Osteocalcin mRNA Expression Research Articles

Effects of Different No-Ozone Cold Plasma Treatment Methods on Mouse Osteoblast Proliferation and Differentiation.

Background and Objectives: Enhanced osteoblast differentiation may be leveraged to prevent and treat bone-related diseases such as osteoporosis. No-ozone cold plasma (NCP) treatment is a promising and safe strategy to enhance osteoblast differentiation. Therefore, this study aimed to determine the effectiveness of direct and indirect NCP treatment methods on osteoblast differentiation. Mouse osteoblastic cells (MC3T3-E1) were treated with NCP using different methods, i.e., no NCP treatment (NT group; control), direct NCP treatment (DT group), direct NCP treatment followed by media replacement (MC group), and indirect treatment with NCP-treated media only (PAM group). Materials and Methods: The MC3T3-E1 cells were subsequently assessed for cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, and ALP and osteocalcin mRNA expression using real-time polymerase chain reaction. Results: Cell proliferation significantly increased in the NCP-treated groups (DT and PAM; MC and PAM) compared to the NT group after 24 h (p < 0.038) and 48 h (p < 0.000). ALP activity was increased in the DT and PAM groups at 1 week (p < 0.115) and in the DT, MC, and PAM groups at 2 weeks (p < 0.000) compared to the NT group. Calcium deposition was higher in the NCP-treated groups than in NT group at 2 and 3 weeks (p < 0.000). ALP mRNA expression peaked in the MC group at 2 weeks compared to the NP group (p < 0.014). Osteocalcin mRNA expression increased in the MC group at 2 weeks (p < 0.000) and was the highest in the PAM group at 3 weeks (p < 0.000). Thus, the effects of direct (DT and MC) and indirect (PAM) treatment varied, with MC direct treatment showing the most significant impact on osteoblast activity. Conclusions: The MC group exhibited enhanced osteoblast differentiation, indicating that direct NCP treatment followed by media replacement is the most effective method for promoting bone formation.

Heraclenin promotes the osteogenic differentiation of bone marrow stromal cells by activating the RhoA/ROCK pathway.

Osteoporosis is a devastating skeletal disease, the pathogenesis of which is related to abnormal bone metabolism, featured by the imbalance between osteoblastic bone formation and osteoclastic bone resorption. Stem cell-based therapies have been demonstrated to improve osteoporosis treatment. Previously, the linear furanocoumarin heraclenin was reported to enhance osteoblast differentiation and mineralization in mouse mesenchymal stem cells (MSCs), suggesting its potential for osteogenic differentiation and bone regeneration. Our study was designed to confirm the promotive role of heraclenin on osteogenic differentiation of human bone MSCs (BMSCs) and explore the underlying mechanisms. Human BMSCs were treated for 24, 48, and 72h with heraclenin (5, 10, 20, 40, and 80 μM), and cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. To further evaluate the cytotoxicity of heraclenin, cell suspension obtained from BMSCs treated with heraclenin (5, 10, and 20 μM) for 72h was subjected to a MUSE™ cell analyzer for cell viability and count assay. BMSCs were incubated in osteogenic induction medium for 7 days. Then, osteogenic differentiation and mineralization of BMSCs were assessed through alkaline phosphatase (ALP) and Alizarin Red S staining. The expression of osteogenesis markers including ALP, osteocalcin (OCN), osterix (OSX), and runt-related transcription factor 2 (RUNX2) was detected via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The effects of heraclenin on the RhoA/ROCK pathway were estimated through western blotting. Y-27632, the ROCK inhibitor, was used to confirm the role of the RhoA/ROCK pathway in heraclenin-mediated osteogenic differentiation of BMSCs. Heraclenin (5-80 μM) was non-toxic on human BMSCs. Heraclenin treatment (5-20 μM) dose-dependently enhanced ALP activity and calcium deposition. Furthermore, heraclenin promoted ALP, OCN, OSX, and RUNX2 mRNA and protein expression. Mechanically, heraclenin treatment increased RhoA and ROCK1 mRNA expression, stimulated the translocation of ROCK from the cytosolic to the membrane fraction, and elevated the protein levels of phosphorylated cofilin (p-cofilin) and active RhoA. Additionally, treatment with Y-27632 overturned the promotion of heraclenin on ALP activity, calcium deposition, the expression of osteogenesis markers, and the RhoA/ROCK signaling pathway. Heraclenin facilitates the osteogenic differentiation of human BMSCs through the activation of the RhoA/ROCK pathway.

Effects of low dietary calcium and lipopolysaccharide challenges on production performance, eggshell quality, and bone metabolism of laying hens.

Dietary calcium supply is essential for bone development and egg production in laying hens. This study investigated the effects of low dietary calcium and lipopolysaccharide (LPS) induced immune challenge in aged laying hens. A total of thirty-two Hy-Line Brown laying hens at 80 weeks old with an average laying rate of 62% were randomly divided into two groups and fed a normal calcium diet (3.57% Ca, NCA) or low calcium diet (2.08% Ca, LCA). At 88 weeks, the experiment was designed using a 2 × 2 factorial arrangement, and hens were intraperitoneally injected with saline (SAL) or LPS (0.5mg/kg, 0.5mg/kg, or 1.5mg/kg body weight) once every 48h intervals over 5 days. Production performance, egg quality, and bone physiology were evaluated. Results showed that LPS challenge decreased the hen-day egg production, egg mass, and eggshell traits (p < 0.05), but increased (p < 0.05) the calcium content of the tibia compared to SAL-injected hens. LCA diet decreased (p < 0.05) the hen-day egg production, and eggshell traits such as weight, percentage, strength, and thickness compared to the NCA diet. LCA diet increased the serum alkaline phosphatase (ALP) activity (p < 0.01) and tibial expression of ALP (p < 0.05) compared to NCA diet. LPS injection suppressed both the serum ALP activity (p < 0.05) and tibial expression of ALP (p < 0.001) compared to SAL injection. Furthermore, LPS injection increased (p < 0.05) the expression of both pro and anti-inflammatory cytokines in the spleen and tibia. The expression of cathepsin K ( Cts K ) and matrix metalloproteinase 9 ( MMP-9 ) were downregulated by LPS injection (p < 0.001). Broken and shell-less egg production and calcium content of eggshell, as well as tibial mRNA expression of osteocalcin ( Ocn ), tumor necrosis factor-alpha ( TNF-α ) and tartrate-resistant acid phosphatase ( TRAP ) were affected by the interaction (p < 0.05) of diet and injection. Therefore, this study demonstrated that to certain extents, low dietary calcium and LPS challenge dysregulated bone homeostasis and metabolism, with detrimental effects on the performance and eggshell quality of aged laying hens.

GREM2 inactivation increases trabecular bone mass in mice

Osteoporosis is a common skeletal disease affecting millions of individuals world-wide, with an increased risk of fracture, and a decreased quality of life. Despite its well-known consequences, the etiology of osteoporosis and optimal treatment methods are not fully understood. Human genetic studies have identified genetic variants within the FMN2/GREM2 locus to be associated with trabecular volumetric bone mineral density (vBMD) and vertebral and forearm fractures, but not with cortical bone parameters. GREM2 is a bone morphogenetic protein (BMP) antagonist. In this study, we employed Grem2-deficient mice to investigate whether GREM2 serves as the plausible causal gene for the fracture signal at the FMN2/GREM2 locus. We observed that Grem2 is moderately expressed in bone tissue and particularly in osteoblasts. Complete Grem2 gene deletion impacted mouse survival and body growth. Partial Grem2 inactivation in Grem2+/− female mice led to increased trabecular BMD of femur and increased trabecular bone mass in tibia due to increased trabecular thickness, with an unchanged cortical thickness, as compared with wildtype littermates. Furthermore, Grem2 inactivation stimulated osteoblast differentiation, as evidenced by higher alkaline phosphatase (Alp), osteocalcin (Bglap), and osterix (Sp7) mRNA expression after BMP-2 stimulation in calvarial osteoblasts and osteoblasts from the long bones of Grem2−/− mice compared to wildtype littermates. These findings suggest that GREM2 is a possible target for novel osteoporotic treatments, to increase trabecular bone mass and prevent osteoporotic fractures.

A novel miR-466l-3p/FGF23 axis promotes osteogenic differentiation of human bone marrow mesenchymal stem cells

BackgroundMicroRNAs (miRNAs) regulate osteogenic differentiation processes and influence the development of osteoporosis (OP). This study aimed to investigate the potential role of miR-466 l-3p in OP. MethodsThe expression levels of miR-466 l-3p and fibroblast growth factor 23 (FGF23) were quantified in the trabeculae of the femoral neck of 40 individuals with or without OP using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The impact of miR-466 l-3p or FGF23 expression on cell proliferation and the expression levels of runt-related transcription factor 2 (RUNX2), type I collagen (Col1), osteocalcin (OCN), osterix (OSX) and dentin matrix protein 1 (DMP1) was quantified in human bone marrow mesenchymal stem cells (hBMSCs) overexpressing miR-466 l-3p. Furthermore, alkaline phosphatase (ALP) staining and alizarin red staining were performed to measure ALP activity and the levels of calcium deposition, respectively. In addition, bioinformatics analysis, luciferase reporter assays, and RNA pull-down assays were conducted to explore the molecular mechanisms underlying the effects of miR-466 l-3p and FGF23 in osteogenic differentiation of hBMSCs. ResultsThe expression levels of miR-466 l-3p were significantly lower in femoral neck trabeculae of patients with OP than in the control cohort, whereas FGF23 levels exhibited the opposite trend. Furthermore, miR-466 l-3p levels were upregulated and FGF23 levels were downregulated in hBMSCs during osteogenic differentiation. Moreover, the high miR-466 l-3p expression enhanced the mRNA expression of RUNX2, Col1, OCN, OSX and DMP1, as well as cell proliferation, ALP activity, and calcium deposition in hBMSCs. FGF23 was found to be a direct target of miR-466 l-3p. FGF23 overexpression downregulated the expression of osteoblast markers and inhibited the osteogenic differentiation induced by miR-466 l-3p overexpression. qRT-PCR and Western blot assays showed that miR-466 l-3p overexpression decreased the expression levels of mRNAs and proteins associated with the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, whereas FGF23 upregulation exhibited the opposite trend. ConclusionIn conclusion, these findings suggest that miR-466 l-3p enhances the osteogenic differentiation of hBMSCs by suppressing FGF23 expression, ultimately preventing OP.

Mogroside V Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells from Diabetic Mice by Altering MicroRNA Profiles.

Mogroside V (MV), a triterpene glycoside, exhibits diverse biological functions. However, its ability to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) under diabetic conditions is yet to be elucidated. To study the regulation of osteogenic differentiation of BMSCs in diabetic mice by MV and determine the potential mechanism. BMSCs were isolated from both normal (referred to as N-BMSCs) and diabetic (referred to as DM-BMSCs) C57BL/6 mice. DM-BMSCs were treated with different concentrations of MV for varying durations, and cell viability was detected using the cell counting kit-8 assay. Following 2 weeks of osteogenic induction, osteogenic differentiation capability was evaluated using alizarin red S staining, alkaline phosphatase (ALP) activity analysis, and quantitative real-time reverse transcription polymerase chain reaction. Furthermore, the microRNA (miRNA) expression profiles of N-BMSCs, DM-BMSCs, and DM-BMSCs treated with MV were tested using high-throughput sequencing. Treatment with MV enhanced the viability of DM-BMSCs and mitigated the reduction of calcium nodule deposition, ALP activity, and mRNA expression of ALP, osteocalcin, and runt-related transcription factor 2. Of the analyzed miRNAs, miR-10b-5p was the only one that exhibited differential expression in N-BMSCs, DM-BMSCs, and DM-BMSCs treated with MV. An analysis of the top four protein clusters based on KEGG suggested that the target genes of differentially expressed miRNAs were closely linked to the PI3K/AKT pathway. MV significantly enhances the viability and osteogenic differentiation of BMSCs under diabetic conditions. The alteration of miRNA profiles provides a foundation for further research into the regulatory role of miRNAs and MV in this process.

Effects of Tenascin-C on Dental Pulp Tissue in Mice In Vivo And on The Proliferation and Differentiation of Dental Pulp Stem Cells into Odontoblasts and Calcification In Vitro

This study aimed to investigate the effects of tenascin-C (TN-C) on dental pulp tissue and on dental pulp stem cells (DPSCs). In in vivo studies, A collagen sponge with phosphate-buffered saline (PBS) for the control group or with TN-C for the experimental group was placed over the dental pulp of mice. The root pulp was excised at 7 and 21 days postoperatively and was observed microscopically using HE staining and immunohistochemistry. Inflammatory cells were found in the entire pulp tissue in the control group but no inflammatory cells were identified in the pulp tissue in the TN-C-treated experimental group. Further, nesting-positive cells at 7 days and dentin sialo phosphoprotein (DSPP)-positive cells at 21 days were seen in the experimental group. In in vitro studies, DPSCs were cultured in a medium with or without TN-C, after which the proliferation rate of DPSCs was measured mRNA expression levels were examined using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the formation of calcified nodules was investigated using alizarin red staining. There was no significant difference in the cell proliferation rate of DPSCs between the experimental and control groups during day 3 or 5, using the proliferation rate on day 1 as the baseline value. The expression of nesting mRNA on day 7 was significantly higher in the experimental group than in the control group (P&lt;0.05), but the expression of osteocalcin (OCN) mRNA was significantly higher in the control group than in the experimental group (P&lt;0.05). More calcified nodules formed and the area of calcified nodules in the control group was significantly greater than in the TN-C-treated experimental group on day 14 and day 21 (P &lt; 0.05). These results suggest that TN-C regulates inflammation during the healing process in the dental pulp and induces the differentiation of dental pulp into odontoblast-like cells. Further, TN-C promotes the early differentiation of DPSCs into odontoblast-like cells, which suggests that TN-C may further contribute to the inhibition of excessive dentin formation. From the above, TN-C is involved in differentiating dental pulp into mineralized tissue-forming cells and may have a beneficial effect in treatments that utilize this function

Experimental Studies on the Effects of Glycitinregulated TGF-β Signaling Pathway on the Proliferation, Osteogenic, and Lipogenic Differentiation of BMSCs

Background Glycitin, generated from the seeds of the legume soybean, has an essential function in antioxidant, obesity inhibition, and wound healing promotion. Furthermore, recent research has shown that it has anti-inflammatory and cartilage-protective properties. Objectives This study was designed to investigate the osteogenic effects of glycitin and the transforming growth factor-β (TGF-β) signaling pathway on bone marrow mesenchymal stem cells (BMSCs), as well as their interaction. Materials and Methods We isolated rabbit BMSCs by whole bone marrow adherent method and cultured, identified, and induced differentiation. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to determine the proliferation ability of BMSCs with different concentrations of glycitin at different times; reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression levels of osteogenesis-related genes runt-related transcription factor 2 (Runx2), collagen type I (Col-1), and osteocalcin (OC) mRNA; and a kit was used to determine the activities of alkaline phosphatase and triglycerides (TG) in BMSCs. A Western blot was used to detect the expression of TGF-β protein in each group. Results On the fifth day of BMSC primary culture, the morphology of long spindle-shaped cells might be plentiful, dominated by a daisy-like or whirling arrangement. They proliferated rapidly and fused to 70%–80% on the eighth day of culture; the third generation of BMSCs was taken for phenotyping, and the detection results of flow cytometry analysis showed that the surface antigenic markers of BMSCs, CD44, and CD90 were positive, whereas CD34 and CD45 were negative; after osteogenic induction of BMSCs, visible calcified nodules were evident. There was cartilage matrix formation after osteogenic induction; glycitin promoted BMSC proliferation in a time-dependent and dose-dependent manner, and BMSC proliferation increased significantly at 20 µM glycitin for 3–5 days; glycitin increased the expression of Runx2, Col-1, and OC mRNA in BMSCs and increased the activity of alkaline phosphatase (ALP) while decreasing the activity of TG. Meanwhile, 20 µM glycitin dramatically increased the expression of TGF-β. Conclusion These findings imply that glycitin increases BMSCs proliferation and osteogenic differentiation. By regulating the TGF-β signaling pathway, glycitin modulates the differentiation of BMSCs into osteoblasts.

Photobiomodulation Effect of Diode Laser on Differentiation of Osteoprogenitor Cells in Rat Bone Marrow.

Bone marrow cells contain nonhematopoietic cells with the ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Mechanical stress influences osteoblast differentiation of bone marrow cells into osteogenic, chondrogenic, and adipogenic lineages, measurable as the abundance of alkaline phosphatase-positive (ALP+) colony-forming unit-fibroblasts (CFU-F); however, the effect of diode laser irradiation on osteoblast differentiation is unknown. The aim of this study was to analyze the effects of photobiomodulation on the osteogenic differentiation of mesenchymal stem cells in the bone marrow, using the CFU-F assay. Bone marrow cells isolated from rat tibiae were cultured and irradiated with a diode laser (wavelength 808 nm) at a total energy of 0 J (control), 50 J, and 150 J. On day 7 after irradiation, ALP+ CFU-F were most abundant in the 50 J group and the least abundant in the 150 J group. Mineralized nodule formation was observed after long-term culture (21 days). Compared with the control group, there were significantly more nodules in the 50 J group and significantly fewer nodules in the 150 J group. Osteocalcin mRNA expression was highest in the 50 J group, and there was no difference between the control and 150 J groups. Irradiation with 50 J was effective in stimulating osteogenesis in bone marrow stem cells. These findings suggest that diode laser irradiation can induce osteogenesis in rat bone marrow cells in an energy-dependent manner, and appears suitable for application in bone regeneration therapy.

Effects of fine particulate matter on bone mass and osteogenic differentiation of bone marrow mesenchymal stem cells in mice

To observe the effects of exposure to fine particulate matter(PM_(2.5)) on bone mass, microstructure, biomechanical properties, and osteogenic differentiation ability of bone marrow mesenchymal stem cells(BMSCs) in mice. A total of 16 C57BL/6J mice aged 8 weeks were randomly divided into control group(NS group) and PM_(2.5) exposure group(PM group). NS group was given normal saline, PM group was given 14 mg/kg PM_(2.5) suspension, 50 μL, poisoning every 3 day. After 10 weeks, the lungs of mice were taken for HE staining, and the left tibia was taken for Micro CT detection to analyze parameters related to cancellous and cortical bone. The right tibia was taken for HE staining to observe changes in bone trabeculae. Immunohistochemical staining was used to detect type I collagen(Col I), osteoprotegerin(OPG), and nuclear factor-κB receptor activating factor ligand(RANKL) protein expression, tartrate resistant acid phosphatase(TRAP) staining for detection of osteoclasts. Extract primary BMSCs from bilateral femurs, induce osteogenesis, and then perform alkaline phosphatase(ALP) staining to detect ALP activity, alizarin red staining to detect bone mineralization ability, real-time PCR to detect osteocalcin(OCN), ALP, OPG, and RANKL mRNA expression, and biomechanical testing to test the mechanical properties of the femur. Compared with the NS group, the pulmonary alveolar structure of the PM group mice was disrupted and a large number of inflammatory cells gathered. Prompt for successful PM_(2.5) poisoning operation. Micro CT result showed that the bone mineral density(BMD) and bone volume fraction(BV/TV) of the PM group mice were 276.959±15.152 mg/cm~3 and 0.208%±0.009%, respectively. The NS group had 316.709±28.205 mg/cm~3 and 0.236%±0.019%, respectively. The PM group was lower than the NS group(P&lt;0.05), but the trabecular number(Tb. N) There was no statistically significant difference in parameters such as trabecular thickness(Tb. Th) and trabecular separation(Tb. SP)(P&gt;0.05). The HE staining result of the tibia showed that the trabeculae in the NS group were thick, dense, and uniform. The bone trabeculae in the PM group were slender, with a decrease in number, widened spacing, and sparse arrangement. The expression of Col I(0.023±0.009) and OPG(0.036±0.010) in the PM group increased compared to the NS group(0.079±0.007, 0.059±0.012), while the expression of RANKL(0.036±0.006) decreased compared to the NS group(0.022±0.002)(P&lt;0.05); The number of TRAP positive particles increased in the PM group. The experimental result after osteoinduction of BMSCs in mice showed that compared with the NS group, the PM group had a decrease in the number of ALP positive cells and a decrease in the number of calcium nodules. The relative expression of ALP, OCN, and OPG mRNA in the PM group(0.375±0.021, 0.585±0.088, 0.768±0.112) was significantly reduced compared to the NS group(1.001±0.043, 1.006±0.132, 1.002±0.086), while the relative expression of RANKL mRNA(1.278±0.118) was increased compared to the NS group(1.001±0.057)(P&lt;0.05). The biomechanical experimental result showed that the maximum deflection of the NS group was 0.337±0.031 mm, while the maximum deflection of the PM group was 0.258±0.041 mm. Compared with the NS group, the maximum deflection of the PM group decreased significantly(P&lt;0.05), and the maximum stress and maximum load showed a decreasing trend, but the difference was not statistically significant(P&gt;0.05). After 10 weeks of exposure to PM_(2.5), it can affect the bone health of mice, and its mechanism may be related to increased osteoclast activity and inhibition of the osteogenic differentiation ability of BMSCs.

Hyaluronic acid enhances cell migration, viability, and mineralized tissue-specific genes in cementoblasts.

It has been repeatedly demonstrated that cementum formation is a crucial step in periodontal regeneration. Hyaluronic acid (HA) is an important component of the extracellular matrix which regulates cells functions and cell-cell communication. Hyaluronic acid/derivatives have been used in regenerative periodontal therapy, but the cellular effects of HA are still unknown. To investigate the effects of HA on cementoblast functions, cell viability, migration, mineralization, differentiation, and mineralized tissue-associated genes and cementoblast-specific markers of the cementoblasts were tested. Cementoblasts (OCCM-30) were treated with various dilutions (0, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128) of HA and examined for cell viability, migration, mineralization, and gene expressions. The mRNA expressions of osteocalcin (OCN), runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), collagen type I (COL-I), alkaline phosphatase (ALP), cementum protein-1 (CEMP-1), cementum attachment protein (CAP), and small mothers against decapentaplegic (Smad) -1, 2, 3, 6, 7, β-catenin (Ctnnb1) were performed with real-time polymerase chain reaction (RT-PCR). Total RNA was isolated on days 3 and 8, and cell viability was determined using MTT assay on days 1 and 3. The cell mineralization was evaluated by von Kossa staining on day 8. Cell migration was assessed 2, 4, 6, and 24 hours following exposure to HA dilutions using an invitro wound healing assay (0, 1:2, 1:4, 1:8). At dilution of 1:2 to 1:128, HA importantly increased cell viability (p < .01). HA at a dilution of 1/2 increased wound healing rates after 4 h compared to the other dilutions and the untreated control group. Increased numbers of mineralized nodules were determined at dilutions of 1:2, 1:4, and 1:8 compared with control group. mRNA expressions of mineralized tissue marker including COL-I, BSP, RunX2, ALP, and OCN significantly improved by HA treatments compared with control group both on 3 days and on 8 days (p < .01). Smad 2, Smad 3, Smad 7, and β-catenin (Ctnnb1) mRNAs were up-regulated, while Smad1 and Smad 6 were not affected by HA administration. Additionally, HA at dilutions of 1:2, 1:4, and 1:8 remarkably enhanced CEMP-1 and CAP expressions in a dilution- and time-dependent manner (p < .01). The present results have demonstrated that HA affected the expression of both mineralized tissue markers and cementoblast-specific genes. Positive effects of HA on the cementoblast functions demonstrated that HA application may play a key role in cementum regeneration.

Lactoferrin-Anchored Tannylated Mesoporous Silica Nanomaterials-Induced Bone Fusion in a Rat Model of Lumbar Spinal Fusion.

Lactoferrin (LF) is a potent antiviral, anti-inflammatory, and antibacterial agent found in cow and human colostrum which acts as an osteogenic growth factor. This study aimed to investigate whether LF-anchored tannylated mesoporous silica nanomaterials (TA-MSN-LF) function as a bone fusion material in a rat model. In this study, we created TA-MSN-LF and measured the effects of low (1 μg) and high (100 μg) TA-MSN-LF concentrations in a spinal fusion animal model. Rats were assigned to four groups in this study: defect, MSN, TA-MSN-LF-low (1 μg/mL), and TA-MSN-LF-high (100 μg/mL). Eight weeks after surgery, a greater amount of radiological fusion was identified in the TA-MSN-LF groups than in the other groups. Hematoxylin and eosin staining showed that new bone fusion was induced in the TA-MSN-LF groups. Additionally, osteocalcin, a marker of bone formation, was detected by immunohistochemistry, and its intensity was induced in the TA-MSN-LF groups. The formation of new vessels was induced in the TA-MSN-LF-high group. We also confirmed an increase in the serum osteocalcin level and the mRNA expression of osteocalcin and osteopontin in the TA-MSN-LF groups. TA-MSN-LF showed effective bone fusion and angiogenesis in rats. We suggest that TA-MSN-LF is a potent material for spinal bone fusion.

Chinese Ecliptae herba (Eclipta prostrata (L.) L.) extract and its component wedelolactone enhances osteoblastogenesis of bone marrow mesenchymal stem cells via targeting METTL3-mediated m6A RNA methylation

Ethnopharmacological relevanceChinese Ecliptae herba (Eclipta prostrata (L.) L.) is an ethnomedicinal herb, which is used mainly to nourish kidney and thus strengthen bones according to traditional Chinese medicine theory. Pharmacological studies have supported the ethnomedicine use, showing that Ecliptae herba extract has an anti-osteoporotic effect in vivo and promoted osteoblast proliferation and activity in vitro. However, the molecular mechanism of Ecliptae herba on osteoblast differentiation from bone marrow mesenchymal stem cells (BMSC), the progenitors of osteoblasts, is still unclear. Aim of the studyN6-methyladenosine (m6A) mRNA epigenetic modification may play a key role in promoting osteoblastic differentiation, and thus treating osteoporosis. This study sought to assess the mechanism through which Eclipate herba and its component wedelolactone influence m6A modification during the process of osteoblastogenesis from BMSC. Material and methodsThe alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were applied to determine osteoblastogenesis from BMSC. Western blot and quantitative real-time PCR were performed. RNA sequencing analysis was used to determine the characteristics of m6A methylation. Stable knocking down of METTL3 using lentiviral-based shRNA was performed. ResultsUpon 9 d treatment of BMSC with ethyl acetate extract of Ecliptae herba (MHL), ALP activity and ossification level increased in comparison with osteogenic medium (OS)-treated control. The expression of methyltransferase METTL3 and METTL14 was significantly increased, but WTAP expression had no change in response to MHL treatment. Knocking down of METTL3 resulted in a decrease in MHL-induced ALP activity, ossification level as well as mRNA expression of Osterix and Osteocalcin, two bone formation-related markers. The level of m6A increased when BMSC was treated with MHL for 9 d. RNA sequencing analysis indicated that MHL treatment altered mRNA m6A modification of genes associated with osteoblastogenesis. By kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, HIF-1α, PI3K/Akt, and Hippo signaling pathways were enriched and associated with m6A modification. The expression of m6A-modified genes including HIF-1α, VEGF-A, and RASSF1, was upregulated by MHL, but the upregulation was reversed after METTL3 knockdown. Additionally, the enhanced expression of METTL3 was also observed after treatment with wedelolactone, a component from MHL. ConclusionsThese results suggested a previously uncharacterized mechanism of MHL and wedelolactone on osteoblastogenesis, by which METTL3-mediated m6A methylation is involved and thus contributes to the enhancement of osteoblastogenesis.

Lrp5 p.Val667Met Variant Compromises Bone Mineral Density and Matrix Properties in Osteoporosis.

Early-onset osteoporosis (EOOP) has been associated with several genes, including LRP5, coding for a coreceptor in the Wnt pathway. Variants in LRP5 were also described in osteoporosis pseudoglioma syndrome, combining severe osteoporosis and eye abnormalities. Genomewide-association studies (GWAS) showed that LRP5 p.Val667Met (V667M) variant is associated with low bone mineral density (BMD) and increased fractures. However, despite association with a bone phenotype in humans and knockout mice, the impact of the variant in bone and eye remains to be investigated. Here, we aimed to evaluate the bone and ocular impact of the V667M variant. We recruited 11 patients carrying the V667M variant or other loss-of-function variants of LRP5 and generated an Lrp5 V667M mutated mice. Patients had low lumbar and hip BMD Z-score and altered bone microarchitecture evaluated by HR-pQCT compared with an age-matched reference population. Murine primary osteoblasts from Lrp5 V667M mice showed lower differentiation capacity, alkaline phosphatase activity, and mineralization capacity in vitro. Ex vivo, mRNA expression of Osx, Col1, and osteocalcin was lower in Lrp5 V667M bones than controls (all p < 0.01). Lrp5 V667M 3-month-old mice, compared with control (CTL) mice, had decreased BMD at the femur (p < 0.01) and lumbar spine (p < 0.01) with normal microarchitecture and bone biomarkers. However, Lrp5 V667M mice revealed a trend toward a lower femoral and vertebral stiffness (p = 0.14) and had a lower hydroxyproline/proline ratio compared with CTL, (p = 0.01), showing altered composition and quality of the bone matrix. Finally, higher tortuosity of retinal vessels was found in the Lrp5 V667M mice and unspecific vascular tortuosity in two patients only. In conclusion, Lrp5 V667M variant is associated with low BMD and impaired bone matrix quality. Retinal vascularization abnormalities were observed in mice. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Expression Changes of Hypoxia-Inducible Factor-1α in G-CSF Induced Hematopoietic Stem Cell Mobilization

To investigate the expression and its relative mechanism of hypoxia-inducible factor-1α(HIF-1α) in bone marrow(BM) of mice during G-CSF mobilization of hematopoietic stem cells (HSC) . Flow cytometry was used to detect the proportion of Lin-Sca-1+ c-kit+ (LSK) cells in peripheral blood of C57BL/6J mice before and after G-CSF mobilization. And the expression of HIF-1α and osteocalcin (OCN) mRNA and protein were detected by RQ-PCR and immunohistochemistry. The number of osteoblasts in bone marrow specimens of mice was counted under the microscope. The proportion of LSK cells in peripheral blood began to increase at day 4 of G-CSF mobilization, and reached the peak at day 5, which was significantly higher than that of control group (P<0.05). There was no distinct difference in the expression of HIF-1α mRNA between bone marrow nucleated cells and osteoblasts of steady-state mice (P=0.073), while OCN mRNA was mainly expressed in osteoblasts, which was higher than that in bone marrow nucleated cells (P=0.034). After mobilization, the expression level of HIF-1α increased, but OCN decreased, and the number of endosteum osteoblasts decreased. The change of HIF-1α expression was later than that of OCN and was consistent with the proportion of LSK cells in peripheral blood. The expression of HIF-1α in bone marrow was increased during the mobilization of HSC mediated by G-CSF, and one of the mechanisms may be related to the peripheral migration of HSC induced by osteoblasts inhibition.

INFLUENCE OF CHOLESTEROL ENRICHED DIET ON GENES EXPRESSION ENCODING BONE MORPHOGENETIC PROTEIN-2 AND OSTEOCALCIN IN MOUSE MANDIBLE.

The aim: To evaluate the mRNA expression of the key regulators of osteogenesis - osteocalcin and BMP-2 in the mouse embryos mandible (17th day of pregnancy) which were borne by females on high-cholesterol diet for 30 days before fertilization and throughout pregnancy. Materials and methods: Experimental hypercholesterolemia (2%) was simulated by adding Cholesterol to the diet for 60 days. In experiment were used 40 mature female white mice that were randomly divided to control and experimental groups. The control group were fed with standard chow diet, the experimental group with diet with cholesterol enriched diet (with addition of 2 grams of Cholesterol per 100 grams of standard chow). The mandibles of mouse embryos (E-17) were examined by using molecular genetic methods. Results: In control group the relative level of BMP-2 mRNA / actin mRNA was 27.0«2.82, the relative level of and osteocalcin mRNA / actin mRNA was 30.5«6,28. In the jaws of animals in the experimental group with cholesterol enriched diet, the expression relative level of BMP-2 was 30.9«5.81 that is by 14,4% higher than in control group. Therefore, the expression level of оsteocalcin, on the contrary, decreased by 22.3% and was 23.7+5.31. Conclusions: Our study report influence of the cholesterol enriched diet (2%) on mRNA expression of BMP-2 and osteocalcin encoding genes. The embryos from mouse on cholesterol enriched diet (2%) had increased level of BMP-2 gene expression, however significantly decreased level of osteocalcin gene expression.

The effect of simvastatin-loaded methoxy poly(ethylene glycol)-polylactic acid nanoparticles on osteoblasts

ObjectiveTo investigate the effect of simvastatin-loaded polymer nanoparticles on osteoblast proliferation and differentiation, and to seek a better pharmaceutical dosage form to improve the osteogenic effect of simvastatin and provide a more practical drug carrier for its clinical application. Methodspolymer nanoparticles were prepared via the modified spontaneous emulsifying solvent diffusion (SESD) method, and the effects of simvastatin-loaded methoxy poly(ethylene glycol)-polylactic acid nanoparticles on osteoblasts were investigated. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was adopted to detect the proliferation abilities of the cells. An enzyme-linked immunosorbent assay was used to detect the amount of alkaline phosphatase (ALP) in the cells. A real-time polymerase chain reaction was used to detect the expression of osteocalcin (OC) and bone morphogenetic protein-2 (BMP-2) messenger ribonucleic acid (mRNA). ResultsThe study found that the proliferation of osteocytes was inhibited in the simvastatin-loaded nanoparticle (Sim-NP) group, and this inhibition was stronger in the Sim-NP than in the simvastatin group; the ALP activity of osteoblasts was increased in both groups, and this increase was stronger in the Sim-NP than in the simvastatin group. The expression of ALP, OC and BMP-2 mRNA was increased in both groups, and this increase was stronger in the Sim-NP than in the simvastatin group. ConclusionThe authors thus posited that the modified Sim-NPs SESD preparation method was simple and reproducible, and the prepared nanoparticles, with uniform distribution, a regular morphology and spherical shape could greatly improve the solubility of simvastatin. The polymer nanoparticle carrier system improved the effect of simvastatin on the proliferation and differentiation of osteoblasts so that the Sim-NPs were systematically applied to target bone tissues. These results provide a new direction for research on the drugs and treatment methods for osteoporosis.

Revealing the mechanisms of Weishi Huogu I capsules used for treating osteonecrosis of the femoral head based on systems pharmacology with one mechanism validated with in vitro experiments

Ethnopharmacological relevanceWeishi Huogu I (WH I) capsules, developed through traditional Chinese medicine, have been used to treat clinical osteonecrosis of the femoral head (ONFH) for decades. However, the mechanisms have not been systematically studied.Aim of the study: In this study, the mechanisms of WH I capsules used in treating ONFH were examined through a systems pharmacology strategy, and one mechanism was validated with in vitro experiments. Materials and methodsWH I capsules compounds were identified by screening databases; then, a database of the potential active compounds was constructed after absorption, distribution, metabolism and excretion (ADME) evaluation. The compounds were identified through a systematic approach in which the probability of an interaction of every candidate compound with each corresponding target in the DrugBank database was calculated. Gene Ontology (GO) and pathway enrichment analyses of the targets was performed with the Metascape and KEGG DISEASE databases. Then, a compound-target network (C-T) and target-pathway network (T-P) of WH I capsule components were constructed, and network characteristics and related information were used for systematically identifying WH I capsule multicomponent-target interactions.Furthermore, the effects of WH I capsule compounds identified through the systematic pharmacology analysis of the osteogenic transformation of human umbilical mesenchymal stem cells (HUMSCs) were validated in vitro. ResultsIn total, 152 potentially important compounds and 176 associated targets were identified. Twenty-two crucial GO biological process (BP) or pathways were related to ONFH, mainly in regulatory modules regulating blood circulation, modulating growth, and affecting pathological processes closely related to ONFH.Furthermore, the GO enrichment analysis showed that corydine, isorhamnetin, and bicuculline were enriched in “RUNX2 regulates osteoblast differentiation”, significantly increased alkaline phosphatase activity and calcium deposition and upregulated runt-related transcription factor 2 mRNA and protein expression and osteocalcin mRNA expression in HUMSCs, suggesting that these compounds promoted the mesenchymal stem cell (MSC) osteogenic transformation. ConclusionsThe study showed that the pharmacological mechanisms of WH I capsule attenuation of ONFH mainly involve three therapeutic modules: blood circulation, modulating growth, and regulating pathological processes. The crosstalk between GOBPs/pathways may constitute the basis of the synergistic effects of the compounds in WH I capsules in attenuating ONFH. One of the pharmacological mechanisms in the WH I capsule effect on ONFH involves enhancement of the osteogenic transformation of MSCs, as validated in experiments performed in vitro; however, more mechanisms should be validated in further studies.

Mechanism of ring finger protein 11 regulating Akt signaling pathway to promote osteogenic differentiation of bone marrow mesenchymal stem cells

To investigate the role and regulatory mechanism of ring finger protein 11 (RNF11) on Akt signaling pathway in the process of osteogenesis of bone marrow mesenchymal stem cells (BMSCs) to provide ideas for further clarifying its osteogenesis mechanism and its use in clinical treatment in the future. BMSCs were isolated and cultured from fresh bone marrow of healthy donors and subcultured. The 4th generation cells were used in experiments after identification by flow cytometry, and osteogenic, chondrogenic, and adipogenic induction. BMSCs were cultured in osteogenic differentiation medium for 0-14 days. The degree of osteogenic differentiation was detected by Alizarin red staining and alkaline phosphatase (ALP) staining, and the protein expression of RNF11 was detected by Western blot. The 4th generation BMSCs were divided into blank control group (group A), empty lentivirus (Lv-NC) group (group B), and knockdown RNF11 (Lv-ShRNF11) group (group C). Osteogenesis was induced and cultured for 0-14 days. The expression of RNF11 protein was detected by Western blot, the degree of osteogenic differentiation was detected by Alizarin red staining and ALP staining, and the relative mRNA expressions of Runx2, osteocalcin (OCN), and osteopontin (OPN) were detected by real-time fluorescence quantitative PCR (qRT-PCR). The protein relative expressions of Akt, Smad1/5/8, and β-catenin signaling pathway were detected by Western blot, expressed as the ratio before and after phosphorylation. In order to study the effect mechanism of RNF11 on Akt signaling pathway, the 4th generation BMSCs were divided into Lv-NC transfection group (group A1), Lv-ShRNF11 transfection group (group B1), and Lv-ShRNF11 transfection supplemented with Akt signaling pathway activator SC79 group (group C1). The protein relative expressions of RNF11 and Akt signaling pathway were detected by Western blot, the related osteogenesis indexes were detected by Alizarin red staining, ALP staining, and qRT-PCR. The flow cytometry, and osteogenic, chondrogenic, adipogenic induction culture identification showed that the isolated and cultured cells were BMSCs. The protein relative expression of RNF11 increased gradually with the extension of osteogenic differentiation time ( P<0.05); after knockdown RNF11, Alizarin red and ALP stainings showed that the degree of osteogenic differentiation of BMSCs in group C were significantly lower than those in groups A and B, and qRT-PCR detection showed that the relative expression of Runx2, OCN, and OPN mRNA significantly decreased ( P<0.05). The protein relative expressions of RNF11 and Akt signaling pathway significantly increased with the extensions of osteogenic differentiation time ( P<0.05). After knockdown RNF11, the protein relative expression of Akt signaling pathway in group C was significantly lower than that in groups A and B ( P<0.05), while Smad1/5/8 and β-catenin signaling pathway had no significant effect ( P>0.05). Compared with group A1, the protein relative expression of RNF11 in groups B1 and C1 significantly decreased ( P<0.05). Compared with groups A1 and C1, the protein relative expression of Akt signaling pathway in group B1 was significantly lower ( P<0.05); Alizarin red and ALP stainings showed that the degree of osteogenic differentiation of BMSCs in group C1 were slightly lower than that of group A1 ( P>0.05), but significantly higher than that of group B1 ( P<0.05); qRT-PCR detection showed that the relative expressions of Runx2, OCN, and OPN mRNA in group C1 were slightly lower than those of group A1 ( P>0.05), but were significantly higher than those of group B1 ( P<0.05). RNF11 promotes the differentiation of BMSCs into osteoblasts by positively regulating the activation level of Akt signaling pathway. RNF11 can be used as a potential target to improve the bone repair efficacy of BMSCs and treat bone metabolic diseases.

Biocompatibility and pro-mineralization effect of tristrontium aluminate cement for endodontic use

Background/purposeTristrontium aluminate (S3A) is a hydraulic cement with setting behavior similar to that of mineral trioxide aggregate (MTA). This study examined the biological effects of S3A on mouse dental papilla cells (MDPs) in vitro and on rat exposed pulps in vivo. Materials and methodsExtracts of S3A and MTA were prepared by immersing each cement in ultrapure water. MDPs were cultured with S3A or MTA extracts, and cell proliferation was evaluated with a tetrazolium-salt assay. Attachment of MDPs on the set cements was examined with scanning electron microscopy (SEM). mRNA expression of bone morphogenic protein (Bmp2), osteocalcin (Oc) and osteopontin (Opn) in MDPs exposed to S3A or MTA extracts was determined with reverse transcription-quantitative polymerase chain reaction. Mineralized nodule formation was evaluated with Alizarin Red S staining. Simulated body fluid (SBF)-dipped S3A was examined with SEM and energy dispersive X-ray analysis (EDX). Exposed molar pulps of male Wistar rats capped with S3A or MTA were histologically examined. ResultsS3A extract did not inhibit proliferation of MDPs. Set S3A and MTA exhibited attachment of MDPs on their surface. S3A extract showed significantly higher mineralized nodule formation and mRNA expression of Bmp2, Oc, and Opn than did MTA extract. SBF-dipped S3A exhibited formation of surface precipitates, which were composed of Ca, P, Sr, and Al. Direct pulp capping with S3A and with MTA induced mineralized tissue repair of the exposed pulp. ConclusionS3A possesses biocompatibility and pro-mineralization effects comparable to those of MTA.