Polydioxanone Enhances Bone Regeneration After Resection and Reconstruction of Rat Femur with rhBMP2.

The aim of this study was to assess the bone regeneration potential of a polydioxanone (PDO) scaffold together with recombinant human bone morphogenetic protein-2 (rhBMP-2) for the reconstruction of large bone defect. In total, 24 male rats (6 months old) were subjected to bilateral femoral stabilization using titanium plates to create a 2 mm gap, and reconstruction using rhBMP-2 (Infuse®; 3.25 µg). The bone defects were covered with PDO (PDO group), or with titanium mesh (Ti group). Animals were euthanized on days 14 and 60. Simultaneously, 16 rats received PDO and Ti in their dorsum for the purpose of biocompatibility analysis at 3, 5, 7, and 10 days postoperatively. X-ray densitometry showed a higher density in the PDO group on day 14. On day 60, coverage of the bone defect with PDO showed a larger quantity of newly formed bone than that found for the Ti group, a lower inflammatory infiltrate value, and a more significant number of blood vessels on day 14. By immunohistochemical assessment, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) showed higher labeling on day 14 in the PDO group. On day 60, bone morphogenetic protein-2 (BMP-2) showed higher labeling in the PDO group, whereas Ti showed higher labeling for osteoprotegerin, nuclear factor kappa B ligand-activating receptor, RUNX2, and OCN. Furthermore, biocompatibility analysis showed a higher inflammatory response in the Ti group. The PDO scaffold enhanced bone regeneration when associated with rhBMP-2 in rat femur reconstruction. Impact statement Regeneration of segmental bone defects is a difficult task, and several techniques and materials have been used. Recent advances in the production of synthetic polymers, such as polydioxanone (PDO), produced by three-dimensional printing, have shown distinct characteristics that could improve tissue regeneration even in an important bone defect. The present preclinical study showed that PDO membranes used as scaffolds to carry recombinant human bone morphogenetic protein-2 (rhBMP-2) improved bone tissue regeneration by more than 8-fold when compared with titanium mesh, suggesting that PDO membranes could be a feasible and useful material for use in guided bone regeneration. (In English, viable is only used for living creatures capable of sustaining life.

Influence of melatonin associated with the Bio-Gide® membrane on osteoblast activity: an in vitro Study.

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.

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.

Influence of melatonin associated with the Bio-Gide® membrane on osteoblast activity: an in vitro Study / Influência da melatonina associada à membrana bio gide® na atividade de osteoblastos: um estudo in vitro

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.

Cytotoxicity and bioactive potential of new root repair materials for use with BMP-2 transfected human osteoblast cells

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.

Análise do potencial do reparo ósseo através da reconstrução de ressecções segmentares por meio da associação de scaffolds de Polidioxanona ou malha de titânio e rhBMP-2: estudo in vivo / Bone potential analysis through segmental resection reconstruction using Polydioxanone or Titanium Mesh scaffolds and rhBMP-2: in vivo study

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)

Effects of ergosteroside combined risedronate on fracture healing and BMP-2, BMP-7 and VEGF expression in rats

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

Biomimetic Mineralization on 3D Printed PLA Scaffolds: On the Response of Human Primary Osteoblasts Spheroids and In Vivo Implantation.

This study aimed to assess the response of 3D printed polylactic acid (PLA) scaffolds biomimetically coated with apatite on human primary osteoblast (HOb) spheroids and evaluate the biological response to its association with Bone Morphogenetic Protein 2 (rhBMP-2) in rat calvaria. PLA scaffolds were produced via 3D printing, soaked in simulated body fluid (SBF) solution to promote apatite deposition, and characterized by physical-chemical, morphological, and mechanical properties. PLA-CaP scaffolds with interconnected porous and mechanical properties suitable for bone repairing were produced with reproducibility. The in vitro biological response was assessed with human primary osteoblast spheroids. Increased cell adhesion and the rise of in vitro release of growth factors (Platelet-Derived Growth Factor (PDGF), Basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF) was observed for PLA-CaP scaffolds, when pre-treated with fetal bovine serum (FBS). This pre-treatment with FBS was done in a way to enhance the adsorption of serum proteins, increasing the number of bioactive sites on the surface of scaffolds, and to partially mimic in vivo interactions. The in vivo analysis was conducted through the implantation of 3D printed PLA scaffolds either alone, coated with apatite (PLA-CaP) or PLA-CaP loaded with rhBMP-2 on critical-sized defects (8 mm) of rat calvaria. PLA-CaP+rhBMP2 presented higher values of newly formed bone (NFB) than other groups at all in vivo experimental periods (p < 0.05), attaining 44.85% of NFB after six months. These findings indicated two new potential candidates as alternatives to autogenous bone grafts for long-term treatment: (i) 3D-printed PLA-CaP scaffold associated with spheroids, since it can reduce the time of repair in situ by expression of biomolecules and growth factors; and (ii) 3D-printed PLA-CaP functionalized rhBMP2 scaffold, a biocompatible, bioactive biomaterial, with osteoconductivity and osteoinductivity.

Synergism between recombinant human bone morphogenetic protein 2/absorbable collagen sponge and bone substitutes favors vertical bone augmentation and the resorption rate of the biomaterials: Histomorphometric and 3D microcomputed tomography analysis.

BACKGROUND: Recombinant human bone morphogenetic protein 2 (rhBMP-2) is an osteoinductor frequently used for bone regeneration in oral and maxillofacial surgery. There is no consensus about the ideal carrier for this growth factor. The aim of this study was to compare the bone augmentation, bone microarchitecture, and biodegradation rate of additional carriers to rhBMP-2/absorbable collagen sponge (ACS) in a vertical guided bone regeneration model. METHODS: Four titanium cylinders were fixed onto the calvaria of rabbits (n = 20) that received (n = 10) or not (n = 10) rhBMP-2/ACS in conjunction with one of the carriers: beta-tricalcium phosphate (ß-TCP), biphasic calcium phosphate (BCP), bovine bone mineral (BBM) or blood clot. The samples were analyzed by means of microcomputed tomography and histomorphology after 14 weeks. RESULTS: All the materials with rhBMP-2/ACS exhibited improvement on bone augmentation, mainly BCP (P = 0.033) and ß-TCP (P = 0.038), in the upper portion of the cylinder. Although trabecular anisotropy was improved in all the materials groups, trabecular connectivity was diminished when the biomaterials received rhBMP-2/ACS. Resorption rate of the remaining biomaterial was improved by rhBMP-2/ACS, mainly in BBM (P <0.01) and ß-TCP (P <0.01). BBM exhibited the highest osteoclast density compared with the other materials groups. CONCLUSIONS: BCP and ß-TCP biomaterials exhibited a synergic effect with rhBMP-2/ACS, acting as suitable and viable carriers for vertical bone augmentation. The addition of rhBMP-2 significantly affected the biodegradation of ß-TCP and BBM, accelerating the resorption of these materials.

Heparin is biocompatible and can induce differentiation of human dental pulp cells.

AIM: To investigate the biocompatibility, osteogenic bioactivity and mRNA expression of the osteo/odontogenic markers bone morphogenetic protein 2 (BMP-2), osteocalcin (OC) and alkaline phosphatase (ALP), induced by heparin in human dental pulp cells (hDPCs). METHODOLOGY: hDPCs were exposed to the heparin, and cell viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT), and cell death was evaluated by flow cytometry. Osteogenic bioactivity was evaluated by the alkaline phosphatase (ALP) assay, and the detection of calcium deposits by alizarin red staining (ARS). The gene expression of BMP-2, OC and ALP was quantified with real-time PCR. Statistical analysis was performed with ANOVA and Bonferroni or Tukey post-test and t-test (α = 0.05). RESULTS: Heparin had no cytotoxic effect and did not induce apoptosis. After 3 days, heparin had significantly higher ALP activity in comparison with the control (P < 0.05). Heparin had a significant (P < 0.05) stimulatory effect on the formation of mineralized nodules. BMP-2 and OC mRNA expressions were significantly higher in cells exposed to heparin than control group after 1 day (P < 0.05). CONCLUSIONS: Heparin was biocompatible in hDPCs, induced osteogenic bioactivity and enhanced mRNA expression of osteo/odontogenic markers BMP-2 and OC. These results suggest that heparin has potential to induce osteo/odontogenic cell differentiation of hDPCs.

Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells

Abstract Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. Results RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

The use of bone morphogenetic proteins (BMP) and pseudarthrosis, a literature review.

Bone morphogenetic proteins (BMP) are multi-functional growth factors to promote bone healing with the proposal of less morbidity compared to the usual methods of bone graft harvest. Pseudoarthrosis occur when the fusion attempt fails, a solid fusion is not achieved, or there is motion across the segment leading to it, and it can be clinically symptomatic as pain, deformity, neurocompression, or hardware failure. BMPs are used at spinal fusion as a tool for the treatment of degenerative, traumatic, neoplastic and infectious conditions of the spine. This review shows that the use of BMPS is effective and secure when compared with iliac crest bone graft (ICGB); however, depending of the location of usage (cervical spine, lumbar spine or sacrum) and the medical status of the patient (presence of comorbidities, tobacco usage), it is more likely to exhibit complications. Therefore, the use of these proteins must be an informed decision of patient and physician preferences.

Proteínas morfogenéticas do osso (Bone morphogenetic proteins [BMP]) são fatores de crescimento multifuncionais que promovem cicatrização óssea, propondo menos comorbidades comparado aos métodos usuais de colheita de enxerto ósseo. Pseudoartroses ocorrem quando a tentativa de fusão óssea falha, uma fusão sólida não é atingida ou quando há movimentação do segmento que leva à pseudoartrose, que pode ser clinicamente sintomática com dor, deformidade, neurocompressão ou falha na colocação de material de síntese. As BMPs são usadas em fusão colunar como ferramenta para o tratamento de trauma degenerativo, condições neoplásicas e infecciosas da coluna. A presente revisão da literatura mostra que o uso de BMPs é efetivo e seguro quando comparado com enxerto ósseo ilíaco. No entanto, a depender do local de uso (coluna cervical ou lombar ou sacro) e do estado médico do paciente (presença de comorbidades, tabagismo) é mais propício o aparecimento de complicações. Portanto, o uso dessas proteínas deve ser decidido após uma decisão conjunta de preferências médicas e do paciente.

The use of bone morphogenetic proteins (BMP) and pseudarthrosis, a literature review / O uso de proteínas morfogenéticas ósseas (BMP) e pseudoartroses, uma revisão de literatura

ABSTRACT Bone morphogenetic proteins (BMP) are multi-functional growth factors to promote bone healing with the proposal of less morbidity compared to the usual methods of bone graft harvest. Pseudoarthrosis occur when the fusion attempt fails, a solid fusion is not achieved, or there is motion across the segment leading to it, and it can be clinically symptomatic as pain, deformity, neurocompression, or hardware failure. BMPs are used at spinal fusion as a tool for the treatment of degenerative, traumatic, neoplastic and infectious conditions of the spine. This review shows that the use of BMPS is effective and secure when compared with iliac crest bone graft (ICGB); however, depending of the location of usage (cervical spine, lumbar spine or sacrum) and the medical status of the patient (presence of comorbidities, tobacco usage), it is more likely to exhibit complications. Therefore, the use of these proteins must be an informed decision of patient and physician preferences.

RESUMO Proteínas morfogenéticas do osso (Bone morphogenetic proteins [BMP]) são fatores de crescimento multifuncionais que promovem cicatrização óssea, propõem menos comorbidades comparada com os métodos usuais de colheita de enxerto ósseo. Pseudoartroses ocorrem quando a tentativa de fusão óssea falha, uma fusão sólida não é atingida ou quando há movimentação do segmento que leva à pseudoartrose, que pode ser clinicamente sintomática com dor, deformidade, neurocompressão ou falha na colocação de material de síntese. As BMPs são usadas em fusão colunar como ferramenta para o tratamento de trauma degenerativo, condições neoplásicas e infecciosas da coluna. A presente revisão da literatura mostra que o uso de BMPs é efetivo e seguro quando comparado com enxerto ósseo ilíaco. No entanto, a depender do local de uso (coluna cervical ou lombar ou sacro) e do estado médico do paciente (presença de comorbidades, tabagismo), é mais propício o aparecimento de complicações. Portanto, o uso dessas proteínas deve ser efetivado após uma decisão conjunta de preferências médicas e do paciente.

Scientific evidence on the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in oral and maxillofacial surgery.

PURPOSE: This study aimed to identify the main indications for the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) for bone repair and maintenance in the maxilla and mandible through a review of clinical trials evaluating the viability of using rhBMP-2 to delay the installation of dental implants, thus allowing satisfactory bone formation and long-term osseointegration. METHODS: Literature search of the PubMed/Medline databases was performed using the following MeSH index terms-"bone morphogenetic protein 2" and "dentistry". Only clinical trials necessarily published in English, related to dentistry, and focused on bone reconstruction in critical defects, post-extraction alveoli, increasing the atrophic alveolar ridge, or surgery for maxillary sinus elevation were included, regardless of the age, sex, ethnicity, associated morbidities, or period of publication. RESULTS: Of the 17 studies identified based on the search filters, 2 were excluded. Therefore, 15 studies were finally included in this review. CONCLUSIONS: Based on the results of our review, we concluded that the use of rhBMP-2 for the preservation of the alveolar ridge after tooth extraction or for increasing the local defects is safe and viable. The use of rhBMP-2/Bio-Oss® for the elevation of the maxillary sinus membrane is unnecessary; however, it can improve and accelerate the maturation process in cases of guided bone regeneration in peri-implant defects. Compounds comprising rhBMP-2, allogenic bone, and plasma-rich platelet (PRP) can act as autograft substitutes in mandibular critical defects.

Effects of ionizing radiation on proteins in lyophilized or frozen demineralized human bone.

OBJECTIVE: The aim was to study the effects of application of ionizing radiation (gamma and electrons) as sterilizing agents at doses of 15 kGy, 25 kGy and 50 kGy, on lyophilized or frozen demineralized bone tissue for use in transplants. METHODS: Five human femoral diaphyses from different donors of musculoskeletal tissue were demineralized and preserved as lyophilized or frozen at -80 °C. The samples were divided into two groups: non-irradiated (control) and irradiated by means of gamma rays or an electron beam. The bone proteins were extracted and used to determine the concentrations of total protein and BMP 2 and 7. RESULTS: Decreases in total protein and BMP 2 and 7 concentrations were observed. The decreases in total protein concentrations, in comparison with the respective control groups, were significant in the lyophilized and frozen samples that were irradiated at a dose of 50 kGy of gamma radiation and electron beam, with reductions of more than 30%. Significant decreases in the levels of BMP 2 and 7 were also observed at higher doses and especially through use of the electron beam. CONCLUSION: The reductions in the concentrations of total proteins and osteoinductive proteins (BMP 2 and 7) were related to the radiation dose, i.e. they increased with higher doses of ionizing radiation type and the type of bone preservation. The largest reductions in concentrations were observed in the bones irradiated by means of an electron beam and at a dose of 50 kGy. However, this type of radiation and this high dose are not usual practices for sterilization of bone tissue.

OBJETIVO: Estudar os efeitos da aplicação das radiações ionizantes (gama e elétrons) como agentes esterilizantes, nas doses de 15 kGy, 25 kGy e 50 kGy, nos tecidos ósseos desmineralizados congelados e liofilizados para uso em transplantes. MÉTODOS: Cinco diáfises femorais humanas de doadores distintos de tecidos musculoesqueléticos foram desmineralizadas e preservadas como liofilizadas ou congeladas a −80 °C. As amostras foram divididas em grupos não irradiados (controle) e irradiados por raios gama ou feixe de elétrons. As proteínas ósseas foram extraídas e dosadas as concentrações de proteínas totais, BMP 2 e 7. RESULTADOS: Foi observada diminuição das concentrações de proteínas totais e BMP 2 e 7. A diminuição das concentrações de proteínas totais, quando comparada com o respectivo controle, foi significativa nos grupos de amostras liofilizadas e congeladas e irradiadas na dose de 50 kGy por radiação gama e feixe de elétrons com redução superiores a 30%. A diminuição significativa nas concentrações das BMP 2 e 7 também foi observada nas maiores doses e principalmente por feixe de elétrons. CONCLUSÃO: As reduções nas concentrações das proteínas totais e em proteínas osteoindutoras (BMP 2 e 7) foram relacionadas à dose de radiação, ou seja, aumentam com maiores doses, tipo de radiação ionizante e ao tipo de preservação dos ossos. As maiores reduções das concentrações foram observadas nos ossos irradiados por feixe de elétrons e na dose de 50 kGy. Porém esse tipo de radiação e essa alta dose não são práticas usuais para a esterilização dos tecidos ósseos.

Effects of ionizing radiation on proteins in lyophilized or frozen demineralized human bone / Efeitos da radiação ionizante nas proteínas presentes em ossos humanos desmineralizados, liofilizados ou congelados

OBJECTIVE: The aim was to study the effects of application of ionizing radiation (gamma and electrons) as sterilizing agents at doses of 15 kGy, 25 kGy and 50 kGy, on lyophilized or frozen demineralized bone tissue for use in transplants. METHODS: Five human femoral diaphyses from different donors of musculoskeletal tissue were demineralized and preserved as lyophilized or frozen at -80 °C. The samples were divided into two groups: non-irradiated (control) and irradiated by means of gamma rays or an electron beam. The bone proteins were extracted and used to determine the concentrations of total protein and BMP 2 and 7. RESULTS: Decreases in total protein and BMP 2 and 7 concentrations were observed. The decreases in total protein concentrations, in comparison with the respective control groups, were significant in the lyophilized and frozen samples that were irradiated at a dose of 50 kGy of gamma radiation and electron beam, with reductions of more than 30%. Significant decreases in the levels of BMP 2 and 7 were also observed at higher doses and especially through use of the electron beam. CONCLUSION: The reductions in the concentrations of total proteins and osteoinductive proteins (BMP 2 and 7) were related to the radiation dose, i.e. they increased with higher doses of ionizing radiation type and the type of bone preservation. The largest reductions in concentrations were observed in the bones irradiated by means of an electron beam and at a dose of 50 kGy. However, this type of radiation and this high dose are not usual practices for sterilization of bone tissue.

OBJETIVO: Estudar os efeitos da aplicação das radiações ionizantes (gama e elétrons) como agentes esterilizantes, nas doses de 15 kGy, 25 kGy e 50 kGy, nos tecidos ósseos desmineralizados congelados e liofilizados para uso em transplantes. MÉTODOS: Cinco diáfises femorais humanas de doadores distintos de tecidos musculoesqueléticos foram desmineralizadas e preservadas como liofilizadas ou congeladas a -80 °C. As amostras foram divididas em grupos não irradiados (controle) e irradiados por raios gama ou feixe de elétrons. As proteínas ósseas foram extraídas e dosadas as concentrações de proteínas totais, BMP 2 e 7. RESULTADOS: Foi observada diminuição das concentrações de proteínas totais e BMP 2 e 7. A diminuição das concentrações de proteínas totais, quando comparada com o respectivo controle, foi significativa nos grupos de amostras liofilizadas e congeladas e irradiadas na dose de 50 kGy por radiação gama e feixe de elétrons com redução superiores a 30%. A diminuição significativa nas concentrações das BMP 2 e 7 também foi observada nas maiores doses e principalmente por feixe de elétrons. CONCLUSÃO: As reduções nas concentrações das proteínas totais e em proteínas osteoindutoras (BMP 2 e 7) foram relacionadas à dose de radiação, ou seja, aumentam com maiores doses, tipo de radiação ionizante e ao tipo de preservação dos ossos. As maiores reduções das concentrações foram observadas nos ossos irradiados por feixe de elétrons e na dose de 50 kGy. Porém esse tipo de radiação e essa alta dose não são práticas usuais para a esterilização dos tecidos ósseos.

Medium modification with bone morphogenetic protein 2 addition for odontogenic differentiation

Abstract The aim of this study was to evaluate whether medium modification improves the odontogenic differentiation of human dental pulp stem cells (DPSC) in vitro and in vivo. DPSC isolated from human impacted third molar teeth were analysed for clusters of differentiation with flow cytometry. Odontogenic differentiation was stimulated by medium modification with the addition of bone morphogenetic protein 2 (BMP2). The expression of dentin sialophosphoprotein, dentin matrix protein 1, enamelysin/matrix metalloproteinase 20 and the phosphate-regulating gene with homologies to endopeptidases on the X chromosome of the cells were analysed with RT-PCR at 7, 14 and 21 days. Then, DPSC were transplanted on the back of immunocompromised mice via a hydroxyapatite tricalcium phosphate scaffold, and the structure of the formed tissue was investigated. The cells were identified as mesenchymal stem cells with a 98.3% CD73 and CD90 double-positive cell rate. The increase in mineralization capacity and expression of human enamel-dentin specific transcripts proportional to the culture period were determined after differentiation. Six weeks after transplantation, an osteo-dentin matrix was formed in the group in which odontogenic differentiation was stimulated, and the odontogenic characteristics of the matrix were confirmed by histological examination and RT-PCR analysis. Odontogenic differentiation of the isolated and characterized human DPSC was improved with medium modification by the addition of BMP2 in vitro and in vivo. The defined medium and applied technique have a potential use for forming reparative dentin in the future, but the effects of the method should be investigated in long-term studies.

O uso da proteína recombinante no aumento ósseo em Implantodontia / Recombinant protein for bone augmentation in Implantology

A reabsorção do osso alveolar ocorre após a perda dentária por vários fatores, tais como: exodontia, traumatismo, processos patológicos ou mesmo reabsorção por desuso. A reabilitação com implantes requer osso em quantidade e qualidade. Os enxertos autógenos são considerados os enxertos tipo "padrão ouro" para regeneração óssea guiada. Contudo, o aumento ósseo dessa região tem sido feito também com biomateriais, que é qualquer substância, droga, combinação de substâncias de origem natural ou sintética que pode ser usado como reposição de algum tecido, órgão ou função do corpo. Embora promissora, a utilização das proteínas morfogenéticas ósseas na Implantodontia e na Cirurgia Bucomaxilofacial ainda necessita de maior respaldo científico. O objetivo desse estudo foi fazer uma breve revisão sobre a utilização das proteínas recombinantes e esclarecer os clínicos sobre suas indicações e reais limitações.

The bone alveolar reabsortion occurs after lost dental by many factors such as: exodontic, traumatism, pathologic process or even atrophy due to disuse. Implant supported rehabilitation requires amount and quality of bone. Autogenous bone has been considered "the gold standard" graft to guided bone regeneration. However, the bone augmentation in this region has been done with biomaterials too, that is any substance, drug, combination of substances of natural or synthetic origin which may be used for a replacement tissue, organ or body function. Although promising, the widespread use of bone morphogenetic proteins in Implantology and in Maxillofacial Surgery demands greater scientific support. The aim of this study was to do a brief review to the use of recombinant proteins and clarify clinical about its indications and true limitations.

Bases genéticas de la formación de fisuras labiales y/o palatinas en humanos / Genetic basis of orofacial cleft formation in humans

La hendidura labio palatina (cleft lip/palate, CL/P) es una alteración craneofacial de alta frecuencia en la población mundial, cuya etiología es multifactorial, y que involucra factores tanto genéticos como ambientales. La mayoría de este tipo de patologías se presenta como un defecto no sindrómico (70%), y el resto de hendiduras se asocian con alteraciones adicionales en los defectos de tipo sindrómico. Gracias al desarrollo de herramientas moleculares se han identificado varios genes que están involucrados en CL/P de tipo no sindrómico, como son el proto-oncogen Bcl3, el gen Tgfb, el gen homeótico Msx1 y el gen Bmp entre otros, los cuales se han podido identificar mediante estudios de ligamiento y el uso de ratones deficientes en estos genes (knock-out). Por otra parte, se han identificado más de 300 síndromes diferentes asociados con CL/P, entre ellos uno de los más frecuentes el síndrome de van der Woude donde también se han identificado mutaciones en genes como el IRF6. Debido a que CL/P es una patología de alta complejidad etiológica donde las alteraciones genéticas juegan un importante papel y donde cada día se amplía el conocimiento sobre alteraciones en diversos genes que contribuyen a la formación de la alteración, el objetivo de este artículo, es revisar la información actualizada sobre la genética del CL/P y los genes reportados que pueden contribuir al desarrollo de esta compleja patología.

Clefts of the lip and palate (CL/P) are a craneofacial alteration of high frequency in the world-wide population, whose etiology is multifactorial, involving genetic factors as much as environmental ones. Most of this type of pathologies (70%) appear as non-syndromic form, although the presence of additional facial alterations is associated with clefts of syndromic type. The development of molecular tools has permitted to identify some of the genes that are involved in non-syndromic CL/P, such as proto-oncogen Bcl3, gene Tgfb, homeotic gene Msx1 and Bmp among others, which have demonstrated a relationships between them using both linkage analysis and knock-out mice. In the other hand, they have been identified more of 300 different syndromes associated with CL/P, being one from most frequent the van der Woude syndrome which also have identified mutations in genes such as IRF6. Because CL / P is a highly complex etiology pathology where genetic alterations play an important role and where every day is more knowledge on alterations in several genes that contribute to the formation of the alteration, the aim of this article is review the current knowledge on the reported genetics of the CL/P and genes that would contribute to the development of this complex pathology.

A rhBMP-2 aumenta e acelera a formação óssea vertical quando associada a biomateriais em calvária de coelhos / rhBMP-2 increases and accelerates vertical bone formation when associated to biomaterials: the rabbit calvaria model

O objetivo deste estudo foi avaliar a formação óssea vertical aposicional obtida com a associação da proteína morfogenética óssea recombinante humana 2 (rhBMP-2) com os materiais beta fosfato tricálcico (CE), fosfato de cálcio bifásico (BC) e osso mineral bovino (BO) em um modelo de regeneração óssea guiada (ROG) em calvária de coelhos; 22 coelhos albinos da raça Nova Zelândia receberam quatro cilindros de titânio fixados na calvária. No grupo 1 (n = 10), os cilindros foram aleatoriamente preenchidos com um dos referidos materiais e coágulo sanguíneo (CO). No grupo 2 (n = 12), foi repetida a mesma distribuição aleatória dos materiais e coágulo, acrescidos da rhBMP-2. Corantes ósseos foram aplicados durante 13 semanas e a eutanásia foi realizada na 14a semana nos dois grupos. Os resultados clínicos mostraram que o volume ósseo e a sua área média no grupo 2 (com rhBMP-2) foram maiores do que no grupo 1 (sem rhBMP-2), independente dos materiais utilizados (p < 0,001). Não houve diferença estatisticamente significante entre os volumes médios de BC e BO (p = 0,625), BC e CE (p = 0,876), BO e CE (p = 0,968). O volume médio do CO foi menor do que todos os materiais (p < 0,001) nos dois grupos. Não houve diferença significativa entre as médias das áreas em BC e BO (p = 0,965), BC e CE (p = 0,866), BO e CE (p = 0,990), BC e CO (p = 0,190), BO e CO (p = 0,060). A área média do CO foi menor em CE (p = 0,028). O estudo histológico revelou quantidades significativas de tecido ósseo neoformado nos dois grupos. No entanto, no grupo com rhBMP-2, foi observado osso mais maduro e lamelar, e com maior taxa de biodegradação em todos os materiais. A análise de fluorescência mostrou que a adição de rhBMP-2 é mais significativa na porção superior do cilindro e que a formação óssea é tempo-dependente. Pôde-se concluir que a utilização da rhBMP-2 associada a qualquer um dos biomateriais gerou maior formação óssea neste modelo de regeneração óssea guiada (ROG) em coelhos.

The aim of this study was to evaluate the appositional vertical bone formation obtained with the association of recombinant human bone morphogenetic protein 2 (rhBMP-2) with the materials Beta Tricalcium Phosphate (CE), Biphasic Calcium Phosphate (BC), and Mineral Bovine Bone (BO) in a guided bone regeneration model (GBR) in rabbit calvaria. Twenty-two New Zealand white rabbits received four titanium cylinders fixed in the calvaria. In group 1 (n = 10), each cylinder was randomly filled with one of these materials and one with a blood clot (CO). In group 2 (n = 12) cylinders were randomly assigned the same materials and blood clot with the addition of rhBMP-2. Bone labels were injected during 13 weeks, and the euthanasia was performed in the 14th week in both groups. The results showed that the average volume and area in group 2 (with rhBMP-2) was higher than that in group 1 (without rhBMP-2), regardless the materials used (p < 0.001). There was no statistically significant difference between the average volumes of BO and BC (p = 0.625), BC and CE (p = 0.876) and BO and CE (p = 0.968). The average volume of CO was lower than all materials (P < 0.001) in both groups. There was no statistically significant difference between the average areas of BC and BO (p = 0,965), BC and CE (p = 0,866), BO and CE (p = 0,990), BC and CO (p = 0,190) and BO and CO (p = 0,060). The average area of CO was lower than CE (p = 0,028). The histological study revealed significant amounts of newly formed bone in both groups. Nevertheless, in the group with rhBMP-2 a more mature and lamellar bone with a greater level of biodegradation in all materials was observed. The fluorescence analysis showed that the addition of rhBMP-2 is more significant in the upper portion of the cylinder and that bone formation is time-dependent...