymal stem cells using an osteoinductive bioink containing alginate and BMP-2-loaded PLGA nanoparticles for bone tissue engineering.

Hydrogels mimicking the physicochemical properties of the native extracellular matrix have attracted great attention as bioinks for three-dimensional (3D) bioprinting in tissue engineering applications. Alginate is a widely used bioink with beneficial properties of fast gelation and biocompatibility; however, bioprinting using alginate-based bioinks has several limitations, such as poor printability, structural instability, and limited biological activities. To address these issues, we formulated various bioinks using bone morphogenetic protein-2 (BMP-2)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles and alginate for mesenchymal stem cell (MSC) printing and induction of osteogenic differentiation. Incorporation of PLGA nanoparticles into alginate could enhance the mechanical properties and printability of the bioink. In particular, Alg/NPN30 (30mg/mL PLGA nanoparticles and 3% w/v alginate) was most suitable for 3D printing with respect to printability and stability. BMP-2-loaded PLGA nanoparticles (NPBMP-2) displayed sustained in vitro release of BMP-2 for up to two weeks. Further in vitro studies indicated that bioinks composed of alginate and NPBMP-2 significantly induced osteogenesis of the MSCs compared with other controls, evidenced by enhanced calcium deposition, alkaline phosphatase activity, and gene expression of osteogenic markers. Our novel bioink consisting of widely used biocompatible components displays good printability, stability, and osteogenic inductivity, and holds strong potential for cell printing and bone tissue engineering applications.

Impact of Fluid Dynamics on the Viability and Differentiation Capacity of 3D-Cultured Jaw Periosteal Cells

Perfused bioreactor systems are considered to be a promising approach for the 3D culturing of stem cells by improving the quality of the tissue-engineered grafts in terms of better cell proliferation and deeper penetration of used scaffold materials. Our study aims to establish an optimal perfusion culture system for jaw periosteal cell (JPC)-seeded scaffolds. For this purpose, we used beta-tricalcium phosphate (β-TCP) scaffolds as a three-dimensional structure for cell growth and osteogenic differentiation. Experimental set-ups of tangential and sigmoidal fluid configurations with medium flow rates of 100 and 200 µL/min were applied within the perfusion system. Cell metabolic activities of 3D-cultured JPCs under dynamic conditions with flow rates of 100 and 200 µL/min were increased in the tendency after 1, and 3 days of culture, and were significantly increased after 5 days. Significantly higher cell densities were detected under the four perfused conditions compared to the static condition at day 5. However, cell metabolic and proliferation activity under dynamic conditions showed flow rate independency in our study. In this study, dynamic conditions increased the expression of osteogenic markers (ALPL, COL1A1, RUNX2, and OCN) compared to static conditions and the tangential configuration showed a stronger osteogenic effect than the sigmoidal flow configuration.

Tissue-specific melt electrowritten polymeric scaffolds for coordinated regeneration of soft and hard periodontal tissues.

Periodontitis is a chronic inflammatory condition that often causes serious damage to tooth-supporting tissues. The limited successful outcomes of clinically available approaches underscore the need for therapeutics that cannot only provide structural guidance to cells but can also modulate the local immune response. Here, three-dimensional melt electrowritten (i.e., poly(ε-caprolactone)) scaffolds with tissue-specific attributes were engineered to guide differentiation of human-derived periodontal ligament stem cells (hPDLSCs) and mediate macrophage polarization. The investigated tissue-specific scaffold attributes comprised fiber morphology (aligned vs. random) and highly-ordered architectures with distinct strand spacings (small 250 μm and large 500 μm). Macrophages exhibited an elongated morphology in aligned and highly-ordered scaffolds, while maintaining their round-shape on randomly-oriented fibrous scaffolds. Expressions of periostin and IL-10 were more pronounced on the aligned and highly-ordered scaffolds. While hPDLSCs on the scaffolds with 500 μm strand spacing show higher expression of osteogenic marker (Runx2) over 21 days, cells on randomly-oriented fibrous scaffolds showed upregulation of M1 markers. In an orthotopic mandibular fenestration defect model, findings revealed that the tissue-specific scaffolds (i.e., aligned fibers for periodontal ligament and highly-ordered 500 μm strand spacing fluorinated calcium phosphate [F/CaP]-coated fibers for bone) could enhance the mimicking of regeneration of natural periodontal tissues.

METTL3 mediates osteoblast apoptosis by regulating endoplasmic reticulum stress during LPS-induced inflammation

Osteoblast apoptosis is a prominent factor for disrupting skeletal homeostasis in multiple inflammatory bone diseases. METTL3, a key methyltransferase that catalyzes the N6-methyladenosine (m6A) modification of mRNA, has recently been shown to exert a critical role in osteogenic differentiation. However, the function of METTL3 in osteoblast apoptosis under inflammatory conditions remains elusive. In the present study, we observed that the total m6A level and METTL3 expression were upregulated in differentiated osteoblasts and downregulated after LPS stimulation. METTL3 knockdown induced a higher apoptotic rate in LPS-treated osteoblasts. The expression of the antiapoptotic protein BCL-2 decreased, and the apoptotic proteins cleaved Caspase-3, cleaved PARP-1 and cleaved Caspase-12 increased following METTL3 knockdown. Meanwhile, METTL3 silencing inhibited osteoblast proliferation and decreased osteogenic marker expression, ALP activity and mineralized nodules. RNA-seq analysis revealed that differentially expressed genes were significantly enriched in unfolded protein response pathways in METTL3-deficient cells. METTL3 depletion upregulated the expression of the ER stress-related markers, including p-PERK, p-eIF2α, p-IRE1α, GRP78, ATF4, CHOP and ATF6. Inhibition of ER stress by 4-PBA remarkably rescued METTL3 knockdown-induced apoptosis and promoted osteoblast proliferation and differentiation. Mechanistically, METTL3 depletion enhanced the expression and mRNA stability of Grp78, and similar results were observed after YTHDF2 knockdown. RIP-qPCR revealed that YTHDF2 directly interacted with Grp78 mRNA and that the interaction relied on METTL3. Taken together, our study demonstrated that METTL3 knockdown enhanced Grp78 expression through YTHDF2-mediated RNA degradation, which elicited ER stress, thereby promoting osteoblast apoptosis and inhibiting cell proliferation and differentiation under LPS-induced inflammatory condition.

A Biomimetic Platelet-Rich Plasma-Based Interpenetrating Network Printable Hydrogel for Bone Regeneration.

Repair of bone defects caused by trauma or diseases is the primary focus of prosthodontics. Hydrogels are among the most promising candidates for bone tissue regeneration due to their unique features such as excellent biocompatibility, similarities to biological tissues, and plasticity. Herein, we developed a type of novel biomimetic interpenetrating polymeric network (IPN) hydrogel by combining methacrylated alginate and 4-arm poly (ethylene glycol)-acrylate (4A-PEGAcr) through photo-crosslinking. Platelet-rich plasma (PRP), a patient-specific source of autologous growth factors, was incorporated into the hydrogel, and thereafter the hydrogels were biological mineralized by simulated body fluid (SBF). Physical properties of hydrogels were comprehensively characterized. In vitro studies demonstrated that the incorporation of PRP and biomineralization promoted the biocompatibility of hydrogel. Strikingly, the osteogenic bioactivities, including ALP activity, mineralized nodule formation, and expression of osteogenic markers were found substantially enhanced by this biomineralized PRP-hydrogel. Finally, a rabbit model of bone defect was employed to assess in vivo bone regeneration, micro-CT analysis showed that the biomineralized PRP-hydrogels could significantly accelerate bone generation. We believed that this novel biomineralized PRP-incorporated IPN hydrogel could be promising scaffolds for bone tissue regeneration.

MiR-146a Protects against Staphylococcus aureus-Induced Osteomyelitis by Regulating Inflammation and Osteogenesis.

Osteomyelitis is a Staphylococcus aureus-caused bone infection. In this study, the effects of miR-146a on osteomyelitis were evaluated. Using the osteoblast cell model and S. aureus-induced osteomyelitis mice model, we monitored the miR-146 expression and explored the effects of miR-146a on cell proliferation of osteoblasts, bone remodeling, osteoclastogenesis, inflammatory cytokine production, and bacterial burden. Upregulated miR-146a was found in mice with S. aureus-induced osteomyelitis. miR-146a attenuated S. aureus-induced cell loss of osteoblasts, rescued the expression of osteogenic markers, altered the bone remodeling, and inhibited inflammatory cytokine production and osteoclastogenesis. miR-146a knockout mice had higher S. aureus burden. In conclusion, miR-146a protects against S. aureus-induced osteomyelitis by regulating inflammation and osteogenesis.

The chitosan/carboxymethyl cellulose/montmorillonite scaffolds incorporated with epigallocatechin-3-gallate-loaded chitosan microspheres for promoting osteogenesis of human umbilical cord-derived mesenchymal stem cell

Epigallocatechin-3-gallate (EGCG) is a plant-derived flavonoid compound with the ability to promote the differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) into osteoblasts. However, the effect of EGCG on the osteogenic differentiation of the human umbilical cord-derived mesenchymal stem cells (HUMSCs) is rarely studied. Therefore, in this study, the osteogenic effects of EGCG are studied in the HUMSCs by detecting cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition and the expression of relevant osteogenic markers. The results showed that EGCG can promote the proliferation and osteogenic differentiation of the HUMSCs in vitro at a concentration of 2.5–5.0 μM. Unfortunately, the EGCG is easily metabolized by cells during cell culture, which reduces its bioavailability. Therefore, in this paper, EGCG-loaded microspheres (ECM) were prepared and embedded in chitosan/carboxymethyl cellulose/montmorillonite (CS/CMC/MMT) scaffolds to form CS/CMC/MMT-ECM scaffolds for improving the bioavailability of EGCG. The HUMSCs were cultured on CS/CMC/MMT-ECM scaffolds to induce osteogenic differentiation. The results showed that the CS/CMC/MMT-ECM scaffold continuously released EGCG for up to 22 days. In addition, CS/CMC/MMT-ECM scaffolds can promote osteoblast differentiation. Taken together, the present study suggested that entrainment of ECM into CS/CMC/MMT scaffolds was a prospective scheme for promotion osteogenic differentiation of the HUMSCs.Graphical

Resveratrol exhibits inhibition effects on osteogenic differentiation of aortic valve interstitial cells by interfering with the AKT pathway

• The expression of osteogenic markers are increased in the aortic valve tissues of patients with calcific aortic valve disease. • Resveratrol inhibits the osteogenic medium-induced osteogenic differentiation of valve interstitial cells. • Resveratrol inhibits the osteogenic differentiation of valve interstitial cells via interfering with AKT signaling pathway activation. • RES may become a potential natural drug for the treatment of calcific aortic valve disease. Resveratrol is a natural active ingredient found in many plants and has a protective effect on cardiovascular diseases. However, it has not been reported that whether resveratrol can inhibit calcific aortic valve disease(CAVD). In this study, we want to explore the effect and mechanism of resveratrol on the osteogenic differentiation of valve interstitial cells（VICs）.We first found that the expression of osteogenic markers in calcified human aortic valve. Western blotting, ALP staining and Alizarin red S staining showed that under osteogenic medium (OM) treatment, resveratrol can inhibit VICs from expressing osteogenic markers, reduce ALP content and calcium Nodule formation. Mechanistically, resveratrol can inhibit the activation of the AKT/Smad1/5/8 pathway induced by OM. We selected the AKT pathway for verification and determined that resveratrol inhibited the osteogenic differentiation of VICs by inhibiting the AKT pathway. Collectively, resveratrol may become a potential natural drug for the treatment of CAVD.

Protective and Osteogenic Effects of Crude Water Extract from Cuscuta japonica Choisy at Gene Expression Level in Human Gingival Cells

Abstract This study focused on observing change of gene expression after long-term treatment with high dose of crude water extract from seeds of Cuscuta japonica Choisy (Japanese dodder). The aim of this study was to study the protective and osteogenic effects of dodder seed extract at gene expression level in human gingival cells. In this research, dodder seeds were blended and boiled in water before freeze-drying to preserve as dried powder. Total phenolic content and antioxidant assay were performed. MTT assay was performed with human gingival cells. The concentrations of 100, 250 and 500 µg / mL had no effect on cell viability when cultivated for 48 hours. The extract at the concentration of 250 µg / mL was chosen to treat human gingival cells for many time points to observe the expressions of osteogenic, inflammatory, antioxidant and cancer gene markers by real-time PCR. The results showed that dodder seed water extract could increase the expressions of osteogenic markers; OPN, OCN and Col-I genes. Moreover, dodder seed water extract induced mineralization of human gingival cells cultured in 3D structure. Change of BCL2, CAS3 and LC3 expression indicated involvement in apoptotic process of dodder seed water extract. Significant changes in gene expression of antioxidant markers (GST1, SOD1 and TXNRD1), inflammatory marker (Cox-2) and epithelial cell marker (α-SMA) without change of important cancer genes were observed. These evidences suggested that seeds of Cuscuta japonica Choisy could benefit the application in regenerative medicine and alternative medicine. Keywords: Cuscuta japonica Choisy, Antioxidant, Osteogenic effects, Gene expression

MiR-153-3p inhibited osteogenic differentiation of human DPSCs through CBFβ signaling.

Dental pulp stem cells (DPSCs) have multilineage differentiation potential and especially show a great foreground in bone regeneration engineering. The mechanism of osteogenic differentiation of DPSCs needs to be explored exactly. As a kind of endogenous and non-coding small RNAs, microRNAs (miRNAs) play an important role in many biological processes including osteogenic differentiation. However, the mechanism of miR-153-3p in osteogenic differentiation of DPSCs is still unknown. Core-binding factors-beta (CBFβ) is a non-DNA-binding factor that combines with the runt-related transcription factor family transcription factors to mediate their DNA-binding affinities, and plays a critical role in regulating osteogenic differentiation. In this study, we explored the mechanisms of miR-153-3p and CBFβ in DPSC osteogenesis. The expression of miR-153-3p and CBFβ was tested under the osteogenic condition, and the influence led by changing the expression of miR-153-3p or CBFβ had also been detected. A luciferase reporter assay confirmed that miR-153-3p directly targeted to CBFβ. The osteogenic markers, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and bone morphogenetic protein 2 (BMP2), were tested in protein level or mRNA level. ALP and Alizarin red staining were used to detect the osteoblast activity and mineral deposition. In osteogenic condition, the expressions of CBFβ and osteogenic markers were upregulated, whereas that of miR-153-3p was downregulated. miR-153-3p negatively regulated the osteogenic differentiation, and overexpression of CBFβ could offset the negative effect of miR-153-3p. Our findings provided a novel strategy for DPSC application in treatment of bone deficiencies and facilitated bone regeneration.

Exendin‐4 enhances osteogenic differentiation of adipose tissue mesenchymal stem cells through the receptor activator of nuclear factor‐kappa B and osteoprotegerin signaling pathway

The capability of mesenchymal stem cells (MSCs) to repair bone damage and defects has long been investigated. The receptor activator of nuclear factor-kappa B (RANK), its ligand (RANKL) and the decoy receptor osteoprotegerin (OPG) axis is crucial to keep the equilibrium between osteoblastic and osteoclastic activity. Exendin-4 utilization increased bone formation and enhanced bone integrity. This study aimed to investigate the mentioned axis and determine the effect of exendin-4 upon adipose mesenchymal stem cells (Ad-MSCs) osteogenic differentiation. Ad-MSCs were isolated from rat epididymal fat, followed by characterization and then differentiation into osteocytes both in the presence or absence of exendin-4. Osteogenic differentiation was evaluated by alizarin red staining and the expression of osteogenic markers; using reverse transcriptase-quantitative polymerase chain reaction, western blotting and enzyme-linked immunoassay. MSCs derived from rat epididymal fat were isolated and characterized, along with their differentiation into osteocytes. The differentiated cells were alizarin red-stained, showing increased staining intensity upon addition of exendin-4. Moreover, the addition of exendin-4 elevated the messenger RNA expression levels of osteogenic markers; runt-related transcription factor-2 (RUNX-2), osteocalcin, and forkhead box protein O-1while reducing the expression of the adipogenic marker peroxisome-proliferator-activated receptor-gamma. Exendin-4 addition elevated OPG levels in the supernatant of osteogenic differentiated cells. Moreover, exendin-4 elevated the protein levels of glucagon-like peptide-1 receptor and RUNX-2, while decreasing both RANK and RANKL. In conclusion, osteogenic differentiation of Ad-MSCs is associated with increased osteoblastic rather than osteoclastic activity. The findings of this study suggest that exendin-4 can enhance Ad-MSCs osteogenic differentiation partially through the RANK/RANKL/OPG axis.

LncRNA KCNQ1OT1 promotes osteogenic differentiation via miR-205-5p/RICTOR axis

Osteoporosis is a prevalent degenerative disease that is characterized by decreased bone density and strength, resulting in gradually increasing bone fragility. Osteoporosis is caused by an imbalance between osteoblastic bone formation and osteoclastic bone resorption. Recently, increasing evidence has suggested that long non-coding RNAs (lncRNAs) participate in the occurrence and development of osteoporosis. Herein, we explored the role of lncRNA KCNQ1OT1 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). QPCR results indicated that KCNQ1OT1 and RICTOR were down-regulated, while miR-205-5p was up-regulated in the osteoporotic patients, as compared with non-osteoporotic controls. During the osteogenic differentiation of BMSCs, the expression of KCNQ1OT1 and RICTOR was upregulated, whereas miR-205-5p was downregulated. The interaction among KCNQ1OT1, miR-205-5p and RICTOR was validated by dual luciferase reporter system. KCNQ1OT1 promoted RICTOR expression via inhibiting miR-205-5p, therefore promoting osteogenesis as demonstrated by ALP assay, alizarin red staining and the increased expression of osteogenic markers (OPN, RUNX2 and OCN). Furthermore, KCNQ1OT1 overexpression or miR-205-5p inhibition could promote ALP activity and mineralization of BMSCs, while overexpressed miR-205-5p could reverse the effects of overexpressed KCNQ1OT1, and knockdown of RICTOR could reverse the effects of miR-205-5p inhibition. In conclusion, our study illustrated that KCNQ1OT1 might inhibit miR-205-5p in BMSCs, thus upregulating the expression of RICTOR and promoting osteogenic differentiation.

Clinical relevance of circulating angiogenic cells in patients with ischemic stroke

BackgroundEndothelial progenitor cells (EPCs) are circulating angiogenic cells with endothelial features associated with risk for stroke. We aimed to delve into their functional characteristics. EPCs were isolated and cultured from Ischemic Stroke (IS) patients and predictors of their variance evaluated.MethodsThis is a single-center observational study evaluating 187 consecutively hospitalized patients with IS. EPCs were isolated from blood samples. The number of circulating angiogenic cells (CACs), colony-forming units (CFU-ECs) and the emergence of late outgrowths endothelial cells (LOECs) were counted. We collected clinical variables and measured the stromal cell-derived factor 1 alpha (SDF1α) serum levels. We also examined the relative telomere length and the expression of osteogenic gene markers in CACs.ResultsCACs counts and CFU-ECs colony numbers were positively correlated (rho = 0.41, p < 0.001, n = 187). We found significant differences according to whether thrombolytic treatment was performed in the distribution of CFU-ECs (odds ratio (OR) = 2.5; 95% confidence interval (CI) 1.01–6.35; p = 0.042) and CACs (OR = 4.45; 95% IC 1.2–15.5; p = 0.012). The main determinants of CACs variation were the number of risks factors, thrombolysis treatment, arterial hypertension, LOECs occurrence, and the vascular endothelial growth factor expression, whereas CFU-ECs variations depended on hemoglobin content and the relative reduction in the National Institutes of Health Stroke Scale (NIHSS) criteria. The main predictors of LOECs appearance were thrombolysis and length of hospital stay.ConclusionsOur study supports the relevance of patient risk factors and treatments in the analysis of the functional properties of EPCs.

BMP-2 Enhances Osteogenic Differentiation of Human Adipose-Derived and Dental Pulp Stem Cells in 2D and 3D In Vitro Models.

Bone tissue provides support and protection to different organs and tissues. Aging and different diseases can cause a decrease in the rate of bone regeneration or incomplete healing; thus, tissue-engineered substitutes can be an acceptable alternative to traditional therapies. In the present work, we have developed an in vitro osteogenic differentiation model based on mesenchymal stem cells (MSCs), to first analyse the influence of the culture media and the origin of the cells on the efficiency of this process and secondly to extrapolate it to a 3D environment to evaluate its possible application in bone regeneration therapies. Two osteogenic culture media were used (one commercial from Stemcell Technologies and a second supplemented with dexamethasone, ascorbic acid, glycerol-2-phosphate, and BMP-2), with human cells of a mesenchymal phenotype from two different origins: adipose tissue (hADSCs) and dental pulp (hDPSCs). The expression of osteogenic markers in 2D cultures was evaluated in several culture periods by means of the immunofluorescence technique and real-time gene expression analysis, taking as reference MG-63 cells of osteogenic origin. The same strategy was extrapolated to a 3D environment of polylactic acid (PLA), with a 3% alginate hydrogel. The expression of osteogenic markers was detected in both hADSCs and hDPSCs, cultured in either 2D or 3D environments. However, the osteogenic differentiation of MSCs was obtained based on the culture medium and the cell origin used, since higher osteogenic marker levels were found when hADSCs were cultured with medium supplemented with BMP-2. Furthermore, the 3D culture used was suitable for cell survival and osteogenic induction.

Aqueous extract of freeze-dried Protaetia brevitarsis larvae promotes osteogenesis by activating β-catenin signaling

Objective: To investigate the effect of an aqueous extract of Protaetia brevitarsis (AEPB) on osteogenesis using preosteoblast MC3T3-E1 cells and zebrafish larvae. Methods: Flow cytometric analysis was used to measure the cytotoxicy. Alkaline phosphatase activity was detetmined using p-nitrophenyl phosphate as a substrate. Calcium deposition was detected using alizarin red staining along with osteogenic marker expression in preosteoblast MC3T3E1 cells. In addition, vertebral formation in zebrafish larvae was detected using calcein staining and osteogenic gene expression. Results: AEPB highly promoted the expression of osteogenic markers including runt-related transcription factor 2, osterix, and alkaline phosphatase, along with elevated levels of mineralization in MC3T3-E1 cells. Moreover, AEPB accelerated vertebral formation in zebrafish larvae accompanied by upregulated expression of osteogenic genes. FH535, an inhibitor of Wnt/β-catenin, suppressed AEPB-induced osteogenic gene expression and vertebral formation, indicating that AEPB stimulates osteogenesis by activating the Wnt/β-catenin signaling pathway. Conclusions: AEPB stimulates osteoblast differentiation and bone formation by activating β-catenin. Therefore, AEPB is a promising material that induces osteogenesis, and is useful for the treatment of bone resorption diseases.

A multicellular bioprinted cell construct for vascularized bone tissue regeneration

• A bioprinting strategy using a biocompatible collagen/β-TCP bioink and the hASC/endothelial cells was proposed. • hASC/HUVEC-laden structures with cell-differentiation niches for bone regeneration were proposed. • The bioprinted structure induced significantly greater new bone and blood vessel formation. In the present study, a bioprinted cell-laden construct consisting of collagen, β-tricalcium phosphate, and two different cell types, human adipose stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs), was prepared for bone tissue regeneration in spinal fusion. By using the appropriate printing parameters and bioceramic/cell compositions in the bioink, a 3D cell-laden construct consisting of microscale struts was successfully generated. To evaluate the contribution of the laden stem cells and endothelial cells in promoting angiogenic and osteogenic activities, differentiation of the cells within the bioink was analyzed by measuring gene expression profiles and immunofluorescence imaging. The hASC/HUVEC-laden structure exhibited a strong angiogenic phenotype and upregulated angiogenic factors, as well as enhanced expression of osteogenic gene markers due to the synergistic crosstalk of the two cell types. The construct laden with hASCs/HUVECs was implanted in a mouse model of posterolateral lumbar spinal fusion, and the structure provided significantly higher new bone formation and angiogenesis compared with constructs laden with only hASCs. The in vitro and in vivo findings suggest that biomimetic cell-laden constructs could perform as potential bone tissue regenerating structures to encourage effective spinal fusion.

Long non-coding RNA KCNQ1OT1 overexpression promotes osteogenic differentiation of staphylococcus aureus-infected human bone mesenchymal stem cells by sponging microRNA miR-29b-3p

ABSTRACT Osteomyelitis (OM) is an orthopedic disease caused by bone infections in the bone cortex, bone marrow, periosteum, and surrounding soft tissues. Recent studies have implicated non-coding RNAs (ncRNAs) in the development of OM. However, little is known about the role of ncRNAs in the osteogenic differentiation during bone infection. In the present study, we investigated the role of KCNQ1OT1/miR-29b-3p axis in osteogenic differentiation in staphylococcus aureus (SpA)-infected human bone mesenchymal stem cells (hBMSCs). We first examined the expression of lncRNA KCNQ1OT1 and miR-29b-3p in the serum samples of OM patients and healthy controls. We also infected hBMSCs with different concentrations of SpA and studied the osteogenic differentiation after infection. Our results revealed that KCNQ1OT1 was downregulated while miR-29b-3p was upregulated in the serum samples of OM patients, as well as in SpA-infected hBMSCs. Overexpression of KCNQ1OT1 ameliorated the damage in hBMSCs caused by SpA infection. KCNQ1OT1 could support hBMSCs osteogenic differentiation by enhancing ALP activity, alizarin red S accumulation, expressions of osteogenic markers, and attenuating inflammatory responses after SpA infection. We further showed that miR-29b-3p was a downstream target of KCNQ1OT1, mediating the osteogenic differentiation of hBMSCs during SpA infection. Our data suggest that KCNQ1OT1 could ameliorate the SpA-induced suppression of osteogenic differentiation in hBMSCs by sponging miR-29b-3p. Modulating KCNQ1OT1 expression may serve as a strategy to ameliorate osteomyelitis.

Fucoidan (Undaria pinnatifida)/Polydopamine Composite-Modified Surface Promotes Osteogenic Potential of Periodontal Ligament Stem Cells.

Fucoidan, a marine-sulfated polysaccharide derived from brown algae, has been recently spotlighted as a natural biomaterial for use in bone formation and regeneration. Current research explores the osteoinductive and osteoconductive properties of fucoidan-based composites for bone tissue engineering applications. The utility of fucoidan in a bone tissue regeneration environment necessitates a better understanding of how fucoidan regulates osteogenic processes at the molecular level. Therefore, this study designed a fucoidan and polydopamine (PDA) composite-based film for use in a culture platform for periodontal ligament stem cells (PDLSCs) and explored the prominent molecular pathways induced during osteogenic differentiation of PDLSCs through transcriptome profiling. Characterization of the fucoidan/PDA-coated culture polystyrene surface was assessed by scanning electron microscopy and X-ray photoelectron spectroscopy. The osteogenic differentiation of the PDLSCs cultured on the fucoidan/PDA composite was examined through alkaline phosphatase activity, intracellular calcium levels, matrix mineralization assay, and analysis of the mRNA and protein expression of osteogenic markers. RNA sequencing was performed to identify significantly enriched and associated molecular networks. The culture of PDLSCs on the fucoidan/PDA composite demonstrated higher osteogenic potency than that on the control surface. Differentially expressed genes (DEGs) (n = 348) were identified during fucoidan/PDA-induced osteogenic differentiation by RNA sequencing. The signaling pathways enriched in the DEGs include regulation of the actin cytoskeleton and Ras-related protein 1 and phosphatidylinositol signaling. These pathways represent cell adhesion and cytoskeleton organization functions that are significantly involved in the osteogenic process. These results suggest that a fucoidan/PDA composite promotes the osteogenic potential of PDLSCs by activation of critical molecular pathways.

Cannabidiol Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in the Inflammatory Microenvironment via the CB2-dependent p38 MAPK Signaling Pathway.

Chronic inflammation of bone tissue often results in bone defects and hazards to tissue repair and regeneration. Cannabidiol (CBD) is a natural cannabinoid with multiple biological activities, including anti-inflammatory and osteogenic potential. This study aimed to investigate the efficacy and mechanisms of CBD in the promotion of bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation in the inflammatory microenvironment. BMSCs isolated from C57BL/6 mice, expressed stem cell characteristic surface markers and presented multidirectional differentiation potential. The CCK-8 assay was applied to evaluate the effects of CBD on BMSCs' vitality, and demonstrating the safety of CBD on BMSCs. Then, BMSCs were stimulated with lipopolysaccharide (LPS) to induce inflammatory microenvironment. We found that CBD intervention down-regulated mRNA expression levels of inflammatory cytokines and promoted cells proliferation in LPS-treated BMSCs, also reversed the protein and mRNA levels downregulation of osteogenic markers caused by LPS treatment. Moreover, CBD intervention activated the cannabinoid receptor 2 (CB2) and the p38 mitogen-activated protein kinase (MAPK) signaling pathway. While AM630, a selective CB2 inhibitor, reduced phosphorylated (p)-p38 levels. In addition, AM630 and SB530689, a selective p38 MAPK inhibitor, attenuated the enhancement of osteogenic markers expression levels by CBD in inflammatory microenvironment, respectively. CBD promoted osteogenic differentiation of BMSCs via the CB2/p38 MAPK signaling pathway in the inflammatory microenvironment.

Hypoxia During the Consolidation Phase of Distraction Osteogenesis Promotes Bone Regeneration.

BackgroundHypoxia is the critical driving force for angiogenesis and can trigger the osteogenic-angiogenic coupling followed by the enhancement of bone regeneration. While lots of studies showed that hypoxia administration can accelerate bone formation during distraction osteogenesis (DO), the therapeutic timing for the osteogenic purpose was concentrated on the distraction phase. The outcomes of hypoxia administration in the consolidation phase stay uncertain. The purpose of this study was to determine the osteogenic effectiveness of hypoxia therapy during the consolidation phase, if any, to enhance bone regeneration in a rat femoral DO model.MethodsA total of 42 adult male Sprague-Dawley rats underwent right femoral mid-diaphysis transverse osteotomy and were randomly divided into Control (NS administration, n = 21) and Group1 (deferoxamine therapy, n = 21) after distraction. During the consolidation phase, Group1 was treated with local deferoxamine (DFO) injection into the distraction zone, while the Control underwent the same dosage of NS. Animals were sacrificed after 2, 4, and 6 weeks of consolidation. The process of bone formation and remodeling was monitored by digital radiographs, and the regenerated bone was evaluated by micro-computed tomography (micro-CT), biomechanical test, and histological analysis. The serum content of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) were measured by enzyme linked immunosorbent assay (ELISA) for further analysis.ResultsBone regeneration was significantly enhanced after hypoxia therapy during the consolidation phase. The digital radiograph, micro-CT, and biomechanical evaluation showed better effects regarding volume, continuity, and mechanical properties of the regenerated bone in Group1. The histomorphological evaluation also revealed the hypoxia treatment contributed to accelerate bone formation and remodeling during DO. The higher positive expression of angiogenic and osteogenic markers were observed in Group1 after hypoxia administration according to the immunohistochemical analysis. The serum content of HIF-1α and VEGF was also increased after hypoxia therapy as evidenced from ELISA.ConclusionHypoxia administration during the consolidation phase of distraction osteogenesis has benefits in enhancing bone regeneration, including accelerates the bone formation and remodeling.