Osteoblast Differentiation Markers Research Articles

A transgenic mouse line with a 58 kb fragment deletion shows skeletal defects

Previously, a transgenic mouse line lacking a 58 kb fragment deletion including the upstream and exon1 of Fam20a gene (58 kb deletion mouse) was generated and showed growth retardation. The aim of this study was to characterize the skeletal phenotype of the homozygous 58 kb deletion mouse (58 kb-/-). Our results showed that body size and bone length of the 58 kb-/- mice were smaller than those of wild-type (WT) mice. The microcomputed tomography (µCT) analyses of trabecular and cortical bones in the 58 kb-/- displayed lower bone volume, thinner trabeculae and thinner bone cortex as compared to WT. Histological examination of the 58 kb-/- growth plate demonstrated disorganized chondrocyte zones and extended hypertrophic zone. The qPCR results showed downregulation of several osteoblast differentiation markers in the 58 kb-/- long bone. Immunohistochemical examination demonstrated reduced chondrocyte proliferation, apoptosis and increased collagen X expression in the 58 kb-/- growth plate. Our data showed a lower number of osteoblasts and osteoclasts in the 58 kb-/- as compared to WT. In vitro cell culture study demonstrated the 58 kb-/- showed a lower number of bone marrow stromal cells and osteoprogenitors. The extent of matrix mineralization was impaired in the 58 kb-/- osteoblast cultures. In conclusion, endochondral ossification defects and reduced number of osteoblasts and their precursors led to the bone phenotype in the 58 kb-/-, indicating that the genes deleted in this 58 kb likely play an important role in skeletal development.

HISTOMORPHOMETRIC AND IMMUNOHISTOCHEMICAL EVALUATION OF BONE HEALING AROUND IMPLANTS PLACED USING PIEZOSURGERY VS CONVENTIONAL DRILLS: A SPLITMOUTH PILOT STUDY.

This pilot study evaluated and compared histomorphometric and immunohistochemical characteristics of peri-implant bone tissue after implant site preparation using piezoelectric tips versus conventional drills. Six patients with bilateral partial edentulism underwent a splitmouth protocol. Twelve alveolar ridges were randomized into six control implant sites prepared using conventional drills (Drill Group); and six test implant sites prepared using piezoelectric implant inserts (Piezo Group). At 28 days after surgery (T1), single-stage "study fixtures" with 0.5 mm of peri-implantbone tissue were explanted and processed for histological, histomorphometric and immunohistochemical analysis in both groups. For each sample inflammatory infiltrates, necrotic bone (Zone 1), woven and newly formed bone (Zone 2), native bone (Zone 3), a vascular endothelium differentiation and neo-osteogenesis marker (CD31) and an osteoblastic cell differentiation and osteogenesis marker (SATB2) were evaluated. According to their histological bone features, the three histologically distinct areas were evident in both groups: Zone 1, Zone 2 and Zone 3 according to their histological bone features. Zone 1 showed lower extension in the Piezo Group than in the Drill Group (p=0.028). Regarding the immunohistochemical markers, in all areas of the Piezo Group, SATB2 and CD31 were statistically higher than in the Drill Group. Implant site preparation using piezo surgery results in less bone necrosis, greater osteoblastic activity and greater vessel proliferation compared to the conventional surgical approach.

Palm Tocotrienol Activates the Wnt3a/β-Catenin Signaling Pathway, Protecting MC3T3-E1 Osteoblasts from Cellular Damage Caused by Dexamethasone and Promoting Bone Formation.

Background and aim: Prolonged glucocorticoid (GC) treatment increases oxidative stress, triggers apoptosis of osteoblasts, and contributes to osteoporosis. Tocotrienol, as an antioxidant, could protect the osteoblasts and preserve bone quality under glucocorticoid treatment. From this study, we aimed to determine the effects of tocotrienol on MC3T3-E1 murine pre-osteoblastic cells treated with GC. Methods: MC3T3-E1 cells were exposed to dexamethasone (150 µM), with or without palm tocotrienol (PTT; 0.25, 0.5, and 1 µg/mL). Cell viability was measured by the MTS assay. Alizarin Red staining was performed to detect calcium deposits. Cellular alkaline phosphatase activity was measured to evaluate osteogenic activity. The expression of osteoblastic differentiation markers was measured by an enzyme-linked immunoassay. Results: Enhanced matrix mineralization was observed in the cells treated with 0.5 µg/mL PTT, especially on day 18 (p < 0.05). The expression of Wnt3a, β-catenin, collagen 1α1, alkaline phosphatase, osteocalcin, low-density lipoprotein receptor-related protein 6, and runt-related transcription factor-2 were significantly increased in the PTT-treated groups compared to the vehicle control group, especially at 0.5 µg/mL of PTT (p < 0.05) and on day 6 of treatment. Conclusions: PTT maintains the osteogenic activity of the dexamethasone-treated osteoblasts by promoting their differentiation.

5,7-Dihydroxy-4-Methylcoumarin enhances osteogenesis and ameliorates osteoporosis via the AKT1 pathway.

Osteoporosis is a chronic disease distinguished by decreased bone density and degradation of bone microstructure, frequently linked with inflammation and oxidative stress, both of which contribute to the acceleration of bone resorption. The compound 5,7-Dihydroxy-4-methylcoumarin (D4M) present in Artemisia dracunculus exhibits significant antioxidant and anti-inflammatory properties. Nonetheless, the potential anti-osteoporotic effects of D4M, along with the molecular targets and mechanisms responsible for these effects, have not been studied. This study aims to assess the impact of D4M on osteoblastogenesis and glucocorticoid-induced osteoporosis while examining the potential underlying mechanisms. We examined the effects of varying concentrations of D4M on the proliferation and differentiation of MC3T3-E1 cells. Additionally, in vivo experiments were carried out using a glucocorticoid-induced zebrafish osteoporosis model to evaluate the effects of D4M on vertebral bone density and osteogenic markers. Target prediction and molecular docking analyses were conducted to investigate the binding interactions between D4M and its target proteins. D4M showed a significant enhancement of MC3T3-E1 cell proliferation and differentiation within the concentration range of 10 to 40 μM, with the greatest increase in mineralization noted at 20 μM. Furthermore, in the zebrafish osteoporosis model, treatment with 20 μM D4M resulted in a significant improvement in vertebral bone density and the restoration of osteoblast-specific marker expression. Ligand-based target prediction identified AKT1 as a potential target for D4M, and molecular docking highlighted the binding interactions between D4M and AKT1 phosphorylation sites. Co-treatment with the AKT1 inhibitor A-443654 abolished the anti-osteoporotic effects of D4M. These findings demonstrate that D4M enhances osteoblast differentiation and mitigates osteoporosis through its interaction with AKT1, suggesting its potential as a therapeutic agent for treating osteoporosis.

Exploring the Anti-Osteoporotic Effects of n-Hexane Fraction from Cotoneaster wilsonii Nakai: Activation of Runx2 and Osteoblast Differentiation In Vivo.

Osteoporosis is characterized by the microstructural depletion of bone tissue and decreased bone density, leading to an increased risk of fractures. Cotoneaster wilsonii Nakai, an endemic species of the Korean Peninsula, grows wild in Ulleungdo. In this study, we aimed to investigate the effects of C. wilsonii and its components on osteoporosis. The alkaline phosphatase (ALP) activity of C. wilsonii extracts and fractions was evaluated in MC3T3-E1 pre-osteoblasts, and the n-hexane fraction (CWH) showed the best properties for ALP activity. The effects of the CWH on bone formation were assessed in MC3T3-E1 cells and ovariectomized mice. Biochemical assays and histological analyses focused on the signaling activation of osteoblast differentiation and osteogenic markers, such as ALP, collagen, and osterix. The CWH significantly activated TGF-β and Wnt signaling, enhancing osteoblast differentiation and bone matrix formation. Notably, CWH treatment improved micro-CT indices, such as femoral bone density, and restored serum osteocalcin levels compared to OVX controls. These results highlight the potential of the C. wilsonii Nakai n-hexane fraction as a promising therapeutic agent for managing osteoporosis.

Inhibition of Mitochondrial Fission Reverses Simulated Microgravity-Induced Osteoblast Dysfunction by Enhancing Mechanotransduction and Epigenetic Modification.

Simulated microgravity (SMG) poses substantial challenges to astronaut health, particularly impacting osteoblast function and leading to disuse osteoporosis. This study investigates the adverse effects of SMG on osteoblasts, focusing on changes in mitochondrial dynamics and their consequent effects on cellular energy metabolism and mechanotransduction pathways. We discovered that SMG markedly reduced the expression of osteoblast differentiation markers and promoted mitochondrial fission, as indicated by an increase in punctate mitochondria, a decrease in mitochondrial length, and a reduction in cristae density. These mitochondrial alterations are linked to elevated reactive oxygen species levels, a decrease in ΔΨm, and a metabolic shift from oxidative phosphorylation to glycolysis, resulting in decreased adenosine triphosphate production, which are all indicative of mitochondrial dysfunction. Our results showed that treatment with mitochondrial division inhibitor-1 (mdivi-1), a mitochondrial fission inhibitor, effectively inhibited these SMG-induced effects, thereby maintaining mitochondrial structure and function and promoting osteoblast differentiation. Furthermore, SMG disrupted critical mechanotransduction processes, by affecting paxillin expression, the RhoA-ROCK-Myosin II pathway, and actin dynamics, which subsequently altered nuclear morphology and disrupted Yes-associated protein signaling. Notably, treatment with mdivi-1 prevented these disruptions in mechanotransduction pathways. Moreover, our study showed that SMG-induced chromatin remodeling and histone methylation, which are epigenetic barriers to osteogenic differentiation, can be reversed by targeting mitochondrial fission, further highlighting the significance of mitochondrial dynamics in osteoblast function in an SMG environment. Therefore, targeting mitochondrial fission emerges as a promising therapeutic strategy to alleviate osteoblast dysfunction under SMG conditions, providing novel approaches to maintain bone health during prolonged space missions and safeguard the astronaut well-being.

Synergistic Effects of Photobiomodulation and Differentiation Inducers on Osteogenic Differentiation of Adipose-Derived Stem Cells in Three-Dimensional Culture.

Osteoporosis, a common metabolic bone disorder, leads to increased fracture risk and significant morbidity, particularly in postmenopausal women and the elderly. Traditional treatments often fail to fully restore bone health and may cause side effects, prompting the exploration of regenerative therapies. Adipose-derived stem cells (ADSCs) offer potential for osteoporosis treatment, but their natural inclination toward adipogenic rather than osteogenic differentiation poses a challenge. This study investigates a novel approach combining differentiation inducers (DIs), three-dimensional (3D) hydrogel scaffolds, and photobiomodulation (PBM) to promote osteogenic differentiation of immortalised ADSCs. A dextran-based 3D hydrogel matrix, supplemented with a DI cocktail of dexamethasone, β-glycerophosphate disodium, and ascorbic acid, was used to foster osteogenesis. PBM was applied using near-infrared (825 nm), green (525 nm), and combined wavelengths at fluences of 3 J/cm2, 5 J/cm2, and 7 J/cm2 to enhance osteogenic potential. Flow cytometry identified osteoblast-specific markers, while inverted light microscopy evaluated cellular morphology. Reactive oxygen species assays measured oxidative stress, and quantitative polymerase chain reaction (qPCR) revealed upregulated gene expression linked to osteogenesis. The findings demonstrate that integrating DIs, 3D hydrogels, and PBM effectively drives osteogenic differentiation in immortalised ADSCs. The PBM enhanced osteogenic marker expression, induced morphological changes, and upregulated gene activity, presenting a promising framework for bone regeneration. Future research should assess the stability and functionality of these differentiated cells and explore their applicability in preclinical models of bone injury or degeneration. This integrative approach demonstrated specific efficacy in promoting the osteogenic differentiation of ADSCs, highlighting its potential application in developing targeted treatments for osteoporosis.

Fabrication and evaluation of Zn-EGCG-loaded chitosan scaffolds for bone regeneration: From cellular responses to in vivo performance.

We have synthesized a flavonoid metal complex (FMC) by chelating zinc to epigallocatechin-3-gallate (EGCG), a flavonoid present in green tea and incorporated into chitosan (CS) to form 3D constructs by freeze drying method. Scanning electron microscopy characterized The scaffolds for surface morphology and pore dimensions and depicted the presence of interconnected porous network. The scaffolds exhibited optimal pore size (>50μm), facilitating bone tissue ingrowth and neovascularization. Inclusion of Zn-EGCG into CS matrix improved the mechanical property by increasing compressive strength (0.53±0.045MPa) and reducing enzymatic degradation with controlled swelling. In addition, increased protein adsorption was observed during the initial hour, which is crucial for cell attachment. Furthermore, the FMC inclusion promoted exogenous biomineralization of CS scaffolds as early as 4d in simulated body fluid. Indirect cytotoxicity measurements indicated the scaffolds with Zn-EGCG had no toxic effects on mouse mesenchymal stem cells (mMSCs). Under osteogenic environment, the scaffold promoted calcium deposition of mMSCs by upregulation of ALP activity and increased expression of osteoblast differentiation markers such as Runx2, ColI, OC and OPN. We found that the involvement of miR-15b/smurf-1 signalling pathway behind the osteogenic potential of the scaffold. In vivo assessments using the chick embryo CAM assay showed enhanced angiogenesis and confirmed the scaffold's biocompatibility with no toxicity. Additionally, in a zebrafish scale regeneration model, the scaffold enhanced calcium deposition and osteoblast marker expression, aligning with the in vitro findings. Overall, form the study it is clear that the osteogenic potential of the scaffold is as follows chitosan<EGCG-Chitosan<Zn-EGCG-Chitosan.

D-limonene suppresses RANKL-induced osteoclast differentiation and promotes osteoblast activity in vitro.

Treatments for osteoporosis are typically given postfracture. Therefore, identifying safe prophylactic interventions to reduce fracture risk would be beneficial. One approach is to utilize the bioactive properties of natural compounds to modify osteoclast and osteoblast activity. d-limonene a well-tolerated, anti-inflammatory monoterpene found in citrus fruits holds promise due to its suppressive effect on NFκB, a key regulator of bone cell activity. We found that limonene promoted osteoblast differentiation and bone nodule formation and inhibited RANKL-induced osteoclast formation and bone resorption in vitro. Limonene also reduced the proresorptive signal provided by osteoblast, augmenting markers of osteoblast differentiation (alkaline phosphatase, osterix, and osteocalcin) and significantly decreasing osteoclastogenic cytokine production (PTHrP, IL-1β, and TNF-α). Therefore, limonene supplementation represents a potential route in combination with current interventions to optimize bone cell activity to maintain or enhance bone mass.

Hypergravity as a Possible Way of Bone Tissue Engineering in Osteoblastic Differentiation from Mesenchymal Stem Cells: A Systematic Review

Background: Tissue engineering development has become a highlight in recent decades. One of the key areas of focus is producing mature bone tissue to overcome orthopedic problems, such as bone defects. Various cultures have been implemented on stem cells; some induce osteoblastic differentiation markers, while others have the opposite effect. Microgravity has been proven in several studies to inhibit the expression of osteogenic differentiation markers. Conversely, hypergravity is expected to have the opposite impact, supporting stem cells in the osteogenesis pathway. Methods: A literature search was conducted using online databases including Sciencedirect, PubMed, and Proquest, covering the period from 2008 to 2022. This search considered only experimental studies published in English. The keywords used in this research were "hypergravity" and "mesenchymal stem cell." All acquired data were processed and analyzed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (2020). Results: Initially, 190 studies were collected from online databases based on relevant keywords. After screening, 5 studies were included in the final analysis, focusing on hypergravity treatment and its effects on mesenchymal stem cells (MSCs). Conclusion: Hypergravity shows a significant and strong impact on osteoblastic differentiation. This study revealed that a gravity force of 30G and a culture duration of 7 to 14 days are the most optimal combination for inducing osteoblastic differentiation in MSCs.

LncRNA MEG3 promotes aortic valve calcification by inducing osteoblast differentiation via Wnt catenin signaling pathway

Abstract Background Calcific aortic valve stenosis (CAVS) is the most common valvular heart disease in elderly population. This study aimed to investigate the role of long non-coding RNAs (lncRNAs) in the development of CAVS. Methods ldlr-/-mice were feeding with western diet for 6 months to induce CAVS. RNA-seq analysis was performed to identify differentially expressed molecular signaling between aortic valves of CAVS mice and control mice. The degree of aortic stenosis was detected by ultrasound. Calcium salt deposits were detected by Haematoxylin and eosin (H&amp;E) staining, Von Kossa staining and alizarin red S staining. PCR and western blotting were employed to detect osteogenic differentiation markers after various interventions. Results A mouse CAVS model was successfully established, as evidenced by increased thickness and calcium deposition in the aortic valve leaflets and increased transvalvular peak jet velocity. RNA-seq analysis showed that lncRNA MEG3 was significantly enriched in aortic valves of CAVS mice as compared to that of control mice. MEG3 expression in aortic valve, assessed by RNA in situ hybridization and PCR, was also significantly upregulated during the osteoblast differentiation of AVICs in vitro. Gain- and loss-of-function experiments indicate that MEG3 could indeed promote osteoblast differentiation of primary AVICs as evidenced by increased calcified nodule formation and expressions of osteoblast differentiation markers (Runx2 and osterix). Consistent with these in vitro data, MEG3 knockdown in vivo through tail injection of antisense oligonucleotide chain MEG3 (ASO-MEG3) at 6 weeks interval for 12 weeks significantly attenuated aortic valve calcification in CAVS mice. Subsequent KEGG analysis demonstrated that the Wnt/β-catenin signaling pathway was significantly enriched during the CAVS progression. Overexpression of MEG3 activated the Wnt/β-catenin signaling pathway and upregulated osteogenic protein expressions, while this upregulation could be significantly ameliorated by Dickkopf 1, a Wnt/β-catenin pathway inhibitor. Conclusion Present study demonstrates that LncRNA MEG3 could promote aortic valve calcification by inducing osteoblast differentiation via activating Wnt/β-catenin signaling pathway. Our results provide experimental evidence of targeting MEG3 and Wnt/β-catenin signaling pathway as potential promising therapeutic option for prevention and treatment of aortic valve calcification.

Effect of recombinant irisin on recombinant human bone morphogenetic protein-2 induced osteogenesis and osteoblast differentiation

Osteoporotic fragility fractures substantially impact aging societies, necessitating long-term care and increasing healthcare costs. Myokine irisin, secreted by skeletal muscle, influences bone metabolism; however, a comprehensive understanding of the mechanisms by which irisin affects bone metabolism is still lacking. Therefore, this study aimed to explore the effects of irisin on osteogenesis and osteoblast differentiation triggered by bone morphogenetic protein-2 (BMP-2). We used 4-week-old male ICR mice and implanted polyethylene glycol pellets containing recombinant human BMP-2 (rh-BMP-2) into the left dorsal muscle pouch. Mice received weekly intraperitoneal injections of either phosphate-buffered saline or recombinant irisin (re-irisin). Ectopic bone formation was evaluated 3 weeks post-surgery using micro-computed tomography (μ-CT) and histological analysis. In vitro experiments, C2C12 cells were treated with or without rh-BMP-2 and re-irisin, and we assessed osteoblast differentiation markers, e.g., runt-related transcription factor 2, alkaline phosphatase, osteocalcin, and osteopontin, using real-time reverse transcription-polymerase chain reaction. The μ-CT analyses showed that re-irisin significantly increased bone mineral content and bone volume of ectopic bones newly formed by rh-BMP-2. The gene expressions of the osteoblast markers were significantly increased by rh-BMP-2 and further upregulated by re-irisin. The treatment of cyclic AMP response element-binding protein (CREB) small interfering RNA attenuated these effects, suggesting that CREB signaling pathway was involved in rh-BMP-2/re-irisin-induced osteoblastic differentiation. This study demonstrates the potential of irisin to enhance osteogenesis through BMP signaling, offering insights for osteoporosis treatment and highlighting irisin as a promising therapeutic target for improving bone health and extending a healthy lifespan.

AEBP1 restores osteoblastic differentiation under dexamethasone treatment by activating PI3K/AKT signalling.

Adipocyte enhancer-binding protein 1 (AEBP1) is closely implicated in osteoblastic differentiation and bone fracture; this research aimed to investigate the effect of AEBP1 on restoring osteoblastic differentiation under dexamethasone (Dex) treatment, and its interaction with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Pre-osteoblastic MC3T3-E1 cells were cultured in osteogenic medium and treated by Dex to mimic steroid-induced osteonecrosis cellular model. They were then further transfected with control or AEBP1-overexpressed lentiviral vectors. Finally, cells were treated with the PI3K inhibitor LY294002, with or without AEBP1-overexpressed lentiviral vectors. AEBP1 expression showed a downward trend in MC3T3-E1 cells under Dex treatment in a dose-dependent manner. AEBP1-overexpressed lentiviral vectors increased relative cell viability, alkaline phosphatase (ALP) staining, Alizarin red staining and osteoblastic differentiation markers including osteocalcin (OCN), osteopontin (OPN), collagen type I alpha 1 (COL1A1), runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2), but decreased cell apoptosis rate in MC3T3-E1 cells under Dex treatment; besides, AEBP1-overexpressed lentiviral vectors positively regulated p-PI3K and p-AKT expressions. Furthermore, LY294002 treatment decreased relative cell viability, Alizarin red staining, osteoblastic differentiation markers including OCN, OPN, RUNX2 and BMP, increased cell apoptosis rate and did not affect ALP staining in MC3T3-E1 cells under Dex treatment; meanwhile, LY294002 treatment weakened the effect of AEBP1 overexpression vectors on the above cell functions. AEBP1 restores osteoblastic differentiation under Dex treatment by activating the PI3K/AKT pathway.

SEV-mediated lipid droplets transferred from bone marrow adipocytes promote ferroptosis and impair osteoblast function

Osteoporosis is linked to increased bone marrow adipocyte (BMAd) proliferation, which displaces bone-forming cells and alters the local environment. The impact of BMAd lipid droplets on bone health and osteoblast function remains unclear. This study investigates the interplay between BMAd-derived lipid droplets and osteoblast functionality, focusing on ferroptosis pathways. Osteoblast cultures were treated with conditioned media from adipocytes to simulate in vivo conditions. High-throughput mRNA sequencing and Western blot analysis were used to profile changes in gene expression and protein levels related to ferroptosis, oxidative phosphorylation, and osteogenic markers. Cellular assays assessed the direct impact of lipid droplets on osteoblast activity. Results showed that osteoblasts exposed to adipocyte-conditioned media had increased intracellular lipid droplet accumulation, upregulation of ferroptosis-related genes and proteins, and downregulation of oxidative phosphorylation and osteoblast differentiation markers. Treatment with ferroptosis inhibitors reversed the detrimental effects on osteoblasts, indicating the functional relevance of this pathway in osteoporosis. BMAd-derived lipid droplets contribute to osteoblast dysfunction through ferroptosis induction. Inhibiting ferroptosis could preserve osteoblast function and combat osteoporosis-related bone issues, suggesting that modulating lipid metabolism and redox balance in bone cells may be promising for future treatments.

Studies on the Anti-Inflammatory Effect of Xiaoyao Pills in The Treatment of Postmenopausal Osteoporosis in Mice.

Osteoporosis is a common metabolic disease of elderly and postmenopausal women, with no obvious symptoms during its early stages. In the latter stages of this condition, the patients are prone to fractures, and this can seriously affect their health and quality of life. The worldwide increase in life expectancy has made osteoporosis a global concern. The Xiaoyao pills were previously used in the treatment of depression. In addition, the drug appeared to have estrogen-like activity, which affected the expression of ALP, an early osteoblast-specific marker, and COL-1, a major component of bone extracellular matrix. Xiaoyao pills were assessed for their effects on postmenopausal osteoporosis (PMOM) in mice. The target information of each herbal component of Xiaoyao pills was accessed through the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Information from GeneCards, OMIM, PharmGkb, TTD, DrugBank, and other websites was used to construct the regulatory network of the herbal complex through Cytoscape and String network to assess the protein interactions. Mice were ovariectomized, and treated with high and low doses of Xiaoyao pills and these were compared to controls. Their symptoms were assessed by immunocytochemistry of bone tissues. The results suggested that Xiaoyao pills had the ability to alleviate the symptoms of PMOM in ovariectomized mice through the IL-17 signaling pathway. This drug has the potential to become a novel therapeutic agent for the treatment of osteoporosis.

The role of lysophosphatidylcholine acyltransferase 2 in osteoblastic differentiation of C2C12 cells.

Glycerophospholipids, a primary component of cellular membranes, play important structural and functional roles in cells. In the remodelling pathway (Lands' cycle), the concerted actions of phospholipase As and lysophospholipid acyltransferases (LPLATs) contribute to the incorporation of diverse fatty acids in glycerophospholipids in an asymmetric manner, which differ between cell types. In this study, the role of LPLATs in osteoblastic differentiation of C2C12 cells was investigated. Gene and protein expression levels of lysophosphatidylcholine acyltransferase 2 (LPCAT2), one of the LPLATs, increased during osteoblastic differentiation in C2C12 cells. LPCAT2 knockdown in C2C12 cells downregulated the expression of osteoblastic differentiation markers and the number and size of lipid droplets (LDs) and suppressed the phosphorylation of Smad1/5/9. In addition, LPCAT2 knockdown inhibited Snail1 and the downstream target of Runx2 and vitamin D receptor (VDR). These results suggest that LPCAT2 modulates osteoblastic differentiation in C2C12 cells through the bone morphogenetic protein (BMP)/Smad signalling pathway.

Combined Effects of Fibroblast Growth Factor-2 and Carbonate Apatite Granules on Periodontal Healing: An In Vivo and In Vitro Study.

The aim of this study was to investigate in vivo and in vitro the effectiveness of the use of fibroblast growth factor (FGF)-2 with carbonate apatite (CO3Ap) on periodontal healing. Periodontal defects created in the maxillary first molars in rats were treated with FGF-2, CO3Ap, FGF-2 + CO3Ap or left unfilled. Healing was evaluated using microcomputed tomography, histological, and immunohistochemical analyses. In vitro experiments were performed to assess cellular behaviors and the expression of osteoblastic differentiation markers in MC3T3-E1 cells. At 4 weeks, the bone volume fraction in the FGF-2 + CO3Ap group was significantly greater than that in the CO3Ap group, but there was no significant difference from the FGF-2 group. The FGF-2 + CO3Ap group demonstrated greater new bone compared with the FGF-2 or CO3Ap group. The FGF-2 + CO3Ap group showed greater levels of osteocalcin-positive cells compared with the CO3Ap group, but there was no significant difference from the FGF-2 group. In vitro, the FGF-2 + CO3Ap group exhibited a greater extent of cell attachment and more elongated cells compared with the CO3Ap group. Compared with the CO3Ap group, the FGF-2 + CO3Ap group showed significantly higher viability/proliferation, but the expressions of Runx2 and Sp7 were reduced. The results indicated that the use of FGF-2 with CO3Ap enhanced healing in the periodontal defects. FGF-2 promoted cell attachment to and proliferation on CO3Ap and regulated osteoblastic differentiation, thereby contributing to novel bone formation.

Glycinamide Facilitates Nanocomplex Formation and Functions Synergistically with Bone Morphogenetic Protein 2 to Promote Osteoblast Differentiation In Vitro and Bone Regeneration in a Mouse Calvarial Defect Model.

This study aimed to identify glycine analogs conducive to the formation of cell-absorbable nanocomplexes, enhancing collagen synthesis and subsequent osteogenesis in combination with BMP2 for improved bone regeneration. Glycine and its derivatives were assessed for their effects on osteogenic differentiation in MC3T3-E1 cells and human bone marrow mesenchymal stem cells (BMSCs) under osteogenic conditions or with BMP2. Osteogenic differentiation was assessed through alkaline phosphatase staining and real-time quantitative polymerase chain reaction (RT-qPCR). Nanocomplex formation was examined via scanning electron microscopy, circular dichroism, and ultraviolet-visible spectroscopy. In vivo osteogenic effects were validated using a mouse calvarial defect model, and bone regeneration was evaluated through micro-computed tomography and histomorphometric analysis. Glycine, glycine methyl ester, and glycinamide significantly enhanced collagen synthesis and ALP activity in conjunction with an osteogenic medium (OSM). GA emerged as the most effective inducer of osteoblast differentiation marker genes. Combining GA with BMP2 synergistically stimulated ALP activity and the expression of osteoblast markers in both cell lines. GA readily formed nanocomplexes, facilitating cellular uptake through strong electrostatic interactions. In an in vivo calvarial defect mouse model, the GA and BMP2 combination demonstrated enhanced bone volume, bone volume/tissue volume ratio, trabecular numbers, and mature bone formation compared to other combinations. GA and BMP2 synergistically promoted in vitro osteoblast differentiation and in vivo bone regeneration through nanocomplex formation. This combination holds therapeutic promise for individuals with bone defects, showcasing its potential for clinical intervention.

Blackcurrant extract promotes differentiation of MC3T3‑E1 pre‑osteoblasts.

Osteoporosis risk increases in menopausal individuals owing to the decrease in estrogen secretion. Blackcurrant extract (BCE) ameliorates osteoporosis; however, the underlying mechanisms are unclear. Furthermore, although BCE has phytoestrogenic activity, its effects on osteoblasts are unknown. In the present study, we investigated BCE-mediated attenuation of osteoporosis using mouse MC3T3-E1 pre-osteoblasts, with a focus on osteogenesis. After treating MC3T3-E1 cells with BCE for 48 h, cell proliferation was assessed using Cell Counting Kit-8. Levels of osteoblast differentiation markers, namely alkaline phosphatase activity and total collagen content in the cells, were evaluated after 3 and 14 days of BCE treatment, respectively. The expression of genes encoding osteoblast differentiation markers, including collagen type I (Col-I), alkaline phosphatase (Alp), bone γ-carboxyglutamate protein (Bglap), and runt-related transcription factor 2 (Runx2), was evaluated using reverse transcription-quantitative polymerase chain reaction. Mineralization of the cells was evaluated using Alizarin Red staining. Femoral tissues of ovariectomized (OVX) rats with or without 3% BCE were stained using ALP to evaluate osteogenic differentiation in femoral tissue. After treating MC3T3-E1 cells with BCE, cell proliferation had increased. BCE treatment increased Alp activity and total collagen content. Moreover, the expression of Col-I, Alp, Bglap, and Runx2 increased in BCE-treated cells. Furthermore, when MC3T3-E1 cells were treated with BCE for 21 days, the levels of calcified nodules increased. Alp staining intensity was stronger in the epiphyses on femoral tissue of OVX rats treated with 3% BCE than in those of untreated OVX rats. The results suggest that BCE may promote osteogenesis by inducing osteoblast differentiation.

A sponge-like nanofiber melatonin-loaded scaffold accelerates vascularized bone regeneration via improving mitochondrial energy metabolism

Electrospun nanofibers have been widely employed in bone tissue engineering for their ability to mimic the micro to nanometer scale network of the native bone extracellular matrix. However, the dense fibrous structure and limited mechanical support of these nanofibers pose challenges for the treatment of critical size bone defects. In this study, we propose a facile approach for creating a three-dimensional scaffold using interconnected electrospun nanofibers containing melatonin (Scaffold@MT). The hypothesis posited that the sponge-like Scaffold@MT could potentially enhance bone regeneration and angiogenesis by modulating mitochondrial energy metabolism. Melatonin-loaded gelatin and poly-lactic-acid nanofibers were fabricated using electrospinning, then fragmented into shorter fibers. The sponge-like Scaffold@MT was created through a process involving homogenization, low-temperature lyophilization, and chemical cross-linking, while maintaining the microstructure of the continuous nanofibers. The incorporation of short nanofibers led to a low release of melatonin and increased Young's modulus of the scaffold. Scaffold@MT demonstrated positive biocompatibility by promoting a 14.2 % increase in cell proliferation. In comparison to the control group, Scaffold@MT significantly enhanced matrix mineralization by 3.2-fold and upregulated the gene expression of osteoblast-specific markers, thereby facilitating osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). Significantly, Scaffold@MT led to a marked enhancement in the mitochondrial energy function of BMMSCs, evidenced by elevated adenosine triphosphate (ATP) production, mitochondrial membrane potential, and protein expression of respiratory chain factors. Furthermore, Scaffold@MT promoted the migration of human umbilical vein endothelial cells (HUVECs) and increased tube formation by 1.3 times compared to the control group, accompanied by an increase in vascular endothelial growth factor (VEGFA) expression. The results of in vivo experiments indicate that the implantation of Scaffold@MT significantly improved vascularized bone regeneration in a distal femur defect in rats. Micro-computed tomography analysis conducted 8 weeks post-surgery revealed that Scaffold@MT led to optimal development of new bone microarchitecture. Histological and immunohistochemical analyses demonstrated that Scaffold@MT facilitated bone matrix deposition and new blood vessel formation at the defect site. Overall, the utilization of melatonin-loaded nanofiber sponges exhibits significant promise as a scaffold that promotes bone growth and angiogenesis, making it a viable option for the repair of critical-sized bone defects.