Uma discussão sobre o efeito da rhBMP-2 no aumento ósseo alveolar / A discussion on the effect of rhBMP-2 in the alveolar bone augmentation

O objetivo deste trabalho foi apresentar uma revisão da literatura sobre a proteína óssea morfogenética tipo 2 (BMP-2) e seu efeito no aumento ósseo alveolar. A proteína óssea morfogenética (BMP) foi identificada, em 1965, por um norte-americano chamado Marshal Urist, que mostrou que essa proteína, extraída da cortical óssea bovina, poderia induzir a formação de novo osso quando implantada em locais não ósseos. Atualmente, muitos trabalhos têm estudado a regeneração óssea através do uso de BMPs em substituição aos enxertos ósseos. O presente estudo conclui que a proteína óssea morfogenética é capaz de induzir neoformação óssea de maneira eficaz, tornando-se uma alternativa na substituição dos enxertos ósseos e a necessidade da descoberta de novos carreadores facilitando a estabilidade mecânica da BMP-2 no leito receptor.

The aim of this paper is to review the literature about bone morphogenetic protein type 2 (BMP-2) and on the effect in the alveolar bone augmentation. In 1965, the BMP was isolated by Marshal Urist, who showed that this protein extracted from bone narrow could induce bone neoformation when implanted in sites without bone cells. Recently, a lot of studies have been looking for bone regeneration using BMPs without bone grafts. The present study it was concluded that the bone morphogenetic protein induces bone neoformation, being an alternative as a substitute to bone grafts and that new carrier discovery is necessary to smooth stability of this carriers in receptor site.

Bovine bone matrix action associated with morphogenetic protein in bone defects in rats submitted to alcoholism / Acción de la matriz ósea bovina asociada a la proteína morfogenéticaen defectos óseos en ratones sometidos a alcoholismo

Extended excessive alcohol use causes changes in bone tissue, thus affecting osteogenesis. The objective of this study was to evaluate if demineralized bone matrix (Gen-ox®) associated with bone morphogenetic protein (Gen-pro®) changes bone neoformation in rats submitted to experimental alcoholism. Forty male rats (Rattus norvegicus) were separated into 2 groups of 20 animals each: Group E1, which received ethyl alcohol at 25 percent and had the surgical cavity filled in only with blood clot; and Group E2, which received ethyl alcohol at 25 percent and had the surgical cavity filled in with demineralized bovine cortical bone associated with bone morphogenetic protein. The animals were submitted to a three-week period of gradual adaptation to alcohol, and then continued receiving alcohol at 25 percent for 90 days, when the surgical cavity was made. After the surgery, the animals continued consuming alcohol until reaching the sacrifice periods of 10, 20, 40, and 60 days, when the tibias were removed for histological processing. Results showed that surgical cavity repair and bone marrow reorganization occurred faster in Group E1 than in Group E2. At the end of the experiment, it was observed that animals in Group E2 had thick bony trabeculae surrounding the implanted material particles and a small area of connective tissue in the surface region. In conclusion, the implanted material did not accelerate bone neoformation, rather it served as a structure for osteogenesis.

El abuso prolongado del alcohol produce alteraciones en el tejido óseo, interfiriendo en el proceso de la osteogénesis. El estudio tiene como objetivo evaluar si la matriz ósea bovina desmineralizada (Gen-ox®) asociada a la proteína morfogenética ósea (Gen-pro®) altera la neoformación ósea en ratones sometidos a alcoholismo experimental. Fueron utilizados 40 ratones machos (Rattus norvegicus), separados en dos grupos de 20 animales cada uno: Grupo E1, que recibió alcohol etílico a 25 por ciento con cavidad quirúrgica rellenada solamente por coágulo sanguíneo, y Grupo E2, que recibió sólo alcohol etílico a 25 por ciento con cavidad quirúrgica rellenada con hueso bovino desmineralizado cortical asociado a proteína morfogenética ósea. Después de 3 semanas de adaptación gradual al alcohol, los animales continuaron recibiéndolo en concentración de 25 por ciento por 90 días, cuando fue realizada la cavidad quirúrgica. Luego de la cirugía, los animales continuaron la ingestión alcohólica hasta los períodos de sacrificio de 10, 20, 40 y 60 días, cuando las tibias fueron removidas para su procesamiento histológico. Los resultados mostraron que en el Grupo E1 hubo reparación de la cavidad quirúrgica y reorganización de la médula ósea en un menor lapso temporal que en el Grupo E2. En el período final del experimento, se observó en los animales del Grupo E2 la presencia de trabéculas óseas espesas alrededor de las partículas de material implantado y pequeña área de tejido conjuntivo en la región superficial. Se puede concluir en que el material implantado no aceleró el proceso de neoformación ósea, sirviendo como estructura de base para generar osteogénesis.

Fosfoproteômica e proteômica quantitativa de células mesenquimais durante a diferenciação osteoblástica mediada por BMP2, expressão e purificação de diferentes tipos de proteínas morfogenéticas ósseas / Quantitative phosphoproteomics and proteomics of mesenchymal stem cells during BMP2-mediated osteoblastic differentiation, expression and purification of different types of bone morphogenetic proteins

As fraturas e perdas ósseas representam altos riscos para o Sistema público de Saúde (SUS), além de afetar a qualidade de vida do paciente, portanto é necessário o entendimento das bases moleculares que envolvem os mecanismos de reparo ósseo. Citocinas secretadas por células do sistema imune presentes no local da inflamação, como as IL-6, IL-10 e TNFα atuam como fatores quimiotáticos para células mesenquimais, que proliferam e se diferenciam em osteoblastos pela ação autócrina e parácrina de Proteínas Morfogenéticas Ósseas (BMPs), principalmente a BMP2. Embora seja conhecido que a ação de BMP2 ocorra através de sua ligação nos receptores ActRI/BMPR, que ativam proteínas SMADS 1/5/8 efetoras, pouco se sabe sobre os mecanismos intracelulares que participam do processo de diferenciação osteoblástico. Neste estudo propôs-se analisar as diferenças no conteúdo de proteínas totais e de proteínas fosforiladas em células mesenquimais de pele induzidas à osteogênese pelo tratamento com BMP2 por diferentes períodos de tempo, utilizando-se de Isótopos Estáveis de Dimetila acoplado ao LC/MS. A partir de 150µg de material inicial, foi possível identificar 2.264 proteínas, as quais foram quantificadas nos diferentes pontos de indução, sendo que 235 são fosforiladas. Análise de motivos de quinases mostrou que diversos substratos possuem sítios fosforilados correspondentes àqueles dos motivos de fosforilação das quinases Casein Kinase, p38, CDK e JNK. A análise da ontologia gênica mostrou um aumento de processos biológicos relacionados com sinalização e diferenciação após a primeira hora de indução com rhBMP2. Além disso, proteínas envolvidas com o rearranjo do citoesqueleto e com vias de sinalização Wnt e Ras foram encontradas como tendo fosforilação diferencial durante todos os períodos estudados. Os dados revelaram novos substratos intracelulares que são fosforilados nos primeiros momentos do comprometimento com a diferenciação osteoblástica mediada pelo tratamento com rhBMP2 em células mesenquimais derivadas da pele. Além disso, clones celulares que superexpressam as proteínas recombinantes humanas BMP2 e BMP4 foram gerados, e sua atividade verificada in vitro. Paralelamente, a rhBMP7, obtida anteriormente, foi purificada por cromatografia de afinidade utilizando-se uma coluna de Heparina-Sepharose, que foi posteriormente utilizada para ensaios in vitro e in vivo, nos quais se mostrou capaz de gerar osteoblastos e tecido ósseo, respectivamente, o que abre novas possibilidades para o uso destas proteínas como biofármacos no Brasil

Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated human skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. From 150 µg of starting material, 2,264 proteins containing two or more peptides were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during commitment to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. Cell clones overexpressing the human BMP 2 and 4 recombinant proteins were also generated, and their biological activity was confirmed in vitro. In parallel, chromatography-affinity purified rhBMP7, obtained using heparin-Sepharose columns, was used for in vivo and in vitro assays to evaluate the ability of this purified protein to generate osteoblasts and bone tissue, respectively, opening new avenues for the use of these proteins as biopharmaceuticals in Brazil

Microarray Analysis of Gene Expression During Differentiation of Human Mesenchymal Stem Cells Treated with Vitamin E in vitro into Osteoblasts

OBJECTIVE: Supplementation with vitamin E is able to protect bone against free radical-induced elevation of bone-resorbing cytokines. We examined gene expression by microarray analysis during the differentiation of human mesenchymal stem cells treated with vitamin E into osteoblasts in vitro. METHODS: Human bone marrow stem cells were cultured in osteogenic differentiation medium and vitamin E was added. A colorimetric immunoassay for the quantification of cell proliferation was used to measure osteoblast differentiation. Gene expression was analyzed using a microarray technique. We also used a real time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: It was found that vitamin E enhanced cell proliferation when compared to cells cultured in media without vitamin E. We focused on 68 genes which are related to osteogenesis and osteoclastogenesis. Alkaline phosphatase, transforming growth factor-beta 1, fibroblast growth factor receptor 1, matrix metalloproteinase 2, muscle segment homeobox 2, bone morphogenetic protein 1, biglycan, vascular endothelial growth factor B, dentin sialophosphoprotein, cartilage oligomeric matrix protein, runt-related transcription factor 2, fibroblast growth factor receptor 3, and SMAD2 were upregulated > 2-fold compared to the control. Conversely, osteopetrosis-associated transmembrane protein 1, microphthalmia-associated transcription factor, and epidermal growth factor receptor were downregulated > 2-fold compared to the control. Vitamin E produced a 1.5-fold increase in the expression of runt-related transcription factor 2 and transforming growth factor-beta 1 as determined by real time RT-PCR. CONCLUSION: Vitamin E had a positive effect on the gene expressions regarding osteogenic differentiation of mesenchymal stem cells.

The cDNA microarray study for the effect of FGF-5 administration on fibroblast

Binding between alpha 1A-adrenergic receptor and segment of bone morphogenetic protein-1 in human embryonic cell 293 / 生理学报

Using matchmaker yeast two-hybrid system, it has been demonstrated that there exists an interaction between the cellular C terminal of alpha(1A)-adrenergic receptor (alpha(1A)-AR) and a segment of bone morphogenetic protein-1 (BMP-1). In the present study binding between the two proteins was further determined in human embryonic cell 293 (HEK293), a mammalian expression system. Mammalian expression vector PCP3HA was constructed by PCR and consisted of segments of BMP-1 cDNA, and vector PDT-alpha(1A) consisted of the full-length cDNA of human alpha(1A)-AR. They were transfected to HEK293 cells and examined by Western blot. alpha(1A)-AR and the segment of BMP-1 could be detected in the lysis of transfected cells. Then binding between alpha(1A)-AR and the segment of BMP-1 in HEK293 cell was determined by enzyme-linked immunosorbent assays (ELISA) and co-immunoprecipitation. In ELISA experiment, the ELISA microwell plate was first coated with anti-FLAG M2 antibody, which recognizes the FLAG-tagged alpha(1A)-AR, then the cell lysis, anti-HA rabbit polyclonal antibody and HRP conjugated anti-rabbit antibody were added in turn. The OD(490) values among the control group, PDT-alpha(1A) transfection group and PCP3HA transfection group, exhibited no significant difference (0.034+/-0.027, 0.042+/-0.019, 0.030+/-0.0096), but the OD(490) values of PDT-alpha(1A) and PCP3HA co-transfection group (0.57+/-0.12) were significantly higher than those of the other groups (P<0.001, respectively). In co-immunoprecipitation experiments, HEK293 cells expressing alpha(1A)-AR or/and segment of BMP-1 were lysed and incubated with anti-FLAG M2 antibody, then the immunoprecipitation pellet was immunoblotted with either the HRP conjugated anti-FLAG antibody or the anti-HA antibody, which recognizes the HA-tagged segment of BMP-1. Segment of BMP-1 was present in the pellet immunoprecipitation of PDT-alpha(1A) and PCP3HA co-transfected group. In conclusion, the results indicate that alpha(1A)-AR and the segment of BMP-1 are present in the same complex in HEK293 cells.