Bone Formation-related Genes Research Articles

Analysis of the Effect of Human Type I Collagen-Derived Peptide on Bone Regenerative Capacity and Comparison with Various Collagen Materials In Vivo.

Background and Objectives: Autologous bone grafting is the first choice for reconstructive surgery in bone defects due to trauma or malignant tumors. However, there is an increasing demand for minimally invasive alternatives involving bone regeneration using artificial materials. Biomimetic materials that replicate the body's microscopic structure, such as Cellnest®, are gaining attention. Cellnest is a xeno-free recombinant peptide based on human type I collagen, containing a rich Arg-Gly-Asp (RGD) motif related to cell adhesion. The aim of this study was to compare the effects of Cellnest with existing collagen materials (Pelnac®, Integra®, Terudermis®) on bone regeneration and elucidate the underlying mechanisms. Materials and Methods: In vivo experiments involved a rat model of calvarial bone defects, in which Cellnest and other collagen materials were implanted into the defect area. Bone formation was assessed after 4 weeks using micro-computed tomography (micro-CT) and histological analysis. In vitro experiments included the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), adhesion, and migration assays, and a real-time polymerase chain reaction using rapidly expanding cells (RECs) to explore the mechanisms of Cellnest's bone regenerative capacity. Results: The micro-CT analysis showed that the regenerated bone area was significantly greater in the Cellnest group (72.3%) than in the Pelnac® (25.5%), Integra® (31.6%), and Terudermis® (38.3%) groups. The histological analysis confirmed similar trends, with Cellnest showing 42.2% bone regeneration, outperforming the other materials. The in vitro assays revealed that Cellnest promoted cell proliferation, adhesion, and migration. Gene expression analysis demonstrated that Cellnest significantly increased the levels of the bone formation markers ALP and COL1. Conclusions: Cellnest, a human type I collagen-like peptide rich in RGD motifs, enhances bone regeneration by promoting MSC adhesion and migration, and bone formation-related gene expression. The findings suggest its potential as an effective material for bone defect reconstruction.

Integrative analysis of bone-formation associated genes and immune cell infiltration in osteoporosis, and the prediction of active ingredients in targeted traditional Chinese medicine

ObjectiveTo explore the differential expression and mechanisms of bone formation-related genes in osteoporosis (OP) leveraging bioinformatics and machine learning methodologies, and to predict the active ingredients of targeted traditional Chinese medicine (TCM) herbs. MethodsThe Gene Expression Omnibus (GEO) and GeneCards databases were employed to conduct a comprehensive screening of genes and disease-associated loci pertinent to the pathogenesis of OP. The R package was utilized as the analytical tool for the identification of differentially expressed genes. Least absolute shrinkage and selection operator (LASSO) logistic regression analysis and support vector machine-recursive feature elimination (SVM-RFE) algorithm were employed in defining the genetic signature specific to OP. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for the selected pivotal genes were conducted. The cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm was leveraged to examine the infiltration patterns of immune cells, with Spearman’s rank correlation analysis utilized to assess the relationship between the expression levels of the genes and the presence of immune cells. Coremine Medical Database was used to screen out potential TCM herbs for the treatment of OP. Comparative Toxicogenomics Database (CTD) was employed for forecasting the TCM active ingredients targeting the key genes. AutoDock Vina 1.2.2 and GROMACS 2020 softwares were employed to conclude analysis results, facilitating the exploration of binding affinity and conformational dynamics between the TCM active ingredients and their biological targets. ResultsTen genes were identified by intersecting the results from the GEO and GeneCards databases. Through the application of LASSO regression and SVM-RFE algorithm, four pivotal genes were selected: coat protein (CP), kallikrein 3 (KLK3), polymerase γ (POLG), and transient receptor potential vanilloid 4 (TRPV4). GO and KEGG pathway enrichment analyses revealed that these trait genes were predominantly engaged in the regulation of defense response activation, maintenance of cellular metal ion balance, and the production of chemokine ligand 5. These genes were notably associated with signaling pathways such as ferroptosis, porphyrin metabolism, and base excision repair. Immune infiltration analysis showed that key genes were highly correlated with immune cells. Macrophage M0, M1, M2, and resting dendritic cell were significantly different between groups, and there were significant differences between different groups (P < 0.05). The interaction counts of resveratrol, curcumin, and quercetin with KLK3 were 7, 3, and 2, respectively. It shows that the interactions of resveratrol, curcumin, and quercetin with KLK3 were substantial. Molecular docking and molecular dynamics simulations further confirmed the robust binding affinity of these bioactive compounds to the target genes. ConclusionPivotal genes including CP, KLK3, POLG, and TRPV4, exhibited commendable significant prognostic value, and played a crucial role in the diagnostic assessment of OP. Resveratrol, curcumin, and quercetin, natural compounds found in TCM, showed promise in their potential to effectively modulate the bone-forming gene KLK3. This study provides a scientific basis for the interpretation of the pathogenesis of OP and the development of clinical drugs.

Neferine alleviates ovariectomy-induced osteoporosis by enhancing osteogenic differentiation of bone marrow mesenchymal stem cells via regulation of the p38MAPK pathway

ABSTRACT Objective Osteoporosis, a skeletal ailment marked by bone metabolism imbalance and disruption of bone microarchitecture, Neferine, a bisbenzylisoquinoline alkaloid with diverse pharmacological activities, has received limited attention in the context of osteoporosis treatment. Methods We employed a bilateral ovariectomy (OVX) rat model to induce osteoporosis and subsequently administered Neferine treatment for four weeks following successful model establishment. Throughout the modeling and treatment phases, we closely monitored rat body weights. We assessed alterations in bone tissue microstructure through micro-CT, HE staining, and safranin O-fast green staining. Levels of bone formation and resorption markers in serum were evaluated using ELISA assay. Western blot analysis was employed to determine the expression levels of p38MAPK, p-p38MAPK, and bone formation-related genes in bone tissue. We isolated and cultured OVX rat BMSCs (OVX-BMSCs) and induced osteogenic differentiation while simultaneously introducing Neferine and the p38MAPK inhibitor SB203580 for intervention. Results Neferine treatment effectively curbed the rapid weight gain in OVX rats, ameliorated bone loss, and decreased serum levels of TRAP, CTX-I, PINP, and BALP. Most notably, Neferine promoted the expression of bone formation-related factors in bone tissue of OVX rats, while concurrently activating the p38MAPK signaling pathway. In in vitro experiments, Neferine facilitated the expression of bone formation-related factors in OVX-BMSCs, increased the osteogenic differentiation potential of OVX-BMSCs, and activated the p38MAPK signaling pathway. Nevertheless, SB203580 partially reversed Neferine’s promotive effect. Conclusion Neferine can boost the osteoblastic differentiation of BMSCs and alleviate OVX-induced osteoporosis in rats by activating the p38MAPK signaling pathway.

Different impact of short-term and long-term hindlimb disuse on bone homeostasis

Disuse osteoporosis is one of the major problems of bone health which commonly occurs in astronauts during long-term spaceflight and bedridden patients. However, the mechanisms underlying such mechanical unloading induced bone loss have not been fully understood. In this study, we employed hindlimb-unloading mice models with different length of tail suspension to investigate if the bone loss was regulated by distinct factors under different duration of disuse. Our micro-CT results showed more significant decrease of bone mass in 6W (6-week) tail-suspension mice compared to the 1W (1-week) tail-suspension ones, as indicated by greater reduction of BV/TV, Tb.N, B.Ar/T.Ar and Ct.Th. RNA-sequencing results showed significant effects of hindlimb disuse on cell locomotion and immune system process which could cause bone loss.Real-time quantitative PCR results indicated a greater number of bone formation related genes that were downregulated in short-term tail-suspension mice compared to the long-term ones. It is, thus, suggested while sustained hindlimb unloading continuously contributes to bone loss, molecular regulation of bone homeostasis tends to reach a balance during this process.

Biological Properties of Bioceramic Sealers on Osteoblastic Cells: A Comparative Study.

This study aimed to assess the biological properties of two ready-to-use bioceramic sealers (EndoSequence BC Sealer - EBCS; Bio-C Sealer - BCS) on osteoblastic lineage cells. MC3T3 osteoblast-like cells were exposed to extracts of bioceramic materials. Cytotoxicity was evaluated using the MTT method, genotoxicity was assessed by the micronucleus test and the expression of BMP1, BMP2 and ALP was measured by RT-qPCR, after 1, 3 and 7 days. Wound healing was monitored at 1, 2 and 3-day intervals using the scratch test. Statistical analysis involved a two-factor ANOVA, followed by the Tukey Test for multiple comparisons (α=0.05). Cytotoxicity assessment revealed no significant differences between the materials and the control group at any of the time points, indicating that neither material exhibited cytotoxic effects. However, both cements induced greater micronuclei formation compared to the control on days 1 and 7 (p<0.05) when evaluating genotoxicity. No significant differences between the groups were observed in wound healing at any of the time intervals. Both biomaterials upregulated the expression of BMP1, BMP2 and ALP. The two bioceramic sealers exhibited comparable biological properties, including cytocompatibility, promotion of wound healing, and upregulation of bone formation-related genes in osteoblast-like cells. While these results suggest the potential for safe clinical application, the observed genotoxicity warrants caution. Therefore, while the null hypothesis-that the two bioceramic sealers exhibit analogous properties-was accepted in terms of cytocompatibility and gene expression, further investigation is essential to fully ensure their safety and efficacy in bone-related procedures.

Short-term dietary magnesium deficiency downregulates the expression of bone formation-related genes in rats.

Dietary magnesium deficiency increases osteoclastic bone resorption and decreases osteoblastic bone formation. Increased bone resorption due to dietary magnesium deficiency can be explained by increased expression of the receptor activator of nuclear factor kB ligand. However, the detailed mechanisms underlying decreased bone formation remain unclear. Thus, in the present study, to determine the mechanism underlying decreased bone formation induced by dietary magnesium deficiency, we investigated the effects of short-term dietary magnesium deficiency on the mRNA expression of genes related to bone formation in rats. Male Wistar rats were fed a control or magnesium-deficient diet for eight days. The mRNA expression level of Runx2, Sp7, Bglap, Alpl, Col1a1, Igf1, and Bmp2 in the femur was significantly lower in magnesium-deficient rats than in control rats. These results suggest that short-term dietary magnesium deficiency decreases the gene expression of insulin-like growth factor-1 and bone morphogenetic protein 2, which, in turn, decreases osteoblastic bone formation through the downregulation of osteoblastogenesis-related gene expression.

Combined computational analysis and cytology show limited depth osteogenic effect on bone defects in negative pressure wound therapy.

Background: The treatment of bone defects remains a clinical challenge. The effect of negative pressure wound therapy (NPWT) on osteogenesis in bone defects has been recognized; however, bone marrow fluid dynamics under negative pressure (NP) remain unknown. In this study, we aimed to examine the marrow fluid mechanics within trabeculae by computational fluid dynamics (CFD), and to verify osteogenic gene expression, osteogenic differentiation to investigate the osteogenic depth under NP. Methods: The human femoral head is scanned using micro-CT to segment the volume of interest (VOI) trabeculae. The VOI trabeculae CFD model simulating the bone marrow cavity is developed by combining the Hypermesh and ANSYS software. The effect of trabecular anisotropy is investigated, and bone regeneration effects are simulated under NP scales of -80, -120, -160, and -200mmHg. The working distance (WD) is proposed to describe the suction depth of the NP. Finally, gene sequence analysis, cytological experiments including bone mesenchymal stem cells (BMSCs) proliferation and osteogenic differentiation are conducted after the BMSCs are cultured under the same NP scale. Results: The pressure, shear stress on trabeculae, and marrow fluid velocity decrease exponentially with an increase in WD. The hydromechanics of fluid at any WD inside the marrow cavity can be theoretically quantified. The NP scale significantly affects the fluid properties, especially those fluid close to the NP source; however, the effect of the NP scale become marginal as WD deepens. Anisotropy of trabecular structure coupled with the anisotropic hydrodynamic behavior of bone marrow; An NP of -120mmHg demonstrates the majority of bone formation-related genes, as well as the most effective proliferation and osteogenic differentiation of BMSCs compared to the other NP scales. Conclusion: An NP of -120mmHg may have the optimal activated ability to promote osteogenesis, but the effective WD may be limited to a certain depth. These findings help improve the understanding of fluid mechanisms behind NPWT in treating bone defects.

A novel CKIP-1 SiRNA slow-release coating on porous titanium implants for enhanced osseointegration.

Osseointegration between implants and bone tissue lays the foundation for the long-term stability of implants. The incorporation of a porous structure and local slow release of siRNA to silence casein kinase-2 interacting protein-1 (CKIP-1), a downregulator of bone formation, is expected to promote osseointegration. Here, porous implants with a porous outer layer and dense inner core were prepared by metal coinjection molding (MIM). Mg-doped calcium phosphate nanoparticles (CaPNPs)-grafted arginine-glycine-aspartate cell adhesion sequence (RGD) and transcribed activator (TAT) (MCPRT)/CKIP-1 siRNA complex and polylysine (PLL) were coated onto the surface of the porous implants by layer-by-layer (LBL) self-deposition. The in vitro results showed that the MCPRT-siRNA coating promoted MG63 cell adhesion and proliferation, enhanced the protein expressions (ALP and OC) and bone formation-related gene expression (OPN, OC and COL-1α) in vitro. The in vivo results demonstrated that the porous structure enhanced bone ingrowth and that the local slow release of MCPRT-siRNA accelerated new bone formation at the early stage. The porous structure coupled with local CKIP-1 siRNA delivery constitutes a promising approach to achieve faster and stronger osseointegration for dental implants.

Ginsenoside Rc Promotes Bone Formation in Ovariectomy-Induced Osteoporosis In Vivo and Osteogenic Differentiation In Vitro.

Ginsenoside Rc is one of the active components used in traditional Chinese medicine. We aim to explore how ginsenoside Rc can be used in the treatment of osteoporosis. Micro-CT demonstrated that the treatment of ovariectomized (OVX) mice with ginsenoside Rc significantly inhibited the decrease in bone mineral density, bone volumetric fraction, and trabecular number, and the increase in trabecular separation. Histological staining, qRT-PCR, and Western blot demonstrated that ginsenoside Rc enhances the microstructure of trabecular bone, and promotes the expression of bone formation-related genes. Alkaline phosphatase (ALP) staining, Alizarin Red staining, qRT-PCR, and Western blotting demonstrated that ginsenoside Rc dose-dependently promoted the osteogenic differentiation of MC3T3-E1 cells. A ginsenoside Rc-induced increase in the expression of β-catenin, p-GSK-3β, collagen-1, ALP, and RUNX-2 family transcription factor-2 was significantly attenuated upon 10 μM XAV-939 treatment, while the decrease in the expression of GSK-3β and p-β-catenin was significantly enhanced. Ginsenoside Rc promotes bone formation in ovariectomy-induced osteoporosis in vivo and promotes osteogenic differentiation in vitro via the Wnt/β-catenin signaling pathway.

Developing a novel calcium magnesium silicate/graphene oxide incorporated silk fibroin porous scaffold with enhanced osteogenesis, angiogenesis and inhibited osteoclastogenesis

Recently, biofunctional ions (Mg2+, Si4+, etc) and graphene derivatives are proved to be promising in stimulating bone formation. In this study, a novel inorganic/organic composite porous scaffold based on silk fibroin (SF), graphene oxide (GO), and calcium magnesium silicate (CMS) was developed for bone repair. The porous scaffolds obtained by lyophilization showed a little difference in pore structure while GO and CMS displayed a good interaction with SF matrix. The addition of CMS with good mineralization potential and sustainedly release ability of biofunctional ions (Ca2+, Mg2+ and Si4+) increased the strength of SF scaffolds a little and facilitated the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) by upregulating bone formation-related genes (ALP, COL1, OC and Runx2). The further incorporation of GO in SF scaffolds enhanced the compressive strength and water retention, and also remarkably promoted the osteogenic differentiation of BMSCs. Besides, the angiogenesis of human umbilical vein endothelial cells was significantly promoted by CMS/GO/SF scaffold extract through the upregulation of angiogenesis genes (eNOs and bFGF). Moreover, the osteoclastic formation ability of RAW264.7 cells was suppressed by the released ions from CMS/GO/SF scaffold through the down-regulation of CAK, MMP9 and TRAP. The promoted osteogenesis, angiogenesis and inhibited osteoclastogenesis functions of CMS/GO/SF composite scaffold may enable it as a novel therapy for bone repair and regeneration.

Hydroxyapatite composited PEEK with 3D porous surface enhances osteoblast differentiation through mediating NO by macrophage.

The adverse immune response mediated by macrophages is one of the main factors that are prone to lead poor osseointegration of polyetheretherketone (PEEK) implants in clinic. Hence, endowing PEEK with immunomodulatory ability to avoid the adverse immune response becomes a promising strategy to promote bone repair. In this work, sulfonation and hydrothermal treatment were used to fabricate a 3D porous surface on PEEK and hydroxyapatite (HA) composited PEEK. The HA composited PEEK with 3D porous surface inhibited macrophages polarizing to M1 phenotype and downregulated inducible nitric oxide synthase protein expression, which led to a nitric oxide concentration reduction in culture medium of mouse bone marrow mesenchymal stem cells (mBMSCs) under co-culture condition. The decrease of nitric oxide concentration could help to increase bone formation-related OSX and ALP genes expressions and decrease bone resorption-related MMP-9 and MMP-13 genes expressions via cAMP–PKA–RUNX2 pathway in mBMSCs. In summary, the HA composited PEEK with 3D porous surface has the potential to promote osteogenesis of PEEK through immunomodulation, which provides a promising strategy to improve the bone repair ability of PEEK.

MiR-26b Regulates Osteoactivin (OA)-Induced Bone Marrow Mesenchymal Cells (BMSCs) Osteogenic Differentiation by Targeting Fms-Like Tyrosine Kinase 3 (FLT3)/AXL

Osteoactivin (OA) plays a key role in osteogenic differentiation. miR-26b is elevated in the bone formation process of BMSCs, but whether it is involved in this process is unclear. Bone formation is regulated by FLT3/AXL signaling pathway, which may be a potential target of miR-26b. qRT-PCR detected miR-26b mRNA levels and bone formation-related genes or FLT3/AXL signaling pathway-related genes. Bone formation was analyzed by staining and FLT3/AXL signaling was evaluated along with analysis of miR-26b’s relation with LT3/AXL. miR-26b was significantly elevated in OA-induced bone formation of BMSCs, which can be promoted by miR-26b mimics. When miR-26b was overexpressed, FLT3/AXL signaling pathway was activated. miR-26b can ameliorate Dex-induced osteo-inhibition. miR-26b promotes bone formation of BMSCs by directly targeting FLT3/AXL signaling pathway, suggesting that miR-26b might be a target for inducing osteogenic differentiation.

The White-Spotted Bamboo Shark Genome Reveals Chromosome Rearrangements and Fast-Evolving Immune Genes of Cartilaginous Fish.

SummaryChondrichthyan (cartilaginous fish) occupies a key phylogenetic position and is important for investigating evolutionary processes of vertebrates. However, limited whole genomes impede our in-depth knowledge of important issues such as chromosome evolution and immunity. Here, we report the chromosome-level genome of white-spotted bamboo shark. Combing it with other shark genomes, we reconstructed 16 ancestral chromosomes of bamboo shark and illustrate a dynamic chromosome rearrangement process. We found that genes on 13 fast-evolving chromosomes can be enriched in immune-related pathways. And two chromosomes contain important genes that can be used to develop single-chain antibodies, which were shown to have high affinity to human disease markers by using enzyme-linked immunosorbent assay. We also found three bone formation-related genes were lost due to chromosome rearrangements. Our study highlights the importance of chromosome rearrangements, providing resources for understanding of cartilaginous fish diversification and potential application of single-chain antibodies.

Sclerostin Regulation, Microarchitecture, and Advanced Glycation End-Products in the Bone of Elderly Women With Type 2 Diabetes.

Increased circulating sclerostin and accumulation of advanced glycation end-products (AGEs) are two potential mechanisms underlying low bone turnover and increased fracture risk in type 2 diabetes (T2D). Whether the expression of the sclerostin-encoding SOST gene is altered in T2D, and whether it is associated with AGEs accumulation or regulation of other bone formation-related genes is unknown. We hypothesized that AGEs accumulate and SOST gene expression is upregulated in bones from subjects with T2D, leading to downregulation of bone forming genes (RUNX2 and osteocalcin) and impaired bone microarchitecture and strength. We obtained bone tissue from femoral heads of 19 T2D postmenopausal women (mean glycated hemoglobin [HbA1c] 6.5%) and 73 age- and BMI-comparable nondiabetic women undergoing hip replacement surgery. Despite similar bone mineral density (BMD) and biomechanical properties, we found a significantly higher SOST (p = .006) and a parallel lower RUNX2 (p = .025) expression in T2D compared with non-diabetic subjects. Osteocalcin gene expression did not differ between T2D and non-diabetic subjects, as well as circulating osteocalcin and sclerostin levels. We found a 1.5-fold increase in total bone AGEs content in T2D compared with non-diabetic women (364.8 ± 78.2 versus 209.9 ± 34.4 μg quinine/g collagen, respectively; p < .001). AGEs bone content correlated with worse bone microarchitecture, including lower volumetric BMD (r = -0.633; p = .02), BV/TV (r = -0.59; p = .033) and increased trabecular separation/spacing (r = 0.624; p = .023). In conclusion, our data show that even in patients with good glycemic control, T2D affects the expression of genes controlling bone formation (SOST and RUNX2). We also found that accumulation of AGEs is associated with impaired bone microarchitecture. We provide novel insights that may help understand the mechanisms underlying bone fragility in T2D. © 2020 American Society for Bone and Mineral Research (ASBMR).

Mechanisms of electroacupuncture underlying treatment of osteoporosis based on HDAC2-mediated osteoblast differentiation pathway

To observe the effect of electroacupuncture (EA) on expression of histone deacetylase 2 (HDAC2), histone H3, bone formation related genes and proteins in osteoporosis rats, so as to reveal its mechanisms underlying improvement of osteoporosis. Female SD rats were randomly divided into 4 groups: sham operation, model, EA and medication (n＝ 10 rats in each group). The osteoporosis model was established by castration. EA (2 Hz, 1 mA) was applied to bilateral "Shenshu" (BL23) and "Pishu" (BL20) for 10 min, once every other day for 8 weeks. Rats of the medication group received subcutaneous injection of 17 β-estradiol (100 µg/kg, 20 µg/mL). The bone quality and quantity including the cortical bone mineral density (CBMD), trabecular bone mineral density (TBMD), ratio of bone volume /total volume (BV /TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb. Sp), trabecular bone pattern factor (Tb.Pf), and structure model index (SMI) of the right thigh-bone were detected by using a micro-computed tomography. Serum alkaline phosphatase (ALP) and estrogen 2 (E2) contents were assayed by using colorimetry and ELISA, expression levels of HDAC2, histone H3 and Runx2 in the thigh-bone were detected using Western blot, and that of Runx2 mRNA was detected using quantitative real-time PCR, separately. The co-expression of Ac-histone H3/Runx2 and Runx2/ALP was observed by using immunofluorescence histochemical staining. After modeling, the levels of TBMD, BV/TV, Tb.Th, and Tb.N, serum E2 and ALP, and expression of Runx2 protein and mRNA, Ac-histone and ALP proteins were significantly lower (P<0.01), and those of Tb.Sp, Tb.Pf and SMI, HDAC2 and histone H3 proteins were significantly higher (P<0.01) in the model group than those in the sham operation group. After the interventions, the decrease of TBMD, BV/TV, Tb.N，Runx2 protein and mRNA，ALP in both EA and medication groups, serum E2 in the medication group, and Ac-histone H3 in the EA group, and the increase of Tb.Sp in the medication group, Tb.Pf, SMI, and HDAC2 in both EA and medication groups, and histone H3 in the EA group were reversed (P<0.01, P<0.05). No significant changes were found in the levels of CBMD after modeling relevant to the sham operation group, and after EA and medication interventions (P>0.05). The effects of EA were significantly superior to 17 β-estradiol in down-regulating the expression of HDAC2 and histone H3 proteins and in up-regulating expression of Ac-histone H3 protein (P<0.01，P<0.05)．. EA treatment can increase bone density, increase bone mass and trabecular bone, and promote trabecular bone rod-like changes in plate shape in osteoporosis rats, which is related to its effect in up-regulating the expression of Ac-histone H3 protein, and down-regulating the expression of bone formation-related proteins.

Regulatory Role of RNA N6-Methyladenosine Modification in Bone Biology and Osteoporosis.

Osteoporosis is a metabolic skeletal disorder in which bone mass is depleted and bone structure is destroyed to the degree that bone becomes fragile and prone to fractures. Emerging evidence suggests that N6-methyladenosine (m6A) modification, a novel epitranscriptomic marker, has a significant role in bone development and metabolism. M6A modification not only participates in bone development, but also plays important roles as writers and erasers in the osteoporosis. M6A methyltransferase METTL3 and demethyltransferase FTO involves in the delicate process between adipogenesis differentiation and osteogenic differentiation, which is important for the pathological development of osteoporosis. Conditional knockdown of the METTL3 in bone marrow stem cells (BMSCs) could suppress PI3K-Akt signaling, limit the expression of bone formation-related genes (such as Runx2 and Osterix), restrain the expression of vascular endothelial growth factor (VEGF) and down-regulate the decreased translation efficiency of parathyroid hormone receptor-1 mRNA. Meanwhile, knockdown of the METTL3 significantly promoted the adipogenesis process and janus kinase 1 (JAK1) protein expression via an m6A-dependent way. Specifically, there was a negative correlation between METTL3 expression and porcine BMSCs adipogenesis. The evidence above suggested that the relationship between METTL3 expression and adipogenesis was inverse, and osteogenesis was positive, respectively. Similarly, FTO regulated for BMSCs fate determination during osteoporosis through the GDF11-FTO-PPARγ axis, prompting the shift of MSC lineage commitment to adipocyte and inhibiting bone formation during osteoporosis. In this systematic review, we summarize the most up-to-date evidence of m6A RNA modification in osteoporosis and highlight the potential role of m6A in prevention, treatment, and management of osteoporosis.

Hypergravity and microgravity exhibited reversal effects on the bone and muscle mass in mice

Spaceflight is known to induce severe systemic bone loss and muscle atrophy of astronauts due to the circumstances of microgravity. We examined the influence of artificially produced 2G hypergravity on mice for bone and muscle mass with newly developed centrifuge device. We also analyzed the effects of microgravity (mostly 0G) and artificial produced 1G in ISS (international space station) on mouse bone mass. Experiment on the ground, the bone mass of humerus, femur and tibia was measured using micro-computed tomography (μCT), and the all bone mass was significantly increased in 2G compared with 1G control. In tibial bone, the mRNA expression of bone formation related genes such as Osx and Bmp2 was elevated. The volume of triceps surae muscle was also increased in 2G compared with 1G control, and the mRNA expression of myogenic factors such as Myod and Myh1 was elevated by 2G. On the other hand, microgravity in ISS significantly induced the loss of bone mass on humerus and tibia, compared with artificial 1G induced by centrifugation. Here, we firstly report that bone and muscle mass are regulated by the gravity with loaded force in both of positive and negative on the ground and in the space.

Antarctic krill oil promotes longitudinal bone growth in adolescent male mice

The effects of dietary supplementation with Antarctic krill oil (AKO) on longitudinal bone growth in adolescent male Institute for Cancer Research (ICR) mice for 21 days were investigated. Histological results showed that AKO promoted longitudinal bone growth, increased skeletal mineral content and strength, enhanced entochondrostosis supported by higher longitudinal bone growth rate and growth plate height. Then, the potential mechanisms responsible for these observations were measured, results showed that AKO increased the secretion of the GH-IGF-1 pathway and entochondrostosis-related factors in serum increased the expression of IGF-1 in the liver and bone, which promoted proliferation and differentiation of chondrocytes, vascular invasion and bone formation related gene expression. AKO promoted longitudinal bone growth, using the GH-IGF-1 pathway, suggesting that AKO might be a potential functional food to improve growth potential of adolescents.

Mettl3 Regulates Osteogenic Differentiation and Alternative Splicing of Vegfa in Bone Marrow Mesenchymal Stem Cells

Bone mesenchymal stem cells (BMSCs) can be a useful cell resource for developing biological treatment strategies for bone repair and regeneration, and their therapeutic applications hinge on an understanding of their physiological characteristics. N6-methyl-adenosine (m6A) is the most prevalent internal chemical modification of mRNAs and has recently been reported to play important roles in cell lineage differentiation and development. However, little is known about the role of m6A modification in the cell differentiation of BMSCs. To address this issue, we investigated the expression of N6-adenosine methyltransferases (Mettl3 and Mettl14) and demethylases (Fto and Alkbh5) and found that Mettl3 was upregulated in BMSCs undergoing osteogenic induction. Furthermore, we knocked down Mettl3 and demonstrated that Mettl3 knockdown decreased the expression of bone formation-related genes, such as Runx2 and Osterix. The alkaline phosphatase (ALP) activity and the formation of mineralized nodules also decreased after Mettl3 knockdown. RNA sequencing analysis revealed that a vast number of genes affected by Mettl3 knockdown were associated with osteogenic differentiation and bone mineralization. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that the phosphatidylinositol 3-kinase/AKT (PI3K-Akt) signaling pathway appeared to be one of the most enriched pathways, and Western blotting results showed that Akt phosphorylation was significantly reduced after Mettl3 knockdown. Mettl3 has been reported to play an important role in regulating alternative splicing of mRNA in previous research. In this study, we found that Mettl3 knockdown not only reduced the expression of Vegfa but also decreased the level of its splice variants, vegfa-164 and vegfa-188, in Mettl3-deficient BMSCs. These findings might contribute to novel progress in understanding the role of epitranscriptomic regulation in the osteogenic differentiation of BMSCs and provide a promising perspective for new therapeutic strategies for bone regeneration.

Endogenous Parathyroid Hormone–Related Protein Compensates for the Absence of Parathyroid Hormone in Promoting Bone Accrual In Vivo in a Model of Bone Marrow Ablation

To assess the effect of hypoparathyroidism on osteogenesis and bone turnover in vivo, bone marrow ablation (BMXs) were performed in tibias of 8-week-old wild-type and parathyroid hormone-null (PTH(-/-)) mice and newly formed bone tissue was analyzed from 5 days to 3 weeks after BMX. At 1 week after BMX, trabecular bone volume, osteoblast numbers, alkaline phosphatase-positive areas, type I collagen-positive areas, PTH receptor-positive areas, calcium sensing receptor-positive areas, and expression of bone formation-related genes were all decreased significantly in the diaphyseal regions of bones of PTH(-/-) mice compared to wild-type mice. In contrast, by 2 weeks after BMX, all parameters related to osteoblastic bone accrual were increased significantly in PTH(-/-) mice. At 5 days after BMX, active tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts had appeared in wild-type mice but were undetectable in PTH(-/-) mice, Both the ratio of mRNA levels of receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) and TRAP-positive osteoclast surface were still reduced in PTH(-/-) mice at 1 week but were increased by 2 weeks after BMX. The expression levels of parathyroid hormone-related protein (PTHrP) at both mRNA and protein levels were upregulated significantly at 1 week and more dramatically at 2 weeks after BMX in PTH(-/-) mice. To determine whether the increased newly formed bones in PTH(-/-) mice at 2 weeks after BMX resulted from the compensatory action of PTHrP, PTH(-/-) PTHrP(+/-) mice were generated and newly formed bone tissue was compared in these mice with PTH(-/-) and wild-type mice at 2 weeks after BMX. All parameters related to osteoblastic bone formation and osteoclastic bone resorption were reduced significantly in PTH(-/-) PTHrP(+/-) mice compared to PTH(-/-) mice. These results demonstrate that PTH deficiency itself impairs osteogenesis, osteoclastogenesis, and osteoclastic bone resorption, whereas subsequent upregulation of PTHrP in osteogenic cells compensates by increasing bone accrual.