ABSTRACT
ObjectiveTo observe the effects of Youguiwan on bone metabolism and bone morphogenetic protein-2 （BMP-2）/Smad signaling pathway in ovaries-removed rats with osteoporosis and study the mechanism of Youguiwan in the prevention and treatment of osteoporosis. MethodA postmenopausal rat model of osteoporosis was prepared by bilateral ovariectomy. The 40 female SD rats were randomly divided into five groups， including sham operation group， model group， alendronate sodium group （0.1 mg·kg-1）， and high-dose and low-dose （5.36 and 2.68 g·kg-1） groups of Youguiwan. The drug was given seven days after modeling， once a day for 12 weeks. After the treatment， the changes in femur tissue structure were observed by micro-CT， including bone mineral density （BMD）， bone volume/total volume （BV/TV）， trabecular number （Tb.N）， trabecular thickness （Tb.Th）， bone surface/bone volume （BS/BV）， and trabecular separation （Tb.Sp）. Ossification was observed by saffrane-solid green staining， and serum levels of bone metabolism markers， including bone alkaline phosphatase （BALP）， osteocalcin （BGP）， type Ⅰ procollagen amino terminal propeptide （PINP）， and tartrate-resistant acid phosphatase 5b （TRACP-5b）， were determined by enzyme-linked immunosorbent assay （ELISA）. The protein and mRNA expression levels of Runx2， BMP-2， and Smad1 in rat femur were detected by Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the sham operation group， bone trabecula in the model group was sparse. BMD， BV/TV， Tb.N， and Tb.Th were decreased （P<0.05， P<0.01）. BS/BV （P<0.05） and Tb.Sp were increased. The content of BGP， BALP， PINP， and TRACP-5b in serum was significantly increased （P<0.01）. The mRNA and protein expressions of Runx2， BMP-2， and Smad1 in rat femur were significantly decreased （P<0.05， P<0.01）. Compared with the model group， the number of bone trabeculae in the high-dose and low-dose groups of Youguiwan was increased， and the bone microstructure was improved. BMD， BV/TV， Tb.N， and Tb.Th were increased significantly （P<0.05， P<0.01）， and BS/BV and Tb.Sp were increased. The content of bone metabolic markers decreased （P<0.05， P<0.01）. ConclusionYouguiwan has certain preventive and therapeutic effects on postmenopausal osteoporosis， and its mechanism may be related to promoting bone formation by regulating the BMP-2/Smad signaling pathway.
ABSTRACT
Berberine is a phytocompound from plants viz. Phellodendri cortex and Coptis rhizome, used to treat a variety of diseases. It is effective in preventing osteoporosis, but it is less effective than drugs currently used in clinical practice. In this study, we used a novel berberine derivative, WJCPR11, to promote osteoblast differentiation and to investigate its use in the prevention and treatment of osteoporosis. WJCPR11 at a safe concentration without toxicity increased alkaline phosphatase (ALP) activity induced by bone morphogenetic protein 2 (BMP2) dose-dependently. The mRNA expression of ALP, osteocalcin (OC), runt-related transcription factor 2 (Runx2), and osterix was increased, with the ALP level increasing the most. In addition, the protein abundance of bone sialoprotein (BSP), collagen, type I, alpha 1, Runx2, and osterix were also increased. Moreover, the transcriptional activity of ALP, BSP, and OC was increased by WJCPR11, with OC showing the most significant increase. The results indicate that osteoblast differentiation is promoted by WJCPR11, and it could play a role in the prevention of osteoporosis.
ABSTRACT
Objective:To explore the mechanism of bone morphogenetic protein 2 (BMP-2) regulating pulmonary vascular remodeling in pulmonary hypertension (PH).Methods:Pulmonary artery smooth muscle cells (PASMC) groups: control group, PH group, PH+BMP-2 group, PH+BMP-2+ small interfering BMP receptor(si-BMPR)-Ⅰa group, PH+BMP-2+ si-BMPR-Ⅰb group, PH+BMP -2+si-BMPR-Ⅱ group. In vitro PH model was induced by hypoxia. The three BMP-2 receptors were silenced by the transfection of si-BMPR-Ⅰa, si-BMPR-Ⅰb and si-BMPR-Ⅱ plasmids, respectively. Cell proliferation and apoptosis in each group were detected, transient receptor potential ion channel C1/6 (TRPC1/6), p21 mRNA and protein levels, and intracellular Ca 2+ concentration were detected. Results:The intracellular Ca 2+ concentration in the PH group was higher than that in the control group: (785.15 ± 44.26) nmol/L vs. (224.15 ± 15.87) nmol/L, the and apoptosis rate was lower than that in the control group: (3.15 ± 0.22)% vs. (7.31 ± 0.45)%, there were statistical differences ( P<0.05). The intracellular Ca 2+ concentration in the PH+BMP-2 group was (297.64 ± 21.46) nmol/L, and was lower than that in the PH group, and apoptosis rate was (6.88 ± 0.75)%, and was higher than that in the PH group, there were statistical differences ( P<0.05). The intracellular Ca 2+ concentration in the PH+BMP-2+si-BMPR-Ⅰa group, PH+BMP-2+ si-BMPR-Ⅰb group, PH+BMP -2+si-BMPR-Ⅱ group was (412.31 ± 29.57), (384.34 ± 30.66), (695.23 ± 39.85) nmol/L, and was higher than that in the PH+BMP-2 group, and apoptosis rate was (4.10 ± 0.27)%, (4.26 ± 0.28)%, (3.33 ± 0.24)%, and was lower than that in the PH+BMP-2 group, there were statistical differences ( P<0.05). The intracellular Ca 2+ concentration in the PH+BMP -2+si-BMPR-Ⅱ group was higher than that in the PH+BMP-2+si-BMPR-Ⅰa group and PH+BMP-2+ si-BMPR-Ⅰb group, the apoptosis rate was lower than that in the PH+BMP-2+si-BMPR-Ⅰa group and PH+BMP-2+ si-BMPR-Ⅰb group, there were statistical differences ( P<0.05). Conclusions:BMP-2 mainly inhibits the expression of TRPC1/6 by interacting with the receptor BMPR-Ⅱ, inhibits the influx of Ca 2+ and promotes the expression of p21, thereby inhibiting the proliferation of PASMC and promoting apoptosis, participating in pulmonary vascular remodeling in PH.
ABSTRACT
ObjectiveTo investigate the mechanism of Xiaonang Tiaojing decoction（XNTJD）in improving polycystic ovary syndrome with insulin resistance（PCOS-IR）model rats based on anti-Müllerian hormone（AMH）/AMH type Ⅱ receptor（AMHRⅡ）signaling pathway. MethodForty-eight adult female SD rats were randomly divided into the blank group， model group， XNTJD group（11.4 g·kg-1·d-1） and Diane-35 group（0.21 g·kg-1·d-1）， PCOS-IR model was established by high-fat and high-sugar diet combined with letrozole in rats of all groups except the blank group， rats in the administration groups were given the corresponding dose of drugs by gavage for 15 days with an interval of 1 d every 4 d， normal saline of the same volume was given to the blank group and the model group. Estrous cycle was recorded daily during treatment. At the end of treatment， body weight and Lee's index were recorded， AMH， luteinizing hormone（LH）， LH/follicle stimulating hormone（FSH）， testosterone（T）and estradiol（E2）levels were measured by enzyme-linked immunosorbent assay（ELISA）， fasting plasma glucose（FPG）was measured by glucometer， fasting insulin（FINS） level was measured by radioimmunoassay（RIA）， and the insulin resistance index（HOMA-IR） and insulin sensitivity index（QUICKI）were calculated， triglyceride（TG）and total cholesterol（TC）levels were measured by automatic biochemical analyzer， hematoxylin-eosin（HE）staining was used to observe the morphological changes of the ovary， the levels of AMHRⅡ， bone morphogenetic protein-15（BMP-15）and Smad5 in ovarian tissues were detected by immunohistochemistry（IHC），Western blot was used to analyze the protein expression levels of AMHRⅡ， BMP-15 and Smad5. ResultCompared with the blank group， a large number of leukocytes were observed in the vaginal exfoliated cells of rats in the model group， mainly in diestrus， the levels of body weight， Lee's index， glucose-lipid metabolism indexes（FPG， FINS， HOMA-IR， TG， TC）， AMH and sex hormones（LH， LH/FSH， T）were significantly increased（P<0.01）， and QUICKI and E2 levels were significantly decreased（P<0.01）， there were more cystic bulges on the ovarian surface， more wet weight， more atretic and cystic dilated follicles in the ovarian tissues， and the thickness of granulosa cell layer was reduced without oocytes， the expression level of AMHRⅡ protein in ovarian tissues was significantly increased（P<0.01）， and the expression levels of BMP-15 and Smad5 proteins were significantly decreased（P<0.01）. Compared with the model group， the exfoliated cells in the vagina of rats treated with XNTJD group showed keratinocytes from the 5th to 6th day of treatment， and a stable estrous cycle gradually appeared， body weight， Lee's index， glucose-lipid metabolism indexes（FPG， FINS， HOMA-IR， TG， TC）， AMH and sex hormones（LH， LH/FSH， T）levels were significantly decreased（P<0.05， P<0.01）， QUICKI and E2 levels were significantly increased（P<0.01）， ovarian surface was smoother and lighter in wet weight， oocytes and mature follicles were observed in ovarian tissues， the thickness of granulosa cell layer increased and the morphology was intact， the expression levels of BMP-15 and Smad5 proteins were significantly increased（P<0.01）and the expression level of AMHRⅡ protein was significantly decreased（P<0.01）in ovarian tissues. ConclusionXNTJD may mediate the up-regulation of BMP-15 and Smad5 in ovarian tissues of PCOS-IR rats by down-regulating AMH/AMHRⅡ， thereby improving ovarian function， sex hormones and glucose-lipid metabolism levels in PCOS-IR rats.
ABSTRACT
Objective@# To investigate the osteogenic effect of β-tricalcium phosphate (β-TCP) and bone morphogenetic protein-2 (BMP-2) in the repair of the alveolar cleft.@*Methods @# Fifty-nine patients with unilateral alveolar cleft who visited Capital Medical University School of Stomatology from January 2016 to May 2021 were included. They were divided into three groups according to the different bone repair materials: autologous bone, β-TCP and BMP-2 +β-TCP. The preoperative and postoperative CBCT data of the patients were imported into Mimics 21.0 software. The preoperative volume of the bone defect and the new volume of bone formation were calculated by the three-dimensional reconstruction method. The osteogenesis rate was calculated to evaluate the osteogenesis effect@*Results@#The wounds in the three groups healed well after the operation, without implant material discharge, infection, dehiscence, rejection or other symptoms. Twelve months after the operation, CBCT scanning and three⁃dimensional reconstruction images of the three groups of patients showed the formation of new bone bridges in the alveolar ridge fissure area. The image density of the new bone tissue was not significantly different from that of normal bone tissue, and the continuity of the maxilla was re⁃ stored to varying degrees. The bone rate of autogenous bone was 65.00% ± 16.66%, β⁃ TCP group and BMP⁃2+ β⁃ The bone composition rate of TCP was 69.82% ± 17.60%, 71.35% ± 17.51%, respectively, and there was no significant dif⁃ ference compared with the autogenous bone group (P = 0.382, P = 0.244). The β⁃TCP and BMP⁃2+ β⁃TCP groups had no significant differences in bone rate (P = 0.789). @*Conclusion@#β⁃TCP could be used to replace autologous bone for alveolar cleft repair. The addition of BMP⁃2 to β⁃TCP did not significantly improve the osteogenesis rate.
ABSTRACT
Objective @#To investigate the role and mechanism of bone formation caused by the ratio of advanced platelet-rich fibrin (A-PRF) and β-tricalcium phosphate (β-TCP) in rabbit femur defect model, which provides a new idea for clinical treatment of bone defect.@*Methods @#Twenty-four New Zealand white rabbits were divided into model group, 1∶1 complex group (A-PRF∶β-TCP=1∶1), 2∶1 complex group (A-PRF∶β- TCP=2∶1) and 4∶1 complex group (A-PRF∶β- TCP=4∶1）, with 6 rabbits in each group. Femoral defect models were constructed in each group. In the composite group, the bone defect was filled with composite material, while in the model group, no material was filled. After 8 weeks, the animals were euthanized and specimens were collected. Bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.SP) and trabecular number (Tb.N) in femoral defect tissue were measured by micro-CT and photographed. Hematoxylin - eosin staining was used to detect the pathological changes of new bone tissue. The morphological changes of the new bone tissue were observed by scanning electron microscopy. Determination of phospho-mitogen activated protein kinase p38 (p-p38MAPK), CCAAT/enhancer binding protein homologous protein (CHOP) and phospho-cysteine aspartic protease-3 (p-Caspase3) in newborn femur by ELISA. The mRNA expressions of osteoprotegerin (OPG), bone morphogenetic protein-2 (BMP-2), receptor activator of nuclear factor kappa-B ligand (RANKL) and p38MAPK were detected by real-time quantitative PCR. The expression of OPG, BMP-2, RANKL, p-p38MAPK and p-Caspase3 protein in the new bone tissue was observed by immunohistochemistry. @*Results @#In the model group, bone formation in the femoral defect area was slow and osteogenic quality was poor. Compared with the model group, the bone formation and neocapillaries of femoral defect area in the complex group was good, BMD, BV.TV, Tb.Th, Tb.N were increased, and Tb.Sp were decreased, the expressions of p-p38MAPK, CHOP and p-Caspase3 were decreased, and the mRNA and protein expressions of OPG and BMP-2 were increased. The mRNA expression of RANKL and p38MAPK was decreased. Apoptosis in new bone tissue of each group showed the lowest apoptosis rate in samples of the 2∶1 complex group (P<0.05); A-PRF: β-TCP=2∶1 ratio has the best osteogenic effect. @*Conclusion@#The complex composed of A-PRF and β-TCP can promote the expression of OPG, inhibit the expression of RANKL and phosphorylation of p38MAPK, reduce the apoptosis of new bone tissue cells, and promote osteogenic differentiation.
ABSTRACT
OBJECTIVES@#To investigate the effect of recombinant human fibroblast growth factor 21 (rhFGF21) on the proliferation and mineralization of cementoblasts and its mechanism.@*METHODS@#Hematoxylin eosin, immunohistochemical staining, and immunofluorescence were used to detect the expression and distribution of fibroblast growth factor 21 (FGF21) in rat periodontal tissues and cementoblasts (OCCM-30), separately. Cell Counting Kit-8 was used to detect the proliferation of OCCM-30 under treatment with rhFGF21. Alkaline phosphatase staining and Alizarin Red staining were used to detect the mineralization state of OCCM-30 after 3 and 7 days of mineralization induction. The transcription and protein expression of the osteogenic-related genes Runx2 and Osterix were detected by real-time quantitative polymerase chain reaction (PCR) and Western blot analysis. The expression levels of genes of transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling pathway in OCCM-30 were detected through PCR array analysis.@*RESULTS@#FGF21 was expressed in rat periodontal tissues and OCCM-30. Although rhFGF21 had no significant effect on the proliferation of OCCM-30, treatment with 50 ng/mL rhFGF21 could promote the mineralization of OCCM-30 cells after 7 days of mineralization induction. The transcriptional levels of Runx2 and Osterix increased significantly at 3 days of mineralization induction and decreased at 5 days of mineralization induction. Western blot analysis showed that the protein expression levels of Runx2 and Osterix increased during mineralization induction. rhFGF21 up-regulated Bmpr1b protein expression in cells.@*CONCLUSIONS@#rhFGF21 can promote the mineralization ability of OCCM-30. This effect is related to the activation of the TGFβ/BMP signaling pathway.
Subject(s)
Humans , Rats , Animals , Dental Cementum , Core Binding Factor Alpha 1 Subunit/metabolism , Cell Differentiation , Bone Morphogenetic Proteins/metabolism , Transforming Growth Factor beta/pharmacologyABSTRACT
Nonalcoholic fatty liver disease (NAFLD) has become one of the most common chronic liver diseases in the world, and it seriously harms human health. Recent studies have found that bone morphogenetic protein 4 (BMP4) might be associated with NAFLD. This article reviews the latest advances in the research on the association between BMP4 and NAFLD in China and globally and explores the potential mechanism of action of BMP4 on NAFLD, in order to provide new ideas for the prevention and treatment of NAFLD.
ABSTRACT
This article reported the genetic analysis of a case diagnosed with fetal micrognathia and cleft palate by mid-trimester ultrasound in two consecutive pregnancies. In the first pregnancy, the pregnant woman delivered a full-term boy transvaginally, who died two weeks after birth and was diagnosed with Pierre Robin sequence (PRS). Chromosome karyotype and genomic copy number variation. In the second pregnancy, the woman underwent amniocentesis due to suspected PRS presenting by fetal cleft palate, micrognathism, and additional ultrasound anomalies. No abnormalities were detected in fetal karyotype or genomic copy number variation. Whole-exome sequencing, bioinformatics analysis, and Sanger sequencing suggested that both the fetus and the firstborn boy inherited a possible pathogenic variant of c.79delG p.E27Sfs*24 in the BMP2 gene from the mother. The pregnancy was terminated after the genetic consultation. Fetal phenotypes in the two fetuses were similar, indicating that short stature, facial dysmorphism, and skeletal anomalies with or without cardiac anomaly in the pedigree were caused by the heterozygous variant of c.79delG p.E27Sfs*24 in the BMP2 gene.
ABSTRACT
Objective @# To investigate the effect of micro/nano hierarchical structures on the adhesion and proliferation of MC3T3-E1 cells, evaluate the drug delivery potential of titanium surfaces, and provide a reference for the modification of selected areas of titanium surfaces to enhance drug delivery and slow drug release. @*Methods @# Pure titanium samples (10 mm in diameter and 2.5 mm in thickness) were randomly divided into a polished group (T), anodized group (TO), and micro/nano hierarchical structure group (FTO) according to the surface treatment of the titanium. The T group was polished, the TO group was treated with anodic oxidation technology, and the FTO group was treated by femtosecond laser etching combined with anodic oxidation technology. The three surface morphologies were observed by scanning electron microscopy (SEM), the wettability of the surface was measured by the contact angle, and the surface chemical composition was analyzed by X-ray energy dispersive spectroscopy (EDS). The depth of the FTO structure and the surface roughness were measured by confocal laser scanning microscopy (CLSM). MC3T3-E1 cell adhesion proliferation and differentiation on the surface of each group of samples was assessed by immunofluorescence staining, CCK-8, and semiquantitative analysis of Alizarin staining. A freeze-drying method was applied to load recombinant human bone morphogenetic protein-2 (rhBMP-2), and an enzyme-linked immunosorbent assay (ELISA) was used to assess the drug-loading potential of different surface structures. @* Results@#SEM revealed that the surface of T group titanium plates showed uniform polishing marks in the same direction. The surface of the TO group was a nanoscale honeycomb-like titanium dioxide (TiO2) nanotube structure, and the FTO group formed a regular and ordered micro/nano layered structure. The contact angle of the FTO group was the smallest at 32° ± 1.7°. Its wettability was the best. The average depth of the first-level structure circular pores was 93.6 μm, and the roughness was 1.5-2 μm. The TO and FTO groups contained a high percentage of oxygen, suggesting TiO2 nanotube formation. The FTO group had the most significant surface cell proliferation (P<0.001) and the largest cell adhesion surface area (P<0.05). rhBMP-2 was slowly released for 14 d after loading in the FTO group and promoted extracellular matrix mineralization (P<0.001). @*Conclusion @#Titanium surface microprepared hierarchical structure has the effect of promoting MC3T3-E1 cell adhesion, proliferation, and osteogenic differentiation with drug loading potential, which is a new method of titanium surface treatment.
ABSTRACT
ABSTRACT Melatonin (MLT) is a hormone responsible for regulating several physiological processes. It has been shown that MLT can be an important mediator in bone formation and stimulation, promoting osteoblast differentiation. In clinical practice, in tissue regeneration procedures, it is necessary to use membranes or barriers, associated with biomaterials, or not. The aim of this in vitro study was to assess the effect of melatonin on the activity of osteoblastic cells, associated, or not, with a resorbable collagen membrane (Bio-Gideä). For this, mice-derived pre-osteoblastic cells MC3T3 obtained from the ATCC (American Type Culture Collection) were used. Cultured cells were subject to the following treatments: MLT with a concentration of 1mM, a Bio-Gideä membrane and a membrane associated with MLT (Bio-Gideä + MLT). Proliferation and cell viability assays and protein lysate (ELISA test) quantification for the BMP-2 protein were carried out, in periods of 72 hours, 7 days and 10 days. After analyzing the data (one-way ANOVA, alpha=5%) it was observed that when MLT was used in isolation, there was an increase in cell proliferation and viability in osteoblastic cells (p<0.05). But, when MLT was associated with resorbable membranes, there was an inverse behavior, both in terms of proliferation and viability (p<0.05). In the case of the ELISA test, no secretion of BMP-2 was detected in any of the analyzed groups. It is concluded that MLT has a stimulatory effect on osteoblasts, but, when associated with Bio-Gideä resorbable membranes, it does not show any viable action in osteoblastic cell stimulation.
RESUMO A melatonina (MLT) é um hormônio responsável pela regulação de diversos processos fisiológicos no nosso organismo. Tem sido demonstrado que a melatonina possa ser um importante mediador na formação e estimulação óssea, promovendo a diferenciação dos osteoblastos. Clinicamente, para o procedimento de regeneração tecidual, faz-se necessário a utilização de membranas ou barreiras, associadas ou não a biomateriais. Assim, o objetivo deste estudo in vitro foi avaliar o efeito da melatonina na atividade de células osteoblásticas, associada ou não a uma membrana de colágeno reabsorvível (Bio-Gide®). Para isto foram utilizadas células pré-osteoblásticas MC3T3 do ATCC (American Type Culture Collection), de camundongos. As células em cultura foram submetidas aos seguintes tratamentos: MLT na concentração de 1mM, membrana Bio Gide® e membrana associada à MLT (Bio-Gide® + MLT). Foram realizados os ensaios de proliferação e viabilidade celular e quantificação do lisado proteico (teste ELISA), para a proteína BMP-2, nos períodos de 72 horas, 7 e 10 dias. Após a análise dos dados (ANOVA um critério, alfa=5%) pode-se observar que a MLT quando utilizada sozinha, resultou em um aumento na proliferação e viabilidade celular nas células osteoblásticas (p<0,05). Entretanto, quando a MLT foi associada à membrana reabsorvível foi observado um comportamento inverso, tanto na proliferação quanto na viabilidade (p<0,05). Para o teste ELISA realizado, não houve secreção detectável de BMP-2 para nenhum grupo analisado. Conclui-se que a melatonina possui uma ação estimuladora nos osteoblastos, mas quando associada à membrana reabsorvível Bio-Gide®, não demonstra uma ação viável na estimulação de células osteoblásticas.
ABSTRACT
O objetivo deste trabalho foi analisar o potencial bioativo de um "scaffold" de Polidioxanona (PDO) com associação da rhBMP-2, nas reconstruções após simulação de ressecção óssea em fêmures de ratos. Para tanto, 24 ratos, machos, adultos, com 6 meses de idade, foram submetidos a ressecção e reconstrução dos fêmures bilateralmente. Inicialmente foi realizada a estabilização com fixação de placas e parafusos de titânio do sistema 1.5mm e em seguida a confecção de um "gap" de 2mm. A reconstrução foi realizada com rhBMP-2 (Infuse) carreada em esponja de colágeno (3,25 µg), tendo uma malha de titânio, para o grupo Titânio (n=24 fêmures) (grupo controle), atuando como um arcabouço. E para o grupo PDO (n=24 fêmures) (grupo teste), a reconstrução foi realizada também com a rhBMP-2 carreada em uma esponja de colágeno (3,25 µg), envolvido por um "scaffold" de PDO. Desses animais, 16 (2 por tempo) receberam em seu dorso, no plano subcutâneo, um fragmento do mesmo material testado em seu fêmur, para análise de biocompatibilidade, que foram removidos sob anestesia local, junto de fragmento do tecido subcutâneo adjacente, aos 3, 5, 7 e 10 dias para análise. Os animais foram submetidos à eutanásia (n=6 por grupo) nos períodos de 14 e 60 dias após a cirurgia de reconstrução tiveram seus órgãos de metabolização (cérebro, rim, fígado e músculo) removidos para análise anatomopatológica e seus fêmures também foram removidos, reduzidos, radiografados para análise da densitometria radiográfica posteriormente os fêmures passaram por descalcificação e em seguida todas as peças foram submetidas ao processamento para obtenção de lâminas com cortes de 5 µm de espessura, para avaliação histológica, com avaliação da área óssea neoformada e perfil inflamatório e para análise imunohistoquimica através das proteínas Runx2, OPG, RANKL, OCN e BMP2. Todos os dados quantitativos foram submetidos ao teste ANOVA-2 fatores e quando p< 0,05, o pós-teste Tukey foi realizado. Os resultados da densitometria radiográfica demonstraram maior densidade para o grupo PDO, especialmente no período de 14 dias (p< 0,05). Na análise histológica observou-se reparo mais favorável para o grupo PDO, especialmente aos 60 dias quando comparado ao Titânio, com diferença estatística significativa (p = 0.002) bem como menor infiltrado inflamatório e maior número de vasos sanguíneos aos 14 dias. Com relação as imunomarcações, BMP-2 não apresentou marcações para Titânio e dados expressivos para PDO, com diferença significativamente estatística aos 60 dias (p< 0.05). OPG e RANKL mostraram maior marcação para titânio, principalmente aos 60 dias (p< 0.05). Já Runx2 e OCN apresentaram resultados superiores para PDO aos 14 dias, entretanto, aos 60 dias titânio demonstrou maior expressão. A análise de biocompatibilidade mostrou maior processo inflamatório para o grupo titânio. Os órgãos de metabolização apresentaram aspectos de higidez dentro da normalidade para ambos grupos. Os resultados deste trabalho demonstram um padrão reparacional mais favorável à associação do "Scaffold" de PDO com a rhBMP-2, quando comparado a reconstrução com malha de titânio(AU)
The objective of this work was to analyze the bioactive potential of a Polydioxanone (PDO) scaffold with rhBMP-2 association, in reconstructions after simulating bone resection in rat femurs. Therefore, 24 male, adult rats, aged 6 months, underwent resection and reconstruction of the femurs bilaterally. Initially, stabilization was performed with fixation of titanium plates and screws of the 1.5mm system and then a 2mm gap was created. The reconstruction was performed with rhBMP-2 (Infuse) loaded in a collagen sponge (3.25 µg), with a titanium mesh, for the Titanium group (n=24 femurs) (control group), acting as a scaffold. And for the PDO group (n=24 femurs) (test group), the reconstruction was also performed with rhBMP-2 carried in a collagen sponge (3.25 µg), surrounded by a PDO scaffold. Of these animals, 16 (2 per time) received on their back, in the subcutaneous plane, a fragment of the same material tested in their femur, for biocompatibility analysis, which was removed under local anesthesia, together with a fragment of the adjacent subcutaneous tissue, at 3, 5, 7 and 10 days for analysis. The animals were euthanized (n=6 per group) in the periods of 14 and 60 days after the reconstruction surgery, had their metabolizing organs (brain, kidney, liver, and muscle) removed for anatomopathological analysis and their femurs were also removed, reduced, radiographed for analysis of radiographic densitometry later the femurs underwent decalcification and then all the pieces were submitted to processing to obtain 5 µm thick slices for histological evaluation, with the evaluation of the newly formed bone area and inflammatory profile and for immunohistochemical analysis through Runx2, OPG, RANKL, OCN, and BMP2 proteins. All quantitative data were submitted to the 2-way ANOVA test and when p< 0.05, the Tukey post-test was performed. The results of radiographic densitometry showed higher density for the PDO group, especially in the 14-day period (p< 0.05). In the histological analysis, a more favorable repair was observed for the PDO group, especially at 60 days when compared to Titanium, with a statistically significant difference (p = 0.002), as well as a lower inflammatory, infiltrate and a greater number of blood vessels at 14 days. Regarding immunostaining, BMP-2 did not show staining for Titanium and expressive data for PDO, with a statistically significant difference at 60 days (p< 0.05). OPG and RANKL showed higher staining for titanium, mainly at 60 days (p< 0.05). On the other hand, Runx2 and OCN showed superior results for PDO at 14 days, however, at 60 days titanium showed greater expression. The biocompatibility analysis showed a greater inflammatory process for the titanium group. The metabolizing organs presented aspects of health within the normal range for both groups. The results of this work demonstrate a more favorable repair pattern for the association of the PDO scaffold with rhBMP-2, when compared to reconstruction with titanium mesh(AU)
Subject(s)
Animals , Rats , Bone Regeneration , Bone Morphogenetic Protein 2 , Polymers , Rats, Wistar , Bone Morphogenetic ProteinsABSTRACT
Abstract: Modified formulations of calcium silicate repair materials with additives have been developed to enhance handling, consistency, biocompatibility and bioactivity. Considering the relevance of osteoblastic cell response to mineralized tissue repair, human osteoblastic cells (Saos-2 cells overexpressing BMP-2) were exposed to mineral trioxide aggregate (MTA) (with calcium tungstate - CaWO4), MTA HP Repair, Bio-C Repair and Bio-C Pulpo. Cell viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR), and cell death, by flow cytometry. Gene expression of bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (RUNX-2), and alkaline phosphatase (ALP) osteogenic markers were evaluated by real-time polymerase chain reaction (RT-qPCR). ALP activity and alizarin red staining (ARS) were used to detect mineralization nodule deposition. Bioactive cements presented no cytotoxic effect, and did not induce apoptosis at the higher dilution (1:12). MTA, Bio-C Repair and Bio-C Pulpo exhibited higher ALP activity than the control group (P < 0.05) after 7 days. MTA, MTA HP and Bio-C Pulpo affected the formation of mineralized nodules (p < 0.05). Exposure to all cement extracts for 1 day increased BMP-2 gene expression. RUNX-2 mRNA was greater in MTA, MTA HP and Bio-C Repair. MTA, MTA HP and Bio-C Pulpo increased the ALP mRNA expression, compared with BMP-2 unexposed cells (P < 0.05). Calcium silicate cements showed osteogenic potential and biocompatibility in Saos-2 cells transfected BMP-2, and increased the mRNA expression of BMP-2, RUNX-2, and ALP osteogenic markers in the BMP-2 transfected system, thereby promoting a cellular response to undertake the mineralized tissue repair.
ABSTRACT
Abstract This study tested the hypothesis that head and neck radiotherapy (HNRT) impacts the immunoexpression of type I collagen, bone sialoprotein (BSP) and bone morphogenetic protein 4 (BMP4), thereby leading to micromorphological changes in the dentin-pulp complex (DPC), and promoting the onset and progression of radiation caries (RC). Twenty-two demineralized sections of carious teeth (a group of 11 irradiated teeth and a control group of 11 non-irradiated teeth) extracted from 19 head and neck cancer patients were analyzed by conventional optical microscopy and immunohistochemistry to investigate the micromorphology (cellular layer hierarchy, blood vessels, odontoblasts, fibroblasts, extracellular matrix, calcification, necrosis, reactionary dentin formation, and chronic inflammation), and the patterns of staining/immunolocalization of type I collagen, BSP and BMP4 in the dental pulp of irradiated and control samples. No significant differences attributable to the direct impact of radiotherapy were detected in DPC micromorphology between the groups. In addition, the patterns of immunohistochemical staining and immunolocalization of the proteins studied did not differ between the irradiated and the control samples for type I collagen, BSP or BMP4. This study rejected the hypothesis that HNRT directly damages dentition by changing the organic components and the microstructure of the DPC, ultimately leading to RC.
ABSTRACT
Objective @#To study the regulatory effect of coiled-coil domain containing 134 (CCDC134) on the osteogenic differentiation of human dental pulp stem cells (hDPSCs).@*Methods @# HDPSCs were isolated and cultured from dental pulp tissue and transfected with NC-CCDC134, shCCDC134 and CCDC134 lentiviruses. They were divided into the control group, negative control group, CCDC134 downregulation (shCCDC134) group and CCDC134 overexpression (CCDC134) group. Surface markers of hDPSCs (Stro-1, CD105, CD34, CD45) were detected by flow cytometry; colony formation was analyzed by toluidine blue staining; ALP expression was estimated by ALP staining; mineralized nodule formation was evaluated by alizarin red staining; lipid droplet formation was examined by oil red staining; and gene and protein expression of CCDC134, Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and mothers against decapentaplegic homolog 1 (SMAD1) was detected by qPCR and western blot, respectively. Further, a BMP-2 activator (BMP-2) and inhibitor (Dorsomorphin) were used to down-regulate and up-regulate CCDC134, respectively (shCCDC134, shCCDC134+BMP-2, CCDC134, CCDC134+Dorsomorphin), in hDPSCs. The hDPSC aggregates were subcutaneously transplanted into nude mice for 2 months, and new bone formation was detected by H&E staining. The BMP-2/SMAD1 signaling in each group was detected by qPCR.@*Results@#hDPSCs showed high expression of mesenchymal markers and low expression of hematopoietic markers. Compared with the control group, the expression of CCDC134 was increased in the osteogenic-induced hDPSCs (P < 0.05). Compared with the negative control group, the expression of CCDC134 was decreased in the shCCDC134 group, whereas it was increased in the CCDC134 group (P < 0.05). The mineralized nodules, osteogenic genes and proteins in the shCCDC134 group were decreased (P < 0.05), while they were increased in the CCDC134 group (P < 0.05). The expression of BMP-2/SMAD1 signaling decreased in the shCCDC134 group, while it increased in the CCDC134 group (P < 0.05). Compared to the shCCDC134 group, osteogenic genes and proteins increased in the shCCDC134+BMP-2 group, and subcutaneous new bone formation increased in nude mice (P < 0.05). The indexes of the CCDC134+Dorsomorphin group decreased compared with the CCDC134 group (P < 0.05).@*Conclusion@#CCDC134 promotes the osteogenic differentiation of hDPSCs by regulating the BMP-2/SMAD1 signaling pathway.
ABSTRACT
Objective:To observe the effect of bone morphogenetic protein 4 (BMP4) on the proliferation and migration of human retinal microvascular endothelial cells (hRMEC) under oxidative stress.Methods:The hRMEC cultured in vitro were divided into control group, 4-hydroxynonenal (HNE) treatment group (4-HNE group), 4-HNE+BMP4 group (BMP4 group). Cell culture medium of 4-HNE treatment group was added with 10 μmmol/L 4-HNE; cell culture of BMP4 group was cultured with 10 μmmol/L 4-HNE, and after stimulation for 6 h, 100 ng/ml recombinant human BMP4 was added. The effects of 4-HNE and BMP4 on hRMEC viability was detected by thiazole blue colorimetric method. The effects of 4-HNE and BMP4 on cell migration was determined by cell scratch test. The relative expression of BMP4 mRNA in the cells of the control group and 4-HNE treatment group and the mRNA expression of the control group, the fibronectin (FN) of BMP4 group, laminin (Laminin), α-smooth muscle contractile protein (α-SMA), and collagen type Ⅰ (Collagen Ⅰ), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) were detected by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the relative expression of BMP4 protein in the control group and 4-HNE group. The control group and 4-HNE group were compared by t test. Results:Compared with the control group, cell viability ( t=12.73, 16.26, P=0.000 2, <0.000 1), cell migration rate ( t=28.17, 37.48, P<0.000 1, <0.000 1) in 4-HNE group and BMP4 group were significantly increased, and the difference was statistically significant; the relative expression of BMP4 mRNA and protein in the 4-HNE group was significantly increased, and the difference was statistically significant ( t=16.36, 69.35, P=0.000 1, <0.000 1). The qRT-PCR test results showed that compared with the control group, the relative expression of VEGF, FN, Laminin, α-SMA, Collagen Ⅰ, and CTGF mRNA in the cells of the BMP4 group was significantly increased, and the difference was statistically significant ( t=10.61, 17.00, 14.85, 7.78, 12.02, 10.61, P=0.0004, <0.000 1, 0.000 1, 0.001 5, 0.000 1, 0.000 4). Conclusion:BMP4 can induce the proliferation and migration of hRMEC; it can also regulate the expression of angiogenesis factors and fibrosis-related factors in hRMEC.
ABSTRACT
Portopulmonary hypertension (POPH) is an increase in pulmonary artery pressure that occurs on the basis of portal hypertension. As a member of the BMP family, bone morphogenetic protein 9 (BMP9) not only has the osteogenic activity, but can also protect endothelial integrity and maintain vascular homeostasis. This article reviews the pathogenesis of POPH, the physiological expression and role of BMP9, and related research advances in the BMP9 signaling pathway and its involvement in pulmonary hypertension and vascular remodeling, thereby exploring the possibility of BMP9 as a new biomarker for POPH to assist in the diagnosis of POPH.
ABSTRACT
Objective:To explore the effect of bone morphogenetic protein 4 (BMP4) on the glycolysis level of human retinal microvascular endothelial cells (hRMECs).Methods:A experimental study. hRMECs cultured in vitro were divided into normal group, 4-hydroxynonenal (HNE) group (4-HNE group) and 4-HNE+BMP4 treatment group (BMP4 group). 4-HNE group cell culture medium was added with 10 μmmol/L 4-HNE; BMP4 group cell culture medium was added with recombinant human BMP4 100 ng/ml after 6 h stimulation with 10 μmol/L 4-HNE. The levels of intracellular reactive oxygen species (ROS) were detected by flow cytometry. The effect of 4-HNE on the viability of cells was detected by thiazole blue colorimetry. Cell scratch test and Transwell cell method were used to determine the effect of 4-HNE on cell migration. The relative expression of BMP4 and SMAD9 mRNA and protein in normal group and 4-HNE group were detected by realtime quantitative polymerase chain reaction and Western blot. Seahorse XFe96 cell energy metabolism analyzer was used to determine the level of intracellular glycolysis metabolism in normal group, 4-HNE group and BMP4 group. One-way analysis of variance was used for comparison between groups.Results:The ROS levels in hRMECs of normal group, 4-HNE group and BMP4 group were 21±1, 815±5, 810±7, respectively. Compared with the normal group, the levels of ROS in the 4-HNE group and the BMP4 group were significantly increased, and the difference was statistically significant ( F=53.40, 50.30; P<0.001). The cell viability in the normal group and 4-HNE group was 1.05±0.05 and 1.28±0.05, respectively; the migration rates were (0.148±0.005)%, (0.376±0.015)%; the number of cells passing through the pores were 109.0±9.6, 318.0±6.4, respectively. Compared with the normal group, the 4-HNE group had significantly higher cell viability, cell migration rate, and the number of cells passing through the pores, and the differences were statistically significant ( F=54.35, 52.84, 84.35; P<0.05). The relative expression levels of BMP4 and SMAD9 mRNA in the cells of the 4-HEN group were 1.680±0.039 and 1.760±0.011, respectively; compared with the normal group, the difference was statistically significant ( F=53.66, 83.54; P<0.05). The relative expression levels of BMP4 and SMAD9 proteins in the cells of the normal group and 4-HEN group were 0.620±0.045, 0.860±0.190, 0.166±0.049, 0.309±0.038, respectively; compared with the normal group, the differences were statistically significant ( F=24.87, 53.84; P<0.05). The levels of intracellular glycolysis, glycolytic capacity and glycolytic reserve in normal group, 4-HNE group and BMP4 group were 1.21±0.12, 2.84±0.24, 1.78±0.36, 2.59±0.11, 5.34±0.32, 2.78±0.45 and 2.64±0.13, 5.20±0.28, 2.66±0.33. Compared with the normal group, the differences were statistically significant (4-HNE group: F=86.34, 69.75, 58.45; P<0.001; BMP4 group: F=56.87, 59.35, 58.35; P<0.05). There was no significant difference in intracellular glycolysis, glycolysis capacity and glycolysis reserve level between 4-HNE group and BMP4 group ( F=48.32, 56.33, 55.01; P>0.05). Conclusion:BMP4 induces the proliferation and migration of hRMECs through glycolysis.
ABSTRACT
ABSTRACT Introduction: In recent years, genetic engineering has made outstanding contributions to sports, and it has played a huge role in promoting the development of sports-related fields. Objective: We analyze the tissue source of bone growth and healing by studying the role of bone morphogenetic protein and transforming growth factors in fracture injuries caused by sports. Methods: We established a human fracture model to express the shape and content of bone morphogenetic protein and transforming growth factor during fracture healing. Results: In the fracture healing stage caused by different sports, the expression levels of the two genes are different. Bone morphogenetic protein has a high content in the osteogenesis stage of the membrane, while transforming growth factor is high in the cartilage ossification stage. Conclusion: Gene therapy for fractures caused by physical exercise has certain advantages. Osteoblasts and chondrocytes are involved in the synthesis of transforming growth factors. Level of evidence II; Therapeutic studies - investigation of treatment results.
RESUMO Introdução: Nos últimos anos, a engenharia genética tem contribuído de forma notável para os esportes, além de ter uma função importante na promoção do desenvolvimento de áreas relacionadas ao esporte. Objetivo: Analisamos a origem de tecidos do crescimento ósseo e sua regeneração através do estudo da proteína morfogenética óssea e fatores de transformação do crescimento em fraturas causadas pela prática do esporte. Métodos: Criamos um modelo de fratura humana para expressar a forma e o conteúdo da proteína morfogenética óssea e de fatores de transformação do crescimento durante a recuperação de fraturas. Resultados: Na fase de recuperação da fratura causada por diversos esportes, os níveis de expressão dos dois genes são diferentes. A proteína morfogenética óssea se apresenta em alta quantidade na fase osteogenetica da membrana, e o fator de transformação de crescimento apresenta alta quantidade na fase de ossificação da cartilagem. Conclusão: A terapia genética para fraturas causadas por exercícios físicos apresenta diversas vantagens. Osteoblastos e condrócitos tem um papel na síntese dos fatores de transformação do crescimento. Nível de evidência II; Estudos terapêuticos - investigação de resultados de tratamento.
RESUMEN Introducción: En los últimos años, la ingeniería genética ha contribuido de forma notable para los deportes, además de tener una función importante en la promoción del desarrollo de áreas relacionadas al deporte. Objetivo: Analizamos el origen de tejidos del crecimiento óseo y su regeneración a través del estudio de la proteína morfogenética ósea y factores de crecimiento transformante en fracturas causadas por la práctica del deporte. Métodos: Creamos un modelo de fractura humana para expresar la forma y el contenido de la proteína morfogenética ósea y de factores de crecimiento transformante durante la recuperación de fracturas. Resultados: En la fase de recuperación de la fractura causada por diversos deportes, los niveles de expresión de los dos genes son diferentes. La proteína morfogenética ósea se presenta en alta cantidad en la fase osteogénica de la membrana, y el factor de crecimiento transformante presenta alta cantidad en la fase de osificación del cartílago. Conclusión: La terapia genética para fracturas causadas por ejercicios físicos presenta diversas ventajas. Osteoblastos y condrocitos tienen un papel en la síntesis de los factores de crecimiento transformante. Nivel de evidencia II; Estudios terapéuticos - investigación de resultados de tratamiento.
ABSTRACT
Purpose To evaluate the effect of ergosterol combined with risedronate on fracture healing. Methods Sixty male Sprague Dawley fracture model rats were assigned into group A (n=20), group B (n=20), and group C (n=20) at random. All rats were fed by gavage until their sacrifice as it follows: group A with ergosteroside and risedronate, group B with risedronate, and group C with saline solution. At weeks 2 and 4, 10 rats of each group were sacrificed. Healing effect and bone tissue changes in the fractures site were assessed by using hematoxylin and eosin stain histology. Enzyme-linked immunosorbent assay was used to detect the expression of serum bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), and vascular endothelial growth factor (VEGF). Reverse transcriptase polymerase chain reaction was applied to detect the expression of osteoprotegerin (OPG) mRNA, osteocalcin (OCN) mRNA and core-binding factor subunit-?1 (CBF-?1) mRNA. Results In terms of serum BMP-2, BMP-7, and VEGF expression at weeks 2 and 4 after gavage, group A < group B < group C (P<0.05). At week 4 after gavage, serum VEGF expression in the three groups harbored positive relationship with serum BMP-2 and BMP-7 expression (P<0.05). Regarding serum OPG, OCN and CBF-?1 mRNA expression at weeks 2 and 4 after gavage, group A Subject(s)
Male
, Animals
, Rats
, Fracture Healing/drug effects
, Ergosterol/analysis
, Vascular Endothelial Growth Factor A
, Osteoprotegerin/isolation & purification
, Risedronic Acid/analysis
, Reverse Transcriptase Polymerase Chain Reaction