Bone Morphogenetic Protein-2 Levels Research Articles

Cyclin A1 (CCNA1) inhibits osteoporosis by suppressing transforming growth factor-beta (TGF-beta) pathway in osteoblasts

BackgroundOsteoporosis is a genetic disease caused by the imbalance between osteoblast-led bone formation and osteoclast-induced bone resorption. However, further gene-related pathogenesis remains to be elucidated.MethodsThe aberrant expressed genes in osteoporosis was identified by analyzing the microarray profile GSE100609. Serum samples of patients with osteoporosis and normal group were collected, and the mRNA expression of candidate genes was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The mouse cranial osteoblast MC3T3-E1 cells were treated with dexamethasone (DEX) to mimic osteoporosis in vitro. Alizarin Red staining and alkaline phosphatase (ALP) staining methods were combined to measure matrix mineralization deposition of MC3T3-E1 cells. Meanwhile, the expression of osteogenesis related genes including alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), Osterix, and bone morphogenetic protein 2 (BMP2) were evaluated by qRT-PCR and western blotting methods. Then the effects of candidate genes on regulating impede bone loss caused by ovariectomy (OVX) in mice were studied.ResultsCyclin A1 (CCNA1) was found to be significantly upregulated in serum of osteoporosis patients and the osteoporosis model cells, which was in line with the bioinformatic analysis. The osteogenic differentiation ability of MC3T3-E1 cells was inhibited by DEX treatment, which was manifested by decreased Alizarin Red staining intensity, ALP staining intensity, and expression levels of ALP, OCN, OPN, Osterix, and BMP2. The effects of CCNA1 inhibition on regulating osteogenesis were opposite to that of DEX. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that genes negatively associated with CCNA1 were enriched in the TGF-beta signaling pathway. Inhibitor of TGF-beta signaling pathway partly reversed osteogenesis induced by suppressed CCNA1. Furthermore, suppressed CCNA1 relieved bone mass of OVX mice in vivo.ConclusionDownregulation of CCNA1 could activate TGF-beta signaling pathway and promote bone formation, thus playing a role in treatment of osteoporosis.

Comparison of the protective effect of the upper zone of the growth plate and unique cartilage matrix-associated protein with hyaluronic acid and corticosteroids on an experimental rat osteoarthritis model.

This study sought to compare the protective effect of the upper zone of the growth plate and unique cartilage matrix-associated protein (UCMA) with hyaluronic acid (HA) and corticosteroids (CS) in a rat model of osteoarthritis (OA). In the experimental animal study, 40 adult male rats were randomly assigned into five groups: control, monosodium iodoacetate (MIA) + vehicle (MIA+V), MIA+HA, MIA+CS, and MIA+UCMA. The OA model was induced by an intra-articular MIA injection to the right knee, and intra-articular injections into the right knee were performed on the treatment groups seven times every three days for 21 days. The knee joints were taken for histopathology and immunohistochemistry (IHC) analyses after the rats were sacrificed. All sections were stained with hematoxylin-eosin, safranin O and fast green FCF, and toluidine blue, and bone morphogenetic protein 2 (BMP-2) and nuclear factor-kappa B (NF-κB) expressions were analyzed with IHC. The Mankin scoring was utilized to determine the histopathological changes in the joint tissues. Mankin score was significantly higher in the MIA group compared to the control group. Histopathologically, in the UCMA-, HA-, and CS-treated groups, degenerations in the articular cartilage were milder than in the MIA+V group. Mankin score was found to be decreased significantly in the UCMA-, HA-, and CS-treated groups compared to the MIA group. Furthermore, IHC analyses revealed that NF-κB and BMP-2 expressions elevated in the MIA-induced OA model, while they were downregulated after UCMA, HA, and CS treatments. Our data revealed that UCMA could be used as a potential protective molecule in the prevention and treatment of OA. Furthermore, the protective effect of UCMA was similar to HA and CS, and its possible beneficial roles against OA may be linked to the reduced BMP-2 and NF-κB levels. Further experimental research would make significant contributions to a better understanding of the therapeutic effect of UCMA on degenerative cartilage tissues.

Expression pattern of the bone morphogenetic protein-4 and its relationship with inflammation, vascular injury in patients suffered the arterial occlusive diseases.

Bone morphogenetic protein-4 (BMP4) has been proved to be an important regulatory factor for the pathological process of atherosclerosis (AS). However, there are few related clinical studies. This study aims to investigate the levels of plasma BMP4 in patients suffering from the arterial occlusive diseases (ACD) characterized by AS, and further to test the relationship between BMP4 and inflammation and vascular injury. A total of 38 ACD patients (the ACD group) and 38 healthy people for the physical examination (the control group) were enrolled. The plasma in each subject from both groups was obtained to test the levels of BMP4, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-10, and vascular endothelial cadherin (VE-cadherin), and the relationship between BMP4 and the detected indicators above were further analyzed. Compared with the control group, the patients in the ACD group displayed significant elevations in the neutrophil to lymphocyte ratio [NLR, 1.63 (1.26, 1.91) vs 3.43 (2.16, 6.61)] and platelet to lymphocyte ratio [PLR, 6.37 (5.26, 7.74) vs 15.79 (7.97, 20.53)], while decrease in the lymphocyte to monocyte ratio [LMR, 5.67 (4.41, 7.14) vs 3.43 (2.07, 3.74)] (all P<0.05). Besides, the ACD patients displayed significant elevations in plasma BMP4 [581.26 (389.85, 735.64) pg/mL vs 653.97(510.95, 890.43) pg/mL], TNF-α [254.16 (182.96, 340.70) pg/mL vs 293.29(238.90, 383.44) pg/mL], and VE-cadherin [1.54 (1.08, 2.13) ng/mL vs 1.85 (1.30, 2.54) ng/mL], and decrease in IL-10 [175.89 (118.39, 219.25) pg/mL vs 135.92 (95.80, 178.04) pg/mL] (all P<0.05). While the levels of IL-1β remained statistically comparable between the 2 groups (P=0.09). Furthermore, the plasma BMP4 levels were further revealed to be positively correlated with the levels of IL-1β (r=0.35), TNF-α (r=0.31) and VE-cadherin (r=0.47), while they were negatively correlated with the levels of IL-10 (r=-0.37; all P<0.01). After ACD occurrence, the patients' plasma concentrations of BMP4 would be upregulated, which may serve as a candidate to indicate the levels of inflammation and vascular injury.

Development of novel hybrid 3D-printed degradable artificial joints incorporating electrospun pharmaceutical- and growth factor-loaded nanofibers for small joint reconstruction

Small joint reconstruction remains challenging and can lead to prosthesis-related complications, mainly due to the suboptimal performance of the silicone materials used and adverse host reactions. In this study, we developed hybrid artificial joints using three-dimensional printing (3D printing) for polycaprolactone (PCL) and incorporated electrospun nanofibers loaded with drugs and biomolecules for small joint reconstruction. We evaluated the mechanical properties of the degradable joints and the drug discharge patterns of the nanofibers. Empirical data revealed that the 3D-printed PCL joints exhibited good mechanical and fatigue properties. The drug-eluting nanofibers sustainedly released teicoplanin, ceftazidime, and ketorolac in vitro for over 30, 19, and 30 days, respectively. Furthermore, the nanofibers released high levels of bone morphogenetic protein-2 and connective tissue growth factors for over 30 days. An in vivo animal test demonstrated that nanofiber-loaded joints released high concentrations of antibiotics and analgesics in a rabbit model for 28 days. The animals in the drug-loaded degradable joint group showed greater activity counts than those in the surgery-only group. The experimental data suggest that degradable joints with sustained release of drugs and biomolecules may be utilized in small joint arthroplasty.

A potential approach for in vitro bone tissue engineering: expression of BMP-2 and FGF-2 on nano Ch-CA scaffolds

Background: Alveolar bone resorption after tooth extraction is a physiological process that cannot be avoided. The traditional approach to treating bone defects is frequently ineffective, but gene therapy, which aims to maintain the local concentration of bone growth factors at a therapeutic level, is a promising method for expediting bone defect repair. One of the polymer materials, which have been developed in bone regeneration is chitosan (Ch). Ch in the form of nanoparticles is neutral, not toxic, and has constant stability. To Enhance the bone regeneration properties of the graft substitution, nano chitosan-carbonate apatite scaffold (nano Ch-CA scaffold) combined with bone morphogenetic protein-2 (BMP-2) and fibroblast growth factors-2 (FGF-2) were developed. BMP-2 and FGF-2 have osteoinducing properties and functions in regulating the process of proliferation, differentiation, and apoptosis of osteoblasts. Purpose: To observe the expression of BMP-2 and FGF-2 on a nano Ch-CA scaffold that promotes osteogenesis in vitro. Materials and Methods: Forty-two nano Ch-CA scaffolds were used and divided into 2 groups, namely group I containing osteoblast cells seeded on nano Ch-CA scaffold with BMP-2, and group II containing osteoblast cells seeded on nano Ch-CA scaffold with FGF-2. Expression of BMP-2 and FGF-2 were examined on days 3, 5, and 7. Each time of examination consisted of 7 nano Ch-CA scaffold. An immunohistochemistry (IHC) test was used to assess the levels of BMP-2 and FGF2 expression on nano Ch-CA scaffold in vitro. Results: The cylindrical nano Ch-CA scaffold that was prepared had a uniform size distribution. The BMP-2 and FGF-2 expression levels were significantly increased on days 3, 5, and 7 of observation. It is possible to infer that encouraging osteogenesis on nano Ch-CA scaffold in vitro. Conclusion: The nano Ch-CA scaffold shows good handling properties when combined with BMP-2 and FGF-2. The osteogenic capabilities of osteoblast cells are effectively promoted in vitro by the expression of BMP-2 and FGF-2 on a nano Ch-CA scaffold.

Effect of Mongolian medicine alcohol spray therapy on fracture healing in rabbits

Purpose: To evaluate the healing effect and mechanism of Mongolian medicine alcohol spray therapy on fracture healing process in rabbits.&#x0D; Methods: Thirty New Zealand white rabbits were randomly divided into normal, fracture, and treatment groups, with 10 rabbits in each group. Fracture and treatment groups underwent bone-fracture modeling, with the latter receiving daily alcohol spraying and stroking for six weeks. Serum calcium and phosphorus were measured using an automatic biochemical analyzer. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of BMP-2, ALP, and TGF-β. Biomechanical testing was performed to assess callus properties, and real-time polymerase chain reaction (PCR) and western blot were performed to assess BMP-2, Runx2, and p-Smad1/5 expressions in callus.&#x0D; Results: There were low serum calcium, phosphorus, BMP-2, ALP, and TGF-β levels in fracture and treatment groups, compared to normal group. Similarly, treatment group showed significantly increased levels of serum calcium, phosphorus, bone morphogenetic protein 2 (BMP-2), alkaline phosphatase (ALP), and transforming growth factor-β (TGF-β) (p &lt; 0.05) compared to fracture group. Biomechanical testing revealed that maximum load, stiffness, and deflection of callus in the fracture and treatment groups were significantly lower compared to normal group (p &lt; 0.05). expressions of BMP-2, Runx2, and p-Smad1/5 mRNA in the callus significantly decreased in both fracture and treatment groups compared to normal group (p &lt; 0.05). However, treatment group showed significantly increased expression levels compared to fracture group (p &lt; 0.05).&#x0D; Conclusion: Mongolian medicine alcohol spray therapy promotes fracture healing in rabbits and increases maximum load of the callus, possibly through upregulation of the BMP-2 signaling pathway. Further research is necessary to confirm the exact mechanism by which alcohol spray therapy regulates BMP-2 signaling pathway.

Bone Morphogenetic Protein-4 Promotes Phenotypic Modulation via SMAD-4/MCT-4 Axis in Vascular Smooth Muscle Cells

Introduction: This study aimed to determine whether bone morphogenetic protein-4 (BMP-4), which increases in response to intimal hyperplasia, promotes phenotype transition in vascular smooth muscle cells (VSMCs). Methods: Balloon injury was used to induce intimal hyperplasia in rats. Hematoxylin-eosin staining was used to detect the alteration of vascular structure. Serum levels of BMP-4 and lactate were detected by ELISA. Human aortic smooth muscle cells (HA-SMCs) were cultured. Protein and mRNA expression levels were detected through Western blot and real-time PCR. Cell migration was measured by transwell assay. Results: Our data showed that serum concentration of BMP-4 was upregulated after balloon injury. Treatment with BMP-4 inhibitor DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo(1,5-a)pyrimidin-3-yl)quinoline) suppressed the abnormal expression of BMP-4 and inhibited the intimal hyperplasia induced by balloon injury. Compared to BMP-4-negative medium, BMP-4-positive medium was associated with higher synthetic VSMC marker expression levels and lower in contractile gene markers in cultured HA-SMCs. Transfection of monocarboxylic acid transporters-4 (MCT-4) siRNA inhibited the excretion of lactate induced by BMP-4. Conclusion: Our analyses provided evidence that BMP-4 and its regulator Smad-4 are key regulators in MCT-4-mediated lactate excretion. This indicates that BMP-4 stimulates the phenotypic transition of VSMCs via SMAD-4/MCT-4 signaling pathway.

MiR-20 promotes cartilage repair in knee osteoarthritis rats via BMP2/Smad1 signaling pathway.

To explore the effect of micro ribonucleic acid (miR)-20b on knee osteoarthritis rats by regulating the bone morphogenetic protein 2 (BMP2)/Smad1 pathway, a total of 36 SD rats were randomly divided into normal group (n=12), model group (n=12) and miR-20b mimics group (n=12). The rats in normal group were fed normally, while those in model group and miR-20b mimics group were used to establish knee osteoarthritis models. After modeling, model group was not given any intervention, but miR-20b mimics group received intra-articular injection of miR-20b mimics once a day for 2 weeks. Basso, Beattie and Bresnahan (BBB) limb motor function scoring was performed at 1, 5, 7 and 14 days after the modeling, and samples were obtained after 2 weeks of intervention. Next, hematoxylin and eosin (H&E) staining was applied to observe tissue morphology, Markin's scoring was utilized to evaluate articular cartilage degeneration, and immunohistochemistry was employed to detect the expressions of BMP2 and Smad1. Thereafter, the expression of miR-20b was detected via qPCR, the content of cartilage oligomeric matrix protein (COMP) and C-telopeptide of type II collagen (CTX-II) was measured via enzyme-linked immunosorbent assay (ELISA), and the expressions of BMP2 and Smad1 proteins were examined via Western blotting (WB). BBB limb motor function scoring showed that compared with that in normal group, the BBB limb motor function score of rats in the other two groups was reduced (P<0.05). In comparison with that in model group, the BBB limb motor function score in miR-20b mimics group was increased from the 7th day after intervention (P<0.05). In addition, H&E staining results manifested that the articular surface in normal group was smooth and flat, with normal morphology, clear structure and no obvious damage. In model group, the articular surface was not smooth and uneven, and more articular cartilage fractures, morphological disorders and structural damages could be observed. Moreover, the articular surface in miR-20b mimics group was slightly damaged and smoother, and its morphology and structure were markedly improved in contrast to that in model group. The Markin's score in normal group was lower than that in model group and miR-20b mimics group (P<0.05), and it was overtly decreased in miR-20b mimics group in comparison with that in model group (P<0.05). Next, immunohistochemistry demonstrated that compared with normal group, the other two groups had lowered positive expressions of BMP2 and Smad1 (P<0.05). In comparison with model group, miR-20b mimics group exhibited notably raised positive expressions of BMP2 and Smad1 (P<0.05). Then it was found from qPCR results that the expression level of miR-20b in the other two groups was overtly reduced compared with that in normal group (P<0.05), and it was prominently elevated in miR-20b mimics group in contrast to that in model group (P<0.05). Besides, ELISA illustrated that the content of COMP and CTX-II in the cartilage tissues in the other two groups was evidently reduced compared with that in normal group (P<0.05), and it was increased prominently in miR-20b mimics group compared with that in model group (P<0.05). Finally, it was revealed by WB examination that the relative expression levels of BMP2 and Smad1 proteins in the other two groups markedly declined in comparison with those in normal group (P<0.05), and they were elevated in contrast to those in model group (P<0.05). MiR-20b can promote cartilage repair and improve articular function in knee osteoarthritis rats by up-regulating the BMP2/Smad1 signaling pathway.

Inokosterone activates the BMP2 to promote the osteogenic differentiation of bone marrow mesenchymal stem cells and improve bone loss in ovariectomized rats

Evidence suggests that enhancing the osteogenic ability of bone marrow-derived mesenchymal stem cells (BMSCs) may be beneficial in the fight against osteoporosis (OP) effects. Inokosterone (IS) is a major active constituent of Achyranthis bidentatae radix (ABR), which stimulates osteogenic differentiation of mouse embryonic osteoblasts. This study aims to investigate effect of IS on OP using osteogenic differentiated BMSCs and ovariectomy (OVX)-induced OP rats. The BMSCs were treated with 50, 100, or 200 mg/L IS and OP rats were given 2 or 4 mg/kg of IS by gavage. Cell viability, the osteogenic differentiation marker protein expression level, and mineralization were observed. This study proved that IS improved cell viability, osteogenic differentiation, and cellular mineralization in BMSCs and raised expression levels of bone morphogenetic protein-2 (BMP2), Smad1, runt-related transcription factor 2 (RUNX2), collagen I, ALP, and OCN. By BMP2 knockdown/overexpression, this study also proved the BMP2 signaling pathway activation is a potential biological mechanism of IS to improve osteogenic differentiation and mineralization in osteogenic differentiated BMSCs. In OVX-induced OP rats, IS was observed to antagonize bone loss, improve osteogenic differentiation marker protein expression levels, and activate BMP-2, smad1, and RUNX2. These findings provide scientific support for further investigation of the biological mechanisms of IS in ameliorating OP.

SAT267 BMP4, GDF11 And TGFβ2 Repress Adrenal Cell Cortisol Production By Blocking CYP17A1 And CYP11B1 Expression

Abstract Disclosure: E.C. Fischbach: None. A. Lopez: None. M. Ullenbruch: None. J. Rege: None. W.E. Rainey: None. Introduction: The adrenal cortex is responsible for the biosynthesis of multiple steroid hormones that regulate electrolyte and carbohydrate balance. Cortisol, the principal glucocorticoid in humans, is primarily produced in the middle zone of the cortex known as the zona fasciculata (ZF). The enzymes responsible for cortisol biosynthesis include cytochrome P450c17 (17α-hydroxylase), encoded by CYP17A1 and cytochrome P450c11 (11β-hydroxylase), encoded by CYP11B1. Several paracrine/autocrine regulators, including members of the transforming growth factor beta (TGFβ) superfamily, have been implicated in controlling adrenocortical cell homeostasis and steroid hormone production. Previously, we reported a bone morphogenetic protein 4 (BMP4) expression gradient in the adrenal cortex with higher levels in the zona glomerulosa (ZG) and outer ZF compared to the zona reticularis (ZR). In this study, we sought to compare adrenal expression of members of the TGFβ family including BMP4, growth differentiation factor 11 (GDF11), and transforming growth factor beta 2 (TGFβ2), and determine their effect on adrenal cell cortisol biosynthesis. Methods: Growth factor mRNA levels were studied using microarray analysis of laser captured ZG, ZF and ZR from human adrenals. The adrenal cell line, HAC15 was used to determine the effects of the TGFβ family members alone and in combination. Output measures included cell cortisol production and mRNA levels of steroidogenic enzymes using ELISA and quantitative RT-PCR, respectively. Results: Microarray analysis indicated higher mRNA levels of BMP4, GDF11 and TGFβ2 in the ZG compared with the ZF and ZR regions of the adrenal. Treatment of HAC15 cells with either BMP4 (50 ng/mL), GDF11 (50 ng/mL) or TGFβ2 (10 ng/mL) significantly inhibited cortisol production, as well as CYP11B1 and CYP17A1 mRNA levels. The inhibition was observed under basal and forskolin-stimulated conditions. Furthermore, the inhibitory effects on cortisol production and enzyme expression were additive when factors were added together. Summary: Overall, our findings suggest that the human adrenal has a gradient of multiple members of the TGFβ family. The additive action of these growth factors on adrenal cell differentiation may explain, in part, the inability of the ZG to produce cortisol and its low expression of CYP11B1 and CYP17A1. Presentation: Saturday, June 17, 2023

Serum BMP-2 and osteocalcin levels, and CT Hounsfield unit post hyperbaric oxygen therapy in patients with cleft lip and palate post alveolar bone graft: A case study

BackgroundThis study investigated the levels of bone morphogenetic protein 2 (BMP-2), osteocalcin, and 3D CT Hounsfield units following hyperbaric oxygen therapy (HBOT) in patients with cleft lip and palate (CLP) undergoing alveolar bone grafts to provide a pilot evaluation of the role of HBOT in osteogenesis. MethodsThis prospective, quasi-experimental, pre–post-intervention study evaluated seven patients with CLP receiving HBOT after single-stage reconstructions with alveolar bone grafts. The outcomes included the serum levels of BMP-2 and osteocalcin and the 3D CT Hounsfield units obtained before and after the surgery, and after the five HBOT sessions, to a total of 12 measurements. The data were analyzed with linear mixed-effects models using the intervention stage (pre-surgery, pre-HBOT, first to fifth HBOT sessions) as covariates and adjusting for several baseline factors. ResultsA significant difference was found in outcome measures across time (ANOVA p < 0.001 for BMP-2 and osteocalcin, p = 0.01 for Hounsfield units), with mean values appearing to steadily increase once HBOT began. Regression analyses indicated that the effect of HBOT was evident in serum osteocalcin after the 1st HBOT session (adjusted b = 1.32; 95% CI 0.39, 2.25) and in serum BMP-2 after the third session (adjusted b = 6.61; 95% CI 1.93, 11.28). After the fifth session, the HBOT effect was fairly pronounced on the two outcomes: the adjusted increase compared to the baseline was 28.06 ng/mL for BMP-2 and 6.27 ng/mL for osteocalcin. Our mixed-effect models also showed a post-HBOT increase in Hounsfield units. ConclusionWe found an increase of BMP-2, osteocalcin, and Hounsfield units following the HBOT intervention. These may suggest an effect of HBOT on osteogenesis.

Differences in levels of bone morphogenetic protein-2 in periodontitis patients with and without type 2 diabetes mellitus

Periodontitis is characterized by gingival bleeding, periodontal pocket formation, damage to the connective tissue attachment, and alveolar bone resorption. One of the risk factors that can increase the occurrence of periodontitis is diabetes mellitus (DM). In Indonesia, the incidence and prevalence of periodontitis are higher in patients with type 2 DM. In the present study, BMP-2 expression in periodontitis patients with and without type 2 DM was evaluated. We performed a descriptive analytical cross-sectional study in 40 respondents with stages II-III grades B-C chronic periodontitis. They were divided into a chronic periodontitis group with type 2 DM and a chronic periodontitis group without DM. Crevicular fluid of the gingival sulcus of the central maxillary anterior teeth from each respondent was taken using paper points for 5 minutes and placed in an Eppendorf tube. Each tube was labelled according to its group. All of the samples were examined by BMP-2 ELISA kit and measured by BioRad microplate reader with a wavelength of 450 nm. Data were analysed using SPSS 16.0 program. The independent sample t-test was used to compare the BMP-2 among two groups. The results of the study revealed the BMP-2 level in periodontitis patients with type 2 DM was 63.1 pg/ml, while in periodontitis patients without DM was 66.8 pg/ml. BMP-2 levels in periodontitis patients with type 2 DM were significantly lower than those in periodontitis patients without DM (p &lt; 0.05).

Exploring the Mechanism of Circ-vgll3 in Osteogenically Differentiated Human Bone Marrow Mesenchymal Stem Cells

Objective: To explore the mechanism of circRNA-vgll3 in osteogenic differentiation of human bone marrow mesenchymal stem cells. Methods: BMSCs cells were transfected with circRNA-vgll3, and divided into circRNA-vgll3 high-level group, circRNA-vgll3 low-level group, and negative control group (circRNA-vgll3 not transfected) according to the amount of transfection. The proliferation and apoptosis of BMSCs osteoblasts in each group were analyzed, and the alkaline phosphatase (ALP) activity, type I collagen gray value, bone morphogenetic protein 2 (BMP-2), Runx2 protein, and mRNA expression levels were detected. Results: The circRNA-vgll3 low-level group had a significant inhibitory effect on the proliferation of BMSCs osteoblasts, and the apoptosis rate of the circRNA-vgll3 low-level group was significantly higher than that of the circRNA-vgll3 high-level group ( P &lt; 0.05 ); ALP activity, type I collagen gray value, BMP-2, Runx2 protein, and mRNA expression levels in the high-level circRNA-vgll3 group were significantly higher than those in the low-level circRNA-vgll3 group, and the difference was statistically significant (P &lt; 0.05). Conclusion: Overexpression of circRNA-vgll3 can promote the osteogenic differentiation ability of BMSCs, while low expression of circRNA-vgll3 can inhibit the osteogenic differentiation ability of BMSCs. The main mechanism of action is that circRNA-vgll3 can affect osteogenic differentiation by regulating the Runx2 protein.

Different doses of vitamin D supplementation to nonsurgical treatment for vitamin-D-insufficient patients with diabetic periodontitis and the effect on gingival BMP-2 levels.

Epidemiological data show people with diabetes mellitus (DM) have three-fold increase in risk of periodontitis. A vitamin D insufficiency can affect the progression of DM and periodontitis. This study evaluated the effects of different-dose vitamin D supplementation to nonsurgical periodontal therapy for vitamin-D-insufficient diabetic patients coexisting with periodontitis and changes of gingival bone morphogenetic protein-2 (BMP-2) levels. The study included 30 vitamin-D-insufficient patients receiving nonsurgical treatment followed by administration of 25,000 international units (IU) vitamin D3 per week (the low-VD group) and 30 patients receiving 50,000 UI vitamin D per week (the high-VD group). The decreases of probing pocket depth, clinical attachment loss, bleeding index, and periodontal plaque index values of patients after the six-month supplementation of 50,000 UI vitamin D3 per week to nonsurgical treatment were more significant than those after the six-month supplementation of 25,000 UI vitamin D3 per week to nonsurgical treatment. It was found that 50,000 IU per week vitamin D supplementation for 6 months could lead to a better glycemic control for vitamin-D-insufficient diabetic patients coexisting with periodontitis after nonsurgical periodontal therapy. Increased levels of serum 25(OH) vitamin D3 and gingival BMP-2 were found in both low- and high-dose VD groups, and the high-dose VD group exhibited higher levels than the low-dose VD group. Vitamin D supplementation in large doses for 6 months tended to improve the treatment of periodontitis and increase gingival BMP-2 levels in diabetic patients coexisting with periodontitis who were vitamin D deficient.

Early adjunctive diagnostic value of contrast-enhanced ultrasound-related quantitative parameter and its relationship with micro-perfusion of nontraumatic necrosis of femoral head.

The aim of this study is to explore the early diagnostic value of contrast-enhanced ultrasound (CEUS)-related quantitative parameter and its relationship with the micro-perfusion of nontraumatic necrosis of the femoral head. According to the random and double-blind method, the patients with non-traumatic femoral head necrosis diagnosed and treated in our hospital from July 2019 to January 2022 were selected as the subjects (the research group). According to the staging of the International Society of Bone Circulation for Femoral Head Necrosis, 89 patients with stage Ⅱ and Ⅲ A were included (39 patients with stage Ⅱ and 50 patients with stage Ⅲ A). 25 patients who conducted physical examination in our hospital during the same time were taken as the control group. Quantitative parameters of CEUS were analyzed. The content of serum vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) were evaluated. The relationship among the quantitative parameters of CEUS, the expression of VEGF and BMP-2 in serum and the patient's condition, and the value for assisting the early diagnosis of nontraumatic femoral head necrosis were analyzed. The body mass, body mass index (BMI), blood lipid, and cholesterol levels were much higher in the research group than in the control group (p < 0.05). The research group had a markedly higher slope of ascending branch (AS), strength enhancement index (EI), and VEGF and obviously lower decay slope (DS), mean transit time (MTT), and time to peak (TTP) than the control group (p < 0.05). In the research group, compared to stage Ⅱ, the levels of AS, EI, and VEGF in stage Ⅲ A patients were memorably higher, and the levels of DS, MTT, TTP and BMP-2 were dramatically lower (p < 0.05). Pearson's correlation test showed that AS, EI, and VEGF were positively correlated with the patients' condition, while DS, MTT, TTP and BMP-2 were negatively correlated with the patients' condition (p < 0.05). The receiver operating characteristic (ROC) curve analysis showed that the diagnostic area under the curve (AUC) of quantitative parameters of CEUS was 0.961, with sensitivity and specificity of 88.0% and 97.4%, respectively. The AUC of the combined detection of VEGF and BMP-2 was 0.945 with sensitivity and specificity of 82.3% and 87.5%, respectively, and the combined detection had a high diagnostic value (p < 0.05). The quantitative parameters of CEUS were of great value in the early diagnosis of nontraumatic necrosis of the femoral head with microvascular perfusion and the patients' condition, and provided a reference for the clinical treatment of non-traumatic necrosis of the femoral head. These parameters were expected to be useful indicators for judging the efficacy before and after treatment.

Overexpression of microRNA-93-5p and microRNA-374a-5p Suppresses the Osteogenic Differentiation and Mineralization of Human Aortic Valvular Interstitial Cells Through the BMP2/Smad1/5/RUNX2 Signaling Pathway.

Aortic valve calcification commonly occurs in patients with chronic kidney disease (CKD). However, the regulatory functions of microRNAs (miRNAs/miRs) in the osteogenic differentiation of human aortic valvular interstitial cells (hAVICs) in patients with CKD remain largely unknown. This study aimed to explore the functional role and underlying mechanisms of miR-93-5p and miR-374a-5p in the osteogenic differentiation of hAVICs. For this purpose, hAVICs calcification was induced with high-calcium/high-phosphate medium and the expression levels of miR-93-5p and miR-374a-5p were determined using bioinformatics assay. Alizarin red staining, intracellular calcium content, and alkaline phosphatase activity were used to evaluate calcification. The expression levels of bone morphogenetic protein-2 (BMP2), runt-related transcription factor 2 (Runx2), and phosphorylated (p)-Smad1/5 were detected by luciferase reporter assay, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blot analysis. The results revealed that the expression levels of miR-93-5p and miR-374a-5p were significantly decreased in hAVICs in response to high-calcium/high-phosphate medium. The overexpression of miR-93-5p and miR-374a-5p effectively suppressed the high-calcium/high-phosphate-induced calcification and osteogenic differentiation makers. Mechanistically, the overexpression of miR-93-5p and miR-374a-5p inhibits osteogenic differentiation by regulating the BMP2/Smad1/5/Runx2 signaling pathway. Taken together, this study indicates that miR-93-5p and miR-374a-5p suppress the osteogenic differentiation of hAVICs associated with calcium-phosphate metabolic dyshomeostasis through the inhibition of the BMP2/Smad1/5/Runx2 signaling pathway.

Mechanism of the Notch1 signaling pathway regulating osteogenic factor influences lumbar disc calcification

To explore the mechanism of the Notch1 signaling pathway in regulating osteogenic factors and influencing lumbar disc calcification. Primary annulus fibroblasts from SD rats were isolated and subcultured in vitro. The calcification-inducing factors bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (b-FGF) were added to separate groups to induce calcification, which were referred to as the BMP-2 group and the b-FGF group, respectively. A control group was also set up, which was cultured in normal medium. Subsequently, cell morphology and fluorescence identification, alizarin red staining, ELISA, and quantitative real-time polymerase chain reaction (QRT-PCR) were performed to determine the effect of calcification induction. Cell grouping was performed again, including the control group, the calcification group (adding the inducer BMP-2), the calcification + LPS group(adding the inducer BMP-2 and the Notch1 pathway activator LPS), and the calcification + DAPT group (adding the inducer BMP-2 and the Notch1 pathway inhibitor DAPT). Alizarin red staining and flow cytometry were used to detect cell apoptosis, ELISA was used to detect the content of osteogenic factors, and Western blot was used to detect the expression of BMP-2, b-FGF, and Notch1 proteins. The induction factor screening results showed that the number of mineralized nodules in fibroannulus cells in BMP-2 group and b-FGF group was significantly increased, and the increase was greater in the BMP-2 group Meanwhile, ELISA and Western blot results showed that BMP-2, b-FGF and mRNA expression levels of BMP-2, b-FGF and Notch1 in the induced group were significantly increased (P<0.01). The results of the mechanism of Notch1 signaling pathway affecting lumbar disc calcification showed that compared to calcified group, the number of fibroannulus cell mineralization nodules, apoptosis rate, BMP-2, b-FGF content, the expression levels of BMP-2, b-FGF, and Notch1 proteins were further increased significantly However, the number of mineralization nodules, apoptosis rate, BMP-2 and b-FGF levels, BMP-2, b-FGF and Notch1 protein expression levels were decreased in the calcified +DAPT group (P<0.05 or P<0.01). Notch1 signaling pathway promotes lumbar disc calcification through positive regulation of osteogenic factors.

Inhibition of BMP4 alleviates diabetic retinal vascular dysfunction via the VEGF and smad1/5 signalling

Objective:The aim of our study was to determine the molecular mechanism of BMP4 (bone morphogenetic protein 4) in DR (diabetic retinopathy).Methods: Human retinal endothelial cell (HRECs) induced by high glucose to simulate one of the pathogenesis in the diabetic retinopathy (DR) model. RT-qPCR and western blot were used to detect the mRNA and protein levels of BMP4 in the STZ/HG group. Flow cytometry and TUNEL staining were performed to detect the apoptosis. Angiogenesis was evaluated by tube formation assay. Transwell assay and wound healing assay were used to detect cell migration ability. H&E staining was used to evaluate the pathological changes.Results: BMP4 was significantly upregulated in the STZ/HG group. Sh-BMP4 significantly inhibited the migration and angiogenesis of RVECs induced by HG. In addition, both in vivo and in vitro experiments confirmed that sh-BMP4 could significantly promote RVECs apoptosis in the HG/STZ group. Western blot results showed that sh-BMP4 could down-regulate the expressions of p-smad1, p-smad5 and VEGF.Conclusions: Inhibition of BMP4 could alleviate the damage of diabetic retinopathy by regulating the p-smad1/5/VEGF signaling axis, inhibiting angiogenesis and promoting apoptosis.

Effect of local application of bone morphogenetic protein -2 on experimental tooth movement and biological remodeling in rats.

Background: This study attempts to detect the potential effects of local bone morphogenetic protein -2 (BMP-2) on orthodontic tooth movement and periodontal tissue remodeling. Methods: Forty adult SD rats were randomly divided into four groups: blank control group, unilateral injection of BMP-2 on the pressure side or tension side of orthodontic teeth and bilateral injection of BMP-2. Their maxillary first molar was moved by a 30g constant force closed coil spring. 60μL of BMP-2 with a concentration of 0.5μg/mL was injected into each part at a time. In addition, three rats were selected as healthy control rats without any intervention. Fluorescent labeled BMP-2 was used to observe the distribution of exogenous BMP-2 in tissues. Micro-CT was used to measure the microscopic parameters of tooth displacement, trabecular bone and root absorption volume. Three different histological methods were used to observe the changes of tissue remodeling, and then the number of osteoclasts and the content of collagen fibers were calculated. Results: Compared with the blank control group, BMP-2 injection reduced the movement distance and increased the collagen fiber content and bone mass (p < 0.01). There was no significant difference in tooth movement distance, BV/TV ratio and BMD between injection sites in unilateral injection group (p > 0.05). In the case of bilateral injection of BMP-2, the osteogenesis is enhanced. Unilateral injection of BMP-2 did not promote root resorption, but double injection showed root resorption (p < 0.01). Conclusion: Our study does show that the osteogenesis of BMP-2 is dose-dependent rather than site-dependent when a certain amount of BMP-2 is applied around orthodontic teeth. Local application of BMP-2 around orthodontic teeth in an appropriate way can enhance bone mass and tooth anchorage without increasing the risk of root absorption volume. However, high levels of BMP-2 may cause aggressive root resorption. These findings are of great significance, that is, BMP-2 is an effective target for regulating orthodontic tooth movement.

MiR-20a promotes osteogenic differentiation in bone marrow-derived mesenchymal stem/stromal cells and bone repair of the maxillary sinus defect model in rabbits.

Introduction: This study aimed to determine whether miR-20 promoted osteogenic differentiation in bone marrow-derived mesenchymal stem/stromal cells (BMSCs) and accelerated bone formation in the maxillary sinus bone defect model in rabbits. Methods: BMSCs were transfected with miR-20a or anti-miR-20a for 12h, followed by detection of RUNX2, Sp7 mRNA, bone morphogenetic protein 2 (BMP2), and RUNX2 protein expression. Alkaline phosphatase (ALP) activity and Alizarin Red S staining were used to detect calcified nodule deposition. In the rabbit maxillary sinus bone defect model, miR-20a loaded with AAV and BMP2 protein were mixed with Bio-Oss bone powder for filling the bone defect. At 4weeks and 8weeks, bone density was detected by cone beam computed tomography (CBCT), and new bone, osteoblasts, and collagen type 1 were evaluated by hematoxylin and eosin (HE) staining and immunohistochemical (IHC) staining. Results: Overexpression of miR-20a enhanced the mRNA and protein levels of BMP2, RUNX2, and SP7, the activity of ALP, and the levels of matrix mineralization, whereas the levels and activity of the aforementioned factors were decreased by anti-miR-20a treatment of BMSCs. Furthermore, miR-20a significantly increased the bone density, the number of osteoblasts, and the secretion of collagen type 1 in bone defects compared with Bio-Oss bone powder in the rabbit maxillary sinus bone defect model. Conclusion: Overall, miR-20a can induce osteogenic differentiation in BMSCs and accelerate bone formation of maxillary sinus defects in rabbits.