Expression Of Bone Morphogenetic Protein Research Articles

Abstract 15554: Smooth Muscle Cells Specific Gene Inactivation of Lysyl Oxidase Reduces Atherosclerosis Burden and Plaque Calcification in Hyperlipidemic Mice

Aim: Dissect the mechanisms by which LOX increases atherogenesis and plaque calcification in hyperlipidemic mice. Methods: LOX conditional knockout [LOX f/f Myh11-CreER T2 ApoE -/- ] and control littermate [LOX wt/wt Myh11-CreER T2 ApoE -/- ] mice were injected with tamoxifen before high fat diet (HFD) feeding for atherosclerosis and calcification assessment and molecular analyses. Results: scRNA sequence showed full Lox gene inactivation in 72.5% of the aortic SMCs without any significant Loxl compensatory regulation. Gene deletion in SMCs decreased global arterial LOX deposition by ~3-fold in relation to control mice. Surprisingly, the loss of vascular LOX neither promoted aneurysm nor affected the aorta's stiffness. However, it reduced atherosclerosis burden (WT: 23±1 vs. KO: 13±2 %, p ≤0.01) and calcium deposition (11.8±3 versus 5±0.4%, p≤ 0.05) within the plaque after 16 weeks of high fat diet (HFD). Using bulk RNA sequencing, we further demonstrated that Lox gene inactivation leads to a marked decrease in Frizzled Related Protein-3 (FRZB) gene expression (127-fold and p≤0.0001), a biphasic modulator of the proatherogenic Wnt signaling that facilitates the association of β-catenin with TCF4 in the nucleus. The compromised FRZB /β-catenin/TCF4 signaling axis reduced the downstream production of Bone Morphogenetic Protein 2 (BMP2), which is responsible for the osteogenic transition of SMCs. Further scRNAseq analysis of atherosclerotic aortas from conditional KO and control mice also showed a reduction of BMP2 in SMCs. Interestingly, genetic inactivation of LOX increased the number of contractile SMCs (KO: 40 vs. WT: 7 %), characterized by the high expression of ACTA2 and TAGLN, in the aorta of HFD fed mice when compared to the control group. BAPN, an irreversible inhibitor of LOX activity, was not able to reduce BMP2 expression and protein levels nor reduce calcium deposition in vitro, suggesting that the pro-calcification effect of LOX is not activity dependent. Conclusions: Vascular gene inactivation of Lox protects mice against atherosclerosis by reducing β-catenin/TCF4/BMP2 proatherogenic signaling.

TRIM44 Promotes Endometrial Carcinoma Progression by Activating the FRS2 Signalling Pathway.

The Tripartite Motif Containing 44 (TRIM44) is highly expressed in a variety of tumours. However, the TRIM44's role in endometrial carcinoma (EC) progression remains unknown. To investigate the TRIM44's role in the development and metastasis of EC, we detected TRIM44 expression in EC cell lines and surgical specimens from patients with EC using immunohistochemistry, real-time reverse transcription-polymerase chain reaction, and western blotting analysis. The biological functions of TRIM44 by loss-of-function analysis in RL95-2 and Ishikawa cells were studied. The effect of TRIM44 on the progression of EC in terms of cell proliferation, apoptosis, and invasion was examined and revealed its underlying mechanism in vitro using EC cell lines and in vivo using mouse xenograft models. The TRIM44's expression was positively correlated with EC progression and poor prognosis. The TRIM44 knockdown reduced the EC cell proliferation and invasion while promoting cell apoptosis. Mechanism experiments showed that the TRIM44 interacts with Fibroblast Growth Factor Receptor Substrate 2 (FRS2) and negatively regulates the expression of Bone Morphogenetic Protein 4(BMP4), β-catenin, and Transforming Growth Factor Beta Receptor 1(TGF-βR1). Moreover, the effect of TRIM44 overexpression on EC cell proliferation, invasion, and apoptosis is reversed by the FRS2 knockdown. Our study may provide a new perspective on targeting the TRIM44/FRS2 signaling pathway in treating EC, which deserves further investigation.

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.

OR13-5 Bone Morphogenetic Protein-4 (BMP4) Inhibits Adrenal Cell Cortisol Synthesis by Blocking CYP17A1 and CYP11B1 Expression.

Abstract Background Cytochrome P450c17 (17α-hydroxylase, encoded by CYP17A1) and cytochrome P450c11 (11β-hydroxylase, encoded by CYP11B1) are essential for production of adrenal cortisol. The expression of CYP17A1 and CYP11B1 in adrenocortical cells is stimulated by adrenocorticotropic hormone (ACTH) through the cAMP and protein kinase A signal transduction pathway. Several growth factors can act as autocrine/paracrine regulators for modulating the steroidogenesis in the adrenal cortex. Among these factors, bone morphogenetic protein-4 (BMP4), a member of the superfamily of transforming growth factor-beta (TGFβ) has been previously reported to have an inhibitory effect on adrenal cell CYP17A1 expression and dehydroepiandrosterone (DHEA) production [1]. The role of BMP4 within the adrenal and its mechanisms regulating steroidogenesis are still unclear. In the present study, we examined human adrenal expression of BMP4 and its chronic effects on the H295R adrenal cell line. Objective To define the autocrine/paracrine role of BMP4 in adrenocortical zonation and adrenal steroidogenesis. Methods BMP4 expression in normal human adrenal glands was studied using microarray analysis with laser-captured adrenal capsule and cortical zones. Adrenal BMP4 protein expression was examined following immunohistochemistry (IHC) with a rabbit polyclonal antibody against human BMP4. In vitro studies were performed using H295R and human adrenal cells treated with or without ACTH (10nM), forskolin (10 µM) and BMP4 (50 ng/mL). The experimental medium was collected every 24 h and analyzed for cortisol. Experiments were terminated at 96 h followed by isolation of RNA and qPCR analysis for PPIA (normalization transcript), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), CYP17A1 and CYP11B1. Results Microarray analysis of adrenal zone RNA demonstrated a BMP4 expression gradient with zona glomerulosa (ZG) expressing 3 and 11-fold higher levels than the zona fasciculata (ZF) and zona reticularis (ZR), respectively. IHC confirmed that BMP4 expression was highest in the ZG and the outer ZF. Administration of BMP4 to cultures of H295R cells caused a significant decrease in basal and forskolin-stimulated cortisol production at 48 h. By 96 h, BMP4 inhibited forskolin-stimulated cortisol by 60%. Cortisol inhibition occurred in conjunction with an inhibition in forskolin-stimulated CYP17A1 (↓75%) and CYP11B1 (↓65%). However, CYP11A1 expression was unaffected. In primary cultures of human adrenal cells BMP4 inhibited the ability of ACTH to increase cortisol production (↓75%), as well as CYP11B1(↓65%) and CYP17A1 (↓57%) expression. Conclusion Our findings indicate that the human adrenal has outer cortex localized expression of BMP4 that could play a paracrine/autocrine role in zone-specific production of aldosterone vs. cortisol through BMP4 selective inhibition of CYP17A1 and CYP11B1. Bibliography: 1. Rege J, Nishimoto HK, Nishimoto K, Rodgers RJ, Auchus RJ, Rainey WE. Bone Morphogenetic Protein-4 (BMP4): A Paracrine Regulator of Human Adrenal C19 Steroid Synthesis. Endocrinology 2015; 156: 2530–2540. Presentation: Sunday, June 12, 2022 12:00 p.m. - 12:15 p.m.

Osteoclast and Sclerostin Expression in Osteocytes in the Femoral Head with Risedronate Therapy in Patients with Hip Fractures: A Retrospective Comparative Study.

Background and Objectives: The majority of research on the effects of osteoporosis drugs has measured the bone mineral density (BMD) of the spine and femur through dual-energy X-ray absorptiometry (DEXA) and compared and analyzed the effects of the drugs through changes in the BMD values. This study aims to compare osteoclast and sclerostin expression in osteocytes after risedronate therapy by obtaining femoral heads from patients with hip fractures. Materials and Methods: We obtained the femoral heads of 10 female patients (age: ≥65 years) who received risedronate therapy for at least 1 year through hip arthroplasty during 2019-2021 (risedronate group). Meanwhile, 10 patients who had never received osteoporosis treatment were selected as controls using propensity scores with age, body mass index, and bone density as covariates (control group). While the osteoclast count was evaluated using tartrate-resistant acid phosphatase (TRAP) staining, the sclerostin expression in osteocytes was assessed using immunohistochemistry. Moreover, Western blotting and polymerase chain reaction (PCR) were performed for receptor activation of nuclear factor kappa-Β ligand (RANKL), RANK, osteoprotegerin (OPG), sclerostin, and bone morphogenetic protein-2 (BMP2). Results: TRAP staining revealed significantly more TRAP-positive cells in the control group (131.75 ± 27.16/mm2) than in the risedronate group (28.00 ± 8.12/mm2). Moreover, sclerostin-positive osteocytes were expressed more in the control group (364.12 ± 28.12/mm2) than in the risedronate group (106.93 ± 12.85/mm2). Western blotting revealed that the expressions of RANKL, RANK, sclerostin, and BMP2 were higher in the control group than in the risedronate group (p < 0.05). Furthermore, RANK, sclerostin, and OPG protein levels were higher in the control group than in the risedronate group. Conclusions: In this study, the risedronate group demonstrated lower osteoclast activity and sclerostin expression in osteocytes in the femoral head than the control group.

Influence of donor age and comorbidities on transduced human adipose-derived stem cell in vitro osteogenic potential.

Human adipose-derived mesenchymal stem cells (ASCs) transduced with a lentiviral vector system to express bone morphogenetic protein 2 (LV-BMP-2) have been shown to reliably heal bone defects in animal models. However, the influence of donor characteristics such as age, sex, race, and medical co-morbidities on ASC yield, growth and bone regenerative capacity, while critical to the successful clinical translation of stem cell-based therapies, are not well understood. Human ASCs isolated from the infrapatellar fat pads in 122 ASC donors were evaluated for cell growth characteristics; 44 underwent additional analyses to evaluate in vitro osteogenic potential, with and without LV-BMP-2 transduction. We found that while female donors demonstrated significantly higher cell yield and ASC growth rates, age, race, and the presence of co-morbid conditions were not associated with differences in proliferation. Donor demographics or the presence of comorbidities were not associated with differences in in vitro osteogenic potential or stem cell differentiation, except that transduced ASCs from healthy donors produced more BMP-2 at day 2. Overall, donor age, sex, race, and the presence of co-morbid conditions had a limited influence on cell yield, proliferation, self-renewal capacity, and osteogenic potential for non-transduced and transduced (LV-BMP-2) ASCs. These results suggest that ASCs are a promising resource for both autologous and allogeneic cell-based gene therapy applications.

Effects of erythropoietin on osteoblast in the tooth extraction socket in mice periodontitis model.

Periodontitis is an excessive inflammatory event in tooth-supporting tissues and can cause tooth loss. We used erythropoietin (EPO), which has been reported to play an important role in bone healing and modulation of angiogenesis, as a therapeutic agent in vivo and in vitro experimental models to analyze its effect on periodontitis. First, EPO was applied to in vitro MC3T3-E1 cells and human periodontal ligament fibroblast (hPDLF) cells to examine its function in altered cellular events and gene expression patterns. In vitro cultivation of MC3T3-E1 and hPDLF cells with 10 IU/ml EPO at 24 and 48 h showed an obvious increase in cell proliferation. Interestingly, EPO treatment altered the expression of osteogenesis-related molecules, including alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OC) in MC3T3-E1 cells but not in hPDLF cells. In particular, MC3T3-E1 cells showed increased expression of ALP, BMP-2, and OC on day 5, while hPDLF cells showed increased expression of BMP-2, and OC on day 14. Based on the in vitro examination, we evaluated the effect of EPO on bone formation using an experimentally-induced animal periodontitis model. After the induction of periodontitis in the maxillary left second M, 10 IU/ml of EPO was locally applied to the extraction tooth sockets. Histomorphological examination using Masson’s trichrome (MTC) staining showed facilitated bone formation in the EPO-treated groups after 14 days. Similarly, stronger positive reactions against vascular endothelial growth factor (VEGF), cluster of differentiation 31 (CD31), runt-related transcription factor 2 (RUNX2), and osteocalcin (OC) were detected in the EPO-treated group compared to the control. Meanwhile, myeloperoxidase, an inflammatory marker, was decreased in the EPO-treated group on days 1 and 5. Overall, EPO facilitates bone healing and regeneration through altered signaling regulation and modulation of inflammation in the osteoblast cell lineage and to a lesser extent in hPDLF cells.

Non-vitamin K antagonist oral anticoagulants (NOACs) attenuate valvular calcification in patients with severe aortic stenosis

Abstract Background In vitro studies has demonstrated that non-vitamin K antagonist oral anticoagulants (NOACs) down-regulated the expression of proteins responsible for valve calcification and inflammation in valve interstitial cells (VICs) cultures. Aims Our goal was to evaluate whether long-term anticoagulation with NOACs, including dabigatran (thrombin inhibitor), rivaroxaban and apixaban (FXa inhibitors) can impair the valvular calcification and inflammation in patients with severe AS. Methods We enrolled 38 patients with severe AS aged 70±5.9 years (mean gradient - PGmean: 50mmHg, max gradient - PGmax: 80mmHg), including 18 individuals with AS and concomitant atrial fibrillation taking NOACs for at least 3 years (51±10 months) (AS-NOACs group). Stenotic aortic valves were obtained during valve replacement surgery. Valvular calcification was estimated ex vivo using micro-computed tomography (micro-CT). Total volume of each calcification (bone volume - BV), the distribution of clods sizes of calcification parts (trabecular thickness -Tbth) and the volume of the hardest / most calcified part (hard CT) of each valve were assessed. Valvular expression of bone morphogenetic protein 2 (BMP-2) and NFκB was evaluated by immunostaining. The fluorescence intensity (FI) was computed as the ratio (%) of positively and negatively stained areas. Results AS patients did not differ from AS-NOACs participants with regard to demographic or risk factors. Micro-CT analysis showed 65.5% lower BV in AS-NOACs valves compared to patients not taking NOACs (87±47 mm3 vs. 252±146 mm3, p=0.0017). Of note, in AS-NOACs patients Tbth showed strong association with transvalvular pressure gradients (PGmean: r=0.76, p=0.033; PGmax: r=0.77, p=0.01), as clinical measures of the AS severity. The duration of anticoagulation was associated with the lower Tbth (from 0.35 mm for 60 months to 0.51 mm for 36 months, p<0.01), which suggests that long NOACs treatment changed the pattern of valvular calcification, leading to formation of calcium deposits with reduced volume. Moreover, in AS-NOACs patients BV correlated with hard CT (r=−0.69, p=0.027), suggesting that the lesions increase their volume but do not ossify. Valvular expression of BMP-2 and NFκB was 44% and 33% decreased in AS-NOACs patients, compared to those without such treatment and the expression of both proteins was positively associated (r=0.34, p=0.001). Moreover, in AS-NOACs patients the expression of BMP-2 and NFκB was observed mainly in the fibrosa layer of the leaflets, but not in the deeper layers of tissue, as in AS patients without NOACs treatment. Conclusions NOACs can inhibit the increase of valvular calcium volume and its ossification via suppressed NFkB expression. Our study may suggest that long-term anticoagulation with NOACs could slow down the rate of AS progression, at least in patients with an indication for anticoagulant therapy. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Polish National Science Centre

Iron-dependent BMP6 Regulation in Liver Sinusoidal Endothelial Cells Is Instructed by Hepatocyte-derived Secretory Signals.

Iron-dependent BMP6 Regulation in Liver Sinusoidal Endothelial Cells Is Instructed by Hepatocyte-derived Secretory Signals.

RNA-Sequence Reveals the Regulatory Mechanism of miR-149 on Osteoblast Skeleton under Mechanical Tension.

Objective Based on RNA-sequencing (RNA-seq), the regulation of miRNAs differentially expressed in dental, periodontal, and alveolar bone tissue of orthodontic tree shrews on osteoblast skeleton under tension was investigated. Methods Tree shrews were used to construct orthodontic models. We used RNA-seq to identify differentially expressed miRNAs in periodontal tissues of the treatment group and control group tree shrews. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for enrichment analysis. Human osteoblast MG63 was treated with 5000 U mechanical tension. Real-time quantitative polymerase chain reaction (RT-qPCR) detected the expression of miR-149 and ARFGAP with SH3 domain, Ankyrin repeat, and Ph domain 3 (ASAP3) mRNA. Western blot detected the protein levels of ASAP3, F-actin, osteogenic markers bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (RUNX2). Rhodamine phalloidin was used to observe the fluorescence intensity of F-actin. Validation of the targeting relationship between miR-149 and ASAP3 by dual luciferase reporter gene assay. Results By performing miRNA-seq analysis on the dental and periodontal tissue of tree shrews in the treatment group and control group, we identified 51 upregulated miRNAs and 13 downregulated miRNAs. The expression of miR-149 in the dental and periodontal tissue of tree shrew and MG63 cells treated with mechanical tension was decreased, and miR-149 targeted ASAP3. Knockdown of ASAP3 inhibited the fluorescence intensity of F-actin in MG63 cells treated with 5000 U tension for 36 h, and overexpression of ASAP3 promoted the expression of F-actin and osteogenic markers BMP2 and RUNX2. Conclusions These findings revealed that miR-149 could modulate osteoblast differentiation under orthodontics mechanical tension through targeting ASAP3.

Comparison of Selected Non-Coding RNAs and Gene Expression Profiles between Common Osteosarcoma Cell Lines.

Simple SummaryOsteosarcoma (OS) is a malignant tumour affecting mainly children and elderly people. Despite significant advances in cancer medicine, osteosarcoma patients’ survival is not improving. The primary treatment methods are established using in vitro models that rely upon the application of well-established cell lines, including U-2 OS, Saos-2 and MG-63. The molecular phenotype of these cell lines is still not fully outlined. Therefore, our study aimed to establish the expression profile of molecular markers related to osteosarcoma survival, progression and metastasis. Non-bone-related cells were used as a reference, i.e. HeLa cell line and human adipose-derived stromal cells (hASCs). Evaluated osteosarcoma cell lines showed characteristic phenotypes with unique patterns related to upregulation of MMP-7, MMP-14, BMP-7, miR-21-5p, miR-124-3p and downregulation of lncRNA MEG3. Our findings may facilitate the selection of the most reliable cellular model for pre-clinical investigations focused on developing new and satisfying methods of osteosarcoma therapy.Osteosarcoma (OS) is a bone tumour affecting adolescents and elderly people. Unfortunately, basic treatment methods are still underdeveloped, which has a high impact on the poor survivability of the patients. Studies designed to understand the underlying mechanisms of osteosarcoma development, as well as preclinical investigations aimed at establishing novel therapeutic strategies, rely significantly upon in vitro models, which apply well-established cell lines such as U-2 OS, Saos-2 and MG-63. In this study, the expression of chosen markers associated with tumour progression, metastasis and survival were identified using RT-qPCR. Levels of several onco-miRs (miR-21-5p, miR-124-3p, miR-223-3p and miR-320a-3p) and long non-coding RNA MEG3 were established. The mRNA expression of bone morphogenetic proteins (BMPs), including BMP-2, BMP-3, BMP-4, BMP-6, BMP-7, as well as their receptors: BMPR-IA, BMPR-IB and BMPR-II was also determined. Other tested markers included metalloproteinases, i.e., MMP-7 and MMP-14 and survivin (BIRC5), C-MYC, as well as CYCLIN D (CCND1). The analysis included comparing obtained profiles with transcript levels established for the osteogenic HeLa cell line and human adipose-derived stromal cells (hASCs). The tested OS cell lines were characterised by a cancer-related phenotype, such as increased expression of mRNA for BMP-7, as well as MMP-7 and MMP-14. Osteosarcoma cells differ considerably in miR-21-5p and miR-124-3p levels, which can be related to uncontrolled tumour growth. The comprehensive examination of osteosarcoma transcriptome profiles may facilitate the selection of appropriate cell models for preclinical investigations aimed at the development of new strategies for OS treatment.

Smoking Affects Bone Healing and Blood Perfusion

Nicotine is the primary component in cigarettes that is responsible for the negative effects that tobacco has on the body. Nicotine is also the primary component responsible for the addiction that can develop from smoking. Tobacco addiction is also primarily caused by nicotine, which is another component of tobacco. Aim: The effect that nicotine has on the rate at which bones can recover after being shattered is still a topic of ongoing debate. The study of bone repair and regeneration is made a great deal easier by the utilisation of a model that is known as distraction osteogenesis. The mandibular extension of a rabbit model is serving as the subject of the inquiry that is now being conducted. Duration & Place of Study: October 2018 to March 2021 Rashid Latif medical college Lahore Method: Using the rabbit as a test subject, the purpose of this study is to evaluate the influence that nicotine has on blood perfusion, angiogenesis, and bone formation. There were a total of forty rabbits, and they were distributed among the control group, which received no nicotine, and the nicotine group, which received nicotine. The bunnies were distributed to the several groups in a haphazard manner. It was discovered that each rat had been exposed to nicotine or a placebo for a total of seven weeks during the course of the experiment. After a period of consolidation time ranging from two to four weeks post osteotomy, the animals were slaughtered and their mandibles were removed. After this, there was a lag time of three days, and then there was active distraction for eleven days. Utilizing the techniques of Laser Doppler monitoring and Collagen IV immunohistochemical labelling, respectively, allowed for the assessment of both vascularization and blood perfusion. Studies that were radiographic, histological, and immunohistochemical in nature were carried out in order to investigate the development of bone. Result : According to the findings, being exposed to nicotine led to an increase in the density of microvessels, but it had a detrimental effect on the flow of blood and the development of bones. In addition to this, it was found that the expression of bone morphogenetic protein (BMP)-2 in osteoblasts was lower than it had been in the past. In the process of distraction regeneration, ischemia and a low oxygen tension were both present, and the creation of cartilage islands on a consistent basis was a sign of both of these circumstances. Result: As a result of our investigation, we got to the realisation that nicotine consumption slows down the process of bone regeneration. We hypothesise that this takes place as a result of the fact that nicotine leads to ischemia and has an effect that is directly inhibitory on osteoblastic cells. It has been demonstrated that nicotine causes an increase in angiogenesis; however, this improvement is not adequate to compensate for the harmful effect of vasoconstriction caused by nicotine.

The regulation of proliferation and apoptosis in hepatocellular carcinoma via insulin-like growth factor 1 receptor

ObjectivesInsulin-like growth factor 1 receptor (IGF-1R) is a transmembrane tyrosine kinase receptor of the insulin receptor family. Its expression is consistently increased in hepatocellular carcinoma (HCC) tissue, and it participates in hepatic carcinogenesis. Targeting IGF-1R may be a potential therapeutic approach against hepatocellular carcinoma. This study therefore aimed to explore the effect of IGF-1R on hepatocellular carcinoma cells. MethodsIGF-1R silencing cell lines were established by small-interfering RNAs in hepatocellular carcinoma cell line SMMC7721, after which the proliferation, invasion, and apoptosis of SMMC7721 was evaluated. The activation of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and the expression of bone morphogenetic protein (BMP)-2 and BMP-7 were measured using Western blot analysis. ResultsThe results indicated that the knockdown of IGF-1R can inhibit the proliferation and invasion of HCC and promote the apoptosis of SMMC7721 by inhibiting the PI3K/AKT signaling pathway. Furthermore, depletion of IGF-1R was found to suppress the expression of BMP-2 and BMP-7. ConclusionsThe findings suggest that IGF-1R plays an important role in the progression of HCC. Therefore, IGF-1R is a potential target for the treatment of HCC in clinic.

Chronic Alcohol Reduces Bone Mass Through Inhibiting Proliferation and Promoting Aging of Endothelial Cells in Type-H Vessels

Alcohol consumption is regarded as one of the leading risk factors for secondary osteopenia. Angiogenesis and osteogenesis coupled by type-H vessels coordinate the biological process of bone homeostasis to prevent osteopenia. This study aimed to determine whether chronic alcohol inhibits type-H vessel-dependent bone formation. Two-month-old mice were fed with 5% (v/v) alcohol liquid diet (28% of calories) or normal liquid diet every day for 2 months. The tibias were isolated and detected with X-ray and microcomputed tomography. Paraffin-embedded or frozen tibial sections were prepared and used for immunohistochemical or immunofluorescence staining, respectively. Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of alcohol, including 0 mM (0%), 8.7 mM (0.5%), 52 mM (3%), or 87 mM (5%) alcohol for 12 h. The conditioned medium of the above HUVEC cells was collected to culture human bone marrow-mesenchymal stem cells (BM-MSCs), which were induced to differentiate into osteoblasts in vitro. The alcoholic diet retarded the bone growth and led to osteoporosis, impaired bone formation of osteoblasts, and decreased CD31hiEMCNhi type-H vessel formation through inhibiting proliferation and promoting aging of endothelial cells in mice. Alcohol treatment obviously increased the expression of p16, while significantly decreased the expression of Bmi-1, CDK6, Cyclin D, E2F1, and bone morphogenetic protein (BMP)2 compared with vehicle. Alcohol inhibited the differentiation of BM-MSCs into osteoblasts through reducing the BMP2 secretion of endothelial cells in type-H vessels. Alcoholic diet impaired CD31hiEMCNhi type-H vessel formation through inhibiting proliferation and promoting aging of endothelial cells through Bmi-1/p16 signaling, and inhibited the differentiation of BM-MSCs into osteoblasts through reducing the BMP2 secretion of endothelial cells in type-H vessels. This study provides a basis for developing a new treatment strategy targeting aging endothelial cells of type-H vessel to prevent alcoholic osteopenia.

Effects of Recombinant Adeno-Associated Virus-Mediated Overexpression of Bone Morphogenetic Protein 3 on the Chondrogenic Fate of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Implantation of genetically modified chondrogenically competent human bone marrow-derived mesenchymal stromal cells (hMSCs) is an attractive strategy to improve cartilage repair. The goal of this study was to examine the potential benefits of transferring a sequence coding for the bone morphogenetic protein 3 (BMP-3) that modulates bone and cartilage formation, using recombinant adeno-associated virus (rAAV) vectors on the chondroreparative activities of hMSCs. Undifferentiated and chondrogenically induced primary human MSCs were treated with an rAAV-hBMP-3 construct to evaluate its effects on the proliferative, metabolic, and chondrogenic activities of the cells compared with control (reporter rAAV-lacZ vector) condition. Effective BMP-3 expression was noted both in undifferentiated and chondrogenically differentiated cells in the presence of rAAV-hBMP-3 relative to rAAV-lacZ, stimulating cell proliferation and extracellular matrix (proteoglycans, type-II collagen) deposition together with higher levels of chondrogenic sex-determining region Y-type high-mobility group box 9 (SOX9) expression. rAAV-hBMP-3 also advantageously decreased terminal differentiation, hypertrophy, and osteogenesis (type-I/-X collagen and alkaline phosphatase expression), with reduced levels of osteoblast-related runt-related transcription factor 2 (RUNX-2) transcription factor and β-catenin (osteodifferentiation mediator) and enhanced parathyroid hormone-related protein expression (inhibitor of hypertrophic maturation, calcification, and bone formation). This study shows the advantage of modifying hMSCs with rAAV-hBMP-3 to trigger adapted chondroreparative activities as a source of improved cells for transplantation protocols in cartilage defects.

Role of molecular and chemical chaperon in assisting refolding of BMP2 in E. coli

Bone morphogenetic protein 2 (BMP2) when expressed in bacteria forms inclusion bodies (IBs) due to its complex disulfide-rich structure. Chaperons are already well known for their role in assisting protein folding. In our studies, we have used two E. coli strains, BL21(DE3) and SHuffle® T7 cells for overexpressing BMP2 in soluble fraction. We observed that SHuffle® T7 cells successfully expressed soluble functionally active BMP2 in presence of molecular and chemical chaperones at low temperature. The combination of chemical and molecular chaperons further increases the yield of protein. The best-suited chaperon system for overexpression of BMP2 is GroES-GroEL at low temperature. The soluble functionally active BMP2 is confirmed by its binding to its receptor ALK3 through Native PAGE and ELISA assay using BMP2 specific antibody. It is possible to obtain BMP2 expression in soluble active form by using molecular and chemical chaperons which work synergistically in bacterial cells to fold disulphide-rich proteins at low temperature in easy and time saving steps (18 ̊C).

Locally Delivered 1.2% Simvastatin Gel and 1% Metformin Gel in Chronic Periodontitis Patients

Background: This study was carried out to investigate the effectiveness of 1.2% Simvastatin gel and 1% Metformin gel in chronic periodontitis pockets. The rationale behind using Statins is that this class of drugs has a potential anti-inflammatory effect on oral epithelial cells, blocking the intermediate metabolites of the mevalonate pathway. Statins also modulate bone formation by increasing the expression of bone morphogenetic protein-2 providing a new direction in the field of periodontal therapy. Statin administration decreases GCF levels of pro-inflammatory mediators which are responsible for much of the host tissue destruction seen in periodontitis. Metformin acts at molecular level via both AMPK (5’adenosine monophosphate activated protein kinase) dependent & AMPK independent pathways. It shows effects on Nuclear Factor-κB (NF-κB), differentiation of monocytes into macrophages as well as suppressing IL-2, interferon (IFN)-γ and TNF-α from these macrophages which thereby improved the periodontal clinical parameters. There is no evidence of literature till now comparing the clinical efficacy of 1.2% Simvastatin gel with 1% Metformin gel in patients with chronic periodontitis. Method: The study population consisted of 48 sites from chronic periodontitis patients, divided into 3 groups which received sub gingival irrigation with Scaling and root planning alone, SRP with 1.2% Simvastatin gel and SRP with1% Metformin gel. Results: 1.2% Simvastatin gel and 1% Metformin gel improve the periodontal health with statistically significant improvement in the PPD, RAL, RGML values as compared to SRP alone. Conclusion: LDD systems using gel formulations are advantageous because of their sustainability, prolonged release, stability in the pockets, clinical effect of 1.2% Simvastatin gel and 1% Metformin gel improves the periodontal health compared to SRP alone.

Guided bone regeneration with extracellular matrix scaffold of small intestinal submucosa membrane.

Guided bone regeneration (GBR) is a promising strategy for repairing bone defects using bioactive membranes. In this study, a new type of GBR membrane based on the small intestinal submucosa (SIS) was created, and its surface structure, cytological characteristics, and bone defect repair ability were compared with commonly used membranes. Our results show that compared to the Heal-all and Dentium membranes, the SIS membrane has an asymmetric structure that does not affect the proliferation of bone marrow mesenchymal stem cells (BMSCs). Instead, it increased their formation of calcium nodules and expression of bone morphogenetic protein-2 (BMP-2), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteopontin (OPN). Six weeks after their insertion into a rat calvarial defect model, increased bone growth was observed in the SIS membrane group. Our results indicate that the SIS membrane has good biocompatibility and is more effective in promoting early bone formation than existing membranes. Given the wide range of source materials and simple preparation processes available, SIS membrane is a promising candidate for guided bone regeneration.

Improved repair of rabbit calvarial defects with hydroxyapatite/chitosan/polycaprolactone composite scaffold-engrafted EPCs and BMSCs.

Endothelial progenitor cells (EPCs) expressing vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) and bone marrow mesenchymal stem cells (BMSCs) expressing endogenous bone morphogenetic protein-2 (BMP-2) play the important role in new bone formation. This study investigated the effects of a porous hydroxyapatite (HA)/chitosan (CS)/polycaprolactone (PCL) composite scaffold-engrafted EPCs and BMSCs on the expression of BMP-2, VEGF, and PDGF in the calvarial defect rabbit model in vivo. It showed that a three-dimensional composite scaffold was successfully constructed by physical interaction with a pore size of 250 μm. The HA/CS/PCL scaffold degraded slowly within 10 weeks and showed non-cytotoxicity. By X-ray, micro-CT examination, and H&E staining, compared with the HA/CS/PCL group, HA/CS/PCL + EPCs, HA/CS/PCL + BMSCs, and HA/CS/PCL + EPCs + BMSCs groups performed a more obvious repair effect, and the dual factor group presented particularly significant improvement on the percentages of bone volume at week 4 and week 8, with evident bone growth. Osteogenesis marker (BMP-2) and vascularization marker (VEGF and PDGF) expression in the dual factor group were much better than those of the HA/CS/PCL control group and single factor groups. Collectively, the HA/CS/PCL composite scaffold-engrafting EPCs and BMSCs is effective to repair calvarial defects by regulating endogenous expression of BMP-2, VEGF, and PDGF. Thus, this study provides important implications for the potential clinical application of biomaterial composite scaffold-engrafted engineering cells.

Value of 3D Printed PLGA Scaffolds for Cartilage Defects in Terms of Repair.

Objective To examine the poly (lactic-co-glycolic acid) and sodium alginate (SA) scaffolds produced by 3D printing technology, access the healing morphology of bones following PLGA/SA implantation within rat cartilage, and examine osteogenesis-related factors in rat serum to determine the efficacy of PLGA/SA scaffolds in healing animal cartilage injuries. To identify the potential of this material to repair a tissue engineering osteochondral injury. Methods Polylactic acid-glycolic acid copolymer and sodium alginate were used as raw materials to create PLGA/SA scaffolds. We observed the scaffold's macrostructure and microstructure, and the scaffold's microstructure was observed through a scanning electron microscope (SEM). The mechanical toughness of a stent was assessed using a biomechanical device. Hematoxylin-eosin staining revealed immune rejection after embedding the scaffolds under the skin of SD rats. The CCK-8 cell proliferation test kit was used to measure cell proliferation. An experimental model of cartilage injury in the knee joint was created in rats. Rats were used to establish an experimental model of cartilage damage in the knee joint. 120 female rats aged 5 weeks were chosen at random from the pool and divided into the experimental and control groups. They were all completely anesthetized with an anesthetic before having the lateral skin of the knee articular cartilage incised. Implanted PLGA/SA scaffolds were not used in the control group and only in the experiment group. Both groups of rats had their muscles and skin sutured and covered in plaster bandages. On the third, seventh, fourteenth, twenty-first, twenty-eighth, and thirty-fifth days after the procedure, the two groups of rats were divided into groups. At various stages, bone tissue, blood samples, and cartilage were examined and evaluated. Immunohistochemistry was used to identify the local bone morphogenetic protein-2 (BMP2). Results (1) PLGA/SA was successfully used to build an artificial cartilage scaffold. (2) Macroscopic and SEM observation results showed the material had increased density and numerous microvoids on the surface. (3) The result of the biomechanical test showed that the PLGA/SA scaffold had superior biomechanical characteristics. (4) The stent did not exhibit any noticeable immunological rejection, according to the results of the subcutaneous embedding experiment performed on rats. (5) The CCK-8 data demonstrated that as the cell development time rose, the number of cells gradually increased. However, there was not statistically significant difference between the growth of the cells in the scaffold extract and the control group (P > 0.05). (6) A successful rat model based on a cartilage defect of the medial knee joint has been built. (7) Observations of specimens revealed that the experimental group's bone tissue score was higher than that of the control group. (8) Using immunohistochemistry, it was found that the experimental group's BMP2 expression was higher on the 7th, 14th, and 28th days than it was in the control group (P < 0.05). Conclusion Strong mechanical and biological properties are present in stable, biodegradable PLGA/SA scaffolds that mimic cartilage. We demonstrated that the cartilage biomimetic PLGA/SA scaffold may repair cartilage and prevent negative reactions such as osteoarthritis in rat knee cartilage, making it suitable as a cartilage scaffolding material for tissue engineering. The PLGA/SA scaffold was also able to promote BMP2 expression in the bone healing zone when inserted into a knee cartilage lesion. Improved cartilage damage is the outcome of BMP2's promotion of bone formation and restriction of bone resorption in the bone healing zone.