Exploring the osteogenic potential of semisynthetic triterpenes from Combretum leprosum: An in vitro and in silico study.

Combretum leprosum Mart. is a plant of the Combretaceae family, widely distributed in the Northeast region of Brazil, popularly used as an anti-inflammatory agent, and rich in triterpenes. This study evaluated in vitro and in silico potential osteogenic of two semisynthetic triterpenes (CL-P2 and CL-P2A) obtained from the pentacyclic triterpene 3ß,6ß,16ß-trihydroxylup-20(29)-ene (CL-1) isolated from C. leprosum. Assays were carried out in cultured murine osteoblasts (OFCOL II), first investigating the possible toxicity of the compounds on these cells through viability assays (MTT). Cell proliferation and activation were investigated by immunohistochemical evaluation of Ki-67, bone alkaline phosphatase (ALP) activity, and mineralization test by Von Kossa. Molecular docking analysis was performed to predict the binding affinity of CL-P2 and CL-P2A to target proteins involved in the regulation of osteogenesis, including: bone morphogenetic protein 2 (BMP-2), proteins related to Wingless-related integration (WNT) pathway (Low-density lipoprotein receptor-related protein 6-LRP6 and sclerostin-SOST), and receptor activator of nuclear factor (NF)-kB-ligand (RANK-L). Next, Western Blot and immunofluorescence investigated BMP-2, WNT, RANK-L, and OPG protein expressions in cultured murine osteoblasts (OFCOL II). None of the CL-P2 and CL-P2A concentrations were toxic to osteoblasts. Increased cell proliferation, ALP activity, and bone mineralization were observed. Molecular docking assays demonstrated interactions with BMP-2, LRP6, SOST, and RANK-L/OPG. There was observed increased expression of BMP-2, WNT, and RANK-L/OPG proteins. These results suggest, for the first time, the osteogenic potential of CL-P2 and CL-P2A.

Effects of photobiomodulation on different phases of in vitro osteogenesis.

The present study aimed to evaluate the effect of photobiomodulation therapy (PBM) on different stages of osteogenesis in vitro. For this, osteoblastic-like cells (Saos-2 cell lineage) were irradiated in two different periods: during the Proliferation phase (PP; from the second to the fourth day) and during the Differentiation phase (DP; from the seventh to the ninth day). The energy density used in the study was 1.5 J/ cm2. The following parameters were evaluated: 1) quantification of collagen type 1 (COL 1), osteopontin (OPN), and bone morphogenetic protein 2 (BMP-2); 2) quantification of alkaline phosphatase (ALP) activity; and 3) quantification of  extracellular matrix (ECM) mineralization. Non-irradiated cultures were used as controls. The data were analyzed using the Student's t-test or one-way ANOVA, considering a significance level of 5%. The results indicated that COL 1 and BMP-2 quantification was higher in Saos-2 irradiated during the DP in relation to the control group at day 10 (p < 0.05). No differences were observed for other comparisons at this time point (p > 0.05). OPN expression was greater in PP compared with the other experimental groups at day 10 (p < 0.05). Irradiation did not affect ALP activity in Saos-2 regardless of the exposure phase and the time point evaluated (p > 0.05). At day 14, ECM mineralization was higher in Saos-2 cultures irradiated during the DP in relation to the PP (p < 0.05). In conclusion, the results suggested that the effects of PBM on osteoblastic cells may be influenced by the stage of cell differentiation.

Single Nucleotide Polymorphisms in RUNX2 and BMP2 contributes to different vertical facial profile.

The vertical facial profile is a crucial factor for facial harmony with significant implications for both aesthetic satisfaction and orthodontic treatment planning. However, the role of single nucleotide polymorphisms (SNPs) in the development of vertical facial proportions is still poorly understood. This study aimed to investigate the potential impact of some SNPs in genes associated with craniofacial bone development on the establishment of different vertical facial profiles. Vertical facial profiles were assessed by two senior orthodontists through pre-treatment digital lateral cephalograms. The vertical facial profile type was determined by recommended measurement according to the American Board of Orthodontics. Healthy orthodontic patients were divided into the following groups: "Normodivergent" (control group), "Hyperdivergent" and "Hypodivergent". Patients with a history of orthodontic or facial surgical intervention were excluded. Genomic DNA extracted from saliva samples was used for the genotyping of 7 SNPs in RUNX2, BMP2, BMP4 and SMAD6 genes using real-time polymerase chain reactions (PCR). The genotype distribution between groups was evaluated by uni- and multivariate analysis adjusted by age (alpha = 5%). A total of 272 patients were included, 158 (58.1%) were "Normodivergent", 68 (25.0%) were "Hyperdivergent", and 46 (16.9%) were "Hypodivergent". The SNPs rs1200425 (RUNX2) and rs1005464 (BMP2) were associated with a hyperdivergent vertical profile in uni- and multivariate analysis (p-value < 0.05). Synergistic effect was observed when evaluating both SNPs rs1200425- rs1005464 simultaneously (Prevalence Ratio = 4.0; 95% Confidence Interval = 1.2-13.4; p-value = 0.022). In conclusion, this study supports a link between genetic factors and the establishment of vertical facial profiles. SNPs in RUNX2 and BMP2 genes were identified as potential contributors to hyperdivergent facial profiles.

Investigation of polymorphisms in BMP2, BMP4, SMAD6 and RUNX2 genes and pulp stones.

This study aimed to assess the association between genetic polymorphisms in BMP2 (rs1005464 and rs235768), BMP4 (rs17563), SMAD6 (rs2119261 and rs3934908) and RUNX2 (rs59983488 and rs1200425) and pulp stones (PS). A total of 117 participants, consisting of 63 individuals with PS and 54 without PS, were included. Digital radiographs and a demographic/clinical questionnaire were used. Genomic DNA from salivary cells was genotyped via real-time polymerase chain reaction. Statistical analyses, including Chi-Square, Fisher's exact tests, Poisson regression and dimensionality reduction, were conducted. The rs2119261 polymorphism in the SMAD6 gene showed an association with genotype distribution in the recessive model (p = 0.049). The T-T haplotype in the SMAD6 gene (rs2119261 and rs3934908) was more prevalent in the control group and significantly linked with PS (p = 0.029). No associations were found between PS risk and genetic polymorphisms in BMP2, BMP4 and RUNX2. Polymorphisms in the SMAD6 gene were associated with PS.

Recombinant Human Peptide Growth Factors, Bone Morphogenetic Protein-7 (rhBMP7), and Platelet-Derived Growth Factor-BB (rhPDGF-BB) for Osteoporosis Treatment in an Oophorectomized Rat Model.

Bone morphogenetic protein (BMP) and platelet-derived growth factor (PDGF) are known to regulate/stimulate osteogenesis, playing vital roles in bone homeostasis, rendering them strong candidates for osteoporosis treatment. We evaluated the effects of recombinant human BMP-7 (rhBMP7) and PDGF-BB (rhPDGF-BB) in an oophorectomy-induced osteoporosis rat model. Forty Sprague Dawley rats underwent oophorectomy surgery; treatments commenced on the 100th day post-surgery when all animals exhibited signs of osteoporosis. These peptide growth factors were administered intraocularly (iv) once or twice a week and the animals were monitored for a total of five weeks. Two weeks after the conclusion of the treatments, the animals were euthanized and tissues were collected for assessment of alkaline phosphatase, X-ray, micro-CT, and histology. The results indicate that the most promising treatments were 20 µg/kg rhPDGF-BB + 30 µg/kg rhBMP-7 twice a week and 30 µg/kg BMP-7 twice a week, showing significant increases of 15% (p < 0.05) and 13% (p < 0.05) in bone volume fraction and 21% (p < 0.05) and 23% (p < 0.05) in trabecular number, respectively. In conclusion, rhPDGF-BB and rhBMP-7 have demonstrated the ability to increase bone volume and density in this osteoporotic animal model, establishing them as potential candidates for osteoporosis treatment.

BMP2 rs1005464 is associated with mandibular condyle size variation.

This study aimed to evaluate the association between single nucleotide polymorphisms (SNPs) in endochondral development-related genes and mandibular condyle shape, size, volume, and symmetry traits. Cone-beam Computed Tomographies and genomic DNA from 118 individuals were evaluated (age range: 15-66 years). Data from twelve 3D landmarks on mandibular condyles were submitted to morphometric analyses including Procrustes fit, principal component analysis, and estimation of centroid sizes and fluctuating asymmetry scores. Condylar volumes were additionally measured. Seven SNPs across BMP2, BMP4, RUNX2 and SMAD6 were genotyped. Linear models were fit to evaluate the effect of the SNPs on the mandibular condyles' quantitative traits. Only the association between BMP2 rs1005464 and centroid size remained significant after adjusting to account for the false discovery rate due to multiple testing. Individuals carrying at least one A allele for this SNP showed larger condylar size than common homozygotes GG (ß = 0.043; 95% CI: 0.014-0.071; P value = 0.028). The model including BMP2 rs1005464, age and sex of the participants explained 17% of the variation in condylar size. Shape, volume, and symmetry were not associated with the evaluated SNPs. These results suggest that BMP2 rs1005464 might be associated with variation in the mandibular condyles size.

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.

Effect of the secretome of mesenchymal stem cells overexpressing BMP-9 on osteoblast differentiation and bone repair.

The secretome present in the conditioned medium (CM) of mesenchymal stem cells (MSCs) is a promising tool to be used in therapies to promote bone regeneration. Considering the high osteogenic potential of the bone morphogenetic protein 9 (BMP-9), we hypothesized that the secretome of MSCs overexpressing BMP-9 (MSCsBMP-9 ) enhances the osteoblast differentiation of MSCs and the bone formation in calvarial defects. CM of either MSCsBMP-9 (CM-MSCsBMP-9 ) or MSCs without BMP-9 overexpression (CM-MSCsVPR ) were obtained at different periods. As the CM-MSCsBMP-9 generated after 1 h presented the highest BMP-9 concentration, CM-MSCsBMP-9 and CM-MSCsVPR were collected at this time point and used to culture MSCs and to be injected into mouse calvarial defects. The CM-MSCsBMP-9 enhanced the osteoblast differentiation of MSC by upregulating RUNX2, alkaline phosphatase (ALP) and osteopontin protein expression, and ALP activity, compared with CM-MSCsVPR . The CM-MSCsBMP-9 also enhanced the bone repair of mouse calvarial defects, increasing bone volume, bone volume/total volume, bone surface, and trabecular number compared with untreated defects and defects treated with CM-MSCsVPR or even with MSCsBMP-9 themselves. In conclusion, the potential of the MSCBMP-9 -secretome to induce osteoblast differentiation and bone formation shed lights on novel cell-free-based therapies to promote bone regeneration of challenging defects.

Monoallelic loss-of-function BMP2 variants result in BMP2-related skeletal dysplasia spectrum.

PURPOSE: Bone morphogenic proteins (BMPs) regulate gene expression that is related to many critical developmental processes, including osteogenesis for which they are named. In addition, BMP2 is widely expressed in cells of mesenchymal origin, including bone, cartilage, skeletal and cardiac muscle, and adipose tissue. It also participates in neurodevelopment by inducing differentiation of neural stem cells. In humans, BMP2 variants result in a multiple congenital anomaly syndrome through a haploinsufficiency mechanism. We sought to expand the phenotypic spectrum and highlight phenotypes of patients harboring monoallelic missense variants in BMP2. METHODS: We used retrospective chart review to examine phenotypes from an international cohort of 18 individuals and compared these with published cases. Patient-derived missense variants were modeled in zebrafish to examine their effect on the ability of bmp2b to promote embryonic ventralization. RESULTS: The presented cases recapitulated existing descriptions of BMP2-related disorders, including craniofacial, cardiac, and skeletal anomalies and exhibit a wide phenotypic spectrum. We also identified patients with neural tube defects, structural brain anomalies, and endocrinopathies. Missense variants modeled in zebrafish resulted in loss of protein function. CONCLUSION: We use this expansion of reported phenotypes to suggest multidisciplinary medical monitoring and management of patients with BMP2-related skeletal dysplasia spectrum.

Bone Morphogenetic Protein 2 Plus Leukocyte and Platelet-Rich Fibrin for the Treatment of MRONJ.

Leukocyte and platelet-rich fibrin is known to contain high concentrations of growth factors and when associated with rhBMP-2, it may increase bone remodeling due to its osteoinductive property. The aim of this case is to report the outcome of surgical treatment of medication-related osteonecrosis of the jaw with prototype plate installation and the use of leukocyte and platelet-rich fibrin in association with rhBMP-2 in a 78-year-old female patient under therapy with alendronate. The present Studies describes that the combination of this treatment presented complete healing of osteonecrosis and represents a promising treatment option to be used for medication-related osteonecrosis of the jaw.

Histological and Immunohistochemical Analysis of the Effects of Topical Melatonin Treatment Associated with Collagen Sponge and rhBMP-2 Protein on Bone Remodeling.

Extensive bone defect healing is an important health issue not yet completely resolved. Different alternative treatments have been proposed but, in face of a critical bone defect, it is still very difficult to reach a complete regeneration, with the new-formed bone presenting all morphological and physiological characteristics of a normal, preinjury bone. Topical melatonin use has shown as a promising adjuvant for bone regeneration due to its positive effects on bone metabolism. Thus, to search for new, safe, biological techniques that promote bone repair and favor defect healing, we hypothesized that there is a synergistic effect of melatonin treatment associated with rhBMP-2 to guide bone regeneration. This study aimed to investigate bone repair effects of topical melatonin administration in different concentrations (1, 10, and 100 µg), associated or not with rhBMP-2. Surgical-induced bone defect healing was qualitatively evaluated through histopathological analysis by light microscopy. Additionally, quantitative stereology was performed in immunohistochemistry-prepared tissue to identify angiogenic, osteogenic, and osteoclastogenic factors. Quantification data were compared between groups by the ANOVA/Tukey test and differences were considered significant when p < 0.05. Our results showed that the presence of the scaffold in the bone defect hindered the process of bone repair because in the group treated with "blood clot + scaffold" the results of bone formation and immunolabeling were reduced in comparison with all other groups (treated with melatonin alone or in association with rhBMP-2). Statistical analysis revealed a significant difference between the control group (bone defect + blood clot), and groups treated with different concentrations of melatonin in association with rhBMP-2, indicating a positive effect of the association for bone repair. This treatment is promising once it becomes a new safe alternative technique for the clinical treatment of fractures, bone defects, and bone grafts. Our results support the hypothesis of the safe use of the association of melatonin and rhBMP-2 and have established a safe and effective dose for this experimental treatment.

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

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.

Assessing the prevalence of S-shaped root canal and associated genes in humans.

BACKGROUND: Multiple signaling molecules have been shown to play crucial roles in dental root development. Therefore, we aimed to investigate the prevalence of S-shaped roots and also to investigate, if single nucleotide polymorphisms (SNPs) in BMP2, BMP4 and SMAD6 are associated with this phenotype in humans. METHODS: This is a cross-sectional phenotype-genotype association study that used radiographs to determine the phenotypes and DNA to investigate SNPs in candidate genes. During the radiographic exam, teeth presenting root canal(s) doubly curved were considered S-shaped roots. SNPs in BMP2 (rs1005464 and rs235768), BMP4 (rs17563) and SMAD6 (rs2119261 and rs3934908) were blindly genotyped by real-time PCR using TaqMan assay. The relative and absolute frequency of S-shaped roots were calculated. Chi-square test was used to compare the genotype distributions between control and S-shaped groups. RESULTS: Among the 578 subjects, 61 (10.6 %) presented at least one tooth with an S-shaped root. The most commonly affected type of tooth was the premolar. rs1005464 in BMP2 was statistically associated with an S-shaped root (p = 0.036). rs235768 in BMP2 was associated with an S-shaped root also in mandibular teeth (p = 0.017). A statistical significance was observed for the rs3934908 in SMAD6 (p = 0.049) for S-shaped root in the mandible. In the analysis stratified according to the type of tooth, rs235768 in BMP2 was associated with S-shaped roots in premolars (p = 0.029). CONCLUSION: The prevalence of S-shaped roots is 10.6 % in permanent teeth. SNPs in BMP2 and SMAD6 could be involved in a higher chance to present S-shaped roots.

Comparison of rhBMP-2 in Combination with Different Biomaterials for Regeneration in Rat Calvaria Critical-Size Defects.

Regeneration of critical bone defects requires the use of biomaterials. The incorporation of osteoinductive agents, such as bone morphogenetic proteins (BMPs), improves bone formation. This study aimed to compare the efficacy of rhBMP-2 in combination with different materials for bone regeneration in critical-sized rat calvarial defects. This was an experimental animal study using 30 rats. In each rat, two 5-mm critical-size defects were made in the calvaria (60 bone defects in total) using a trephine. All rats were randomized to one of the six groups: control (C), autograft + rhBMP-2 (A), absorbable collagen sponge + rhBMP-2 (ACS), ß-tricalcium phosphate + rhBMP-2 (B-TCP), bovine xenograft + rhBMP-2 (B), and hydroxyapatite + rhBMP-2 (HA). The outcome was assessed after 4 and 8 weeks using histological description and the histological bone healing scale. Statistical analysis was performed using the Kruskal-Wallis and Mann-Whitney U tests, with a p-value set at 0.05. The average bone healing scores per group were as follows: C group, 12.5; A group, 26.5; ACS group, 18.8; B-TCP group, 26.2; HA group, 20.9; and B group, 20.9. The C group showed a significant difference between weeks 4 and 8 (p = 0.032). Among the 4-week groups, the C group showed a significant difference compared to A (p = 0.001), ACS (p = 0.017), and B-TCP (p = 0.005) groups. The 8-week experimental group did not show any significant differences between the groups. The 5-mm critical size defect in rat calvaria requires the use of bone biomaterials to heal at 4 and 8 weeks. rhBMP-2, as applied in this study, showed no difference in new bone formation when combined with bovine, B-TCP, or HA biomaterials.

The Rise and Fall of Bone Morphogenetic Protein 2 Throughout the United States.

STUDY DESIGN: Retrospective Database Study. OBJECTIVE: Investigate utilization of bone morphogenetic protein (BMP-2) between 2004 and 2014. SUMMARY OF BACKGROUND DATA: The utilization, particularly off-label utilization, of BMP-2 has been controversial and debated in the literature. Given the concerns regarding cancer and potential complications, the risk benefit profile of BMP must be weighed with each surgical case. The debate regarding the costs and potential side effects of BMP-2 compared with autologous iliac crest bone harvest has continued. METHODS: The National Inpatient Sample (NIS) database was queried for the use of BMP-2 (ICD-9-CM 84.52) between 2004 and 2014 across 44 states. The NIS database represents a 20% sample of discharges, weighted to provide national estimates. BMP-2 utilization rates in spine surgery fusion procedures were calculated as a fraction of the total number of thoracic, lumbar, and sacral spinal fusion surgeries performed each year. RESULTS: Between 2004 and 2014, BMP-2 was utilized in 927,275 spinal fusion surgeries. In 2004, BMP-2 was utilized in 28.3% of all cases (N=48,613). The relative use of BMP-2 in spine fusion surgeries peaked in 2008 at 47.0% (N=112,180). Since then, it has continued to steadily decline with an endpoint of 23.6% of cases in 2014 (N=60,863). CONCLUSIONS: Throughout the United States, the utilization of BMP-2 in thoracolumbar fusion surgeries increased from 28.3% to 47.0% between 2004 and 2008. However, from 2008 to 2014, the utilization of BMP-2 in thoracolumbar spine fusion surgeries decreased significantly from 47.0% to 23.4%. While this study provides information on the utilization of BMP-2 for the entire United States over an 11-year period, further research is needed to the determine the factors affecting these trends.

BMP-2 and asporin expression regulate 5-aza-dC-mediated osteoblast/cementoblast differentiation of periodontal dental ligament mesenchymal progenitor cells.

Periodontal dental ligament (PDL) is composed of heterogeneous population of mesenchymal progenitor cells. The mechanisms that regulate the differentiation of these cells towards osteoblast/cementoblast phenotype are not fully understood. Some studies have demonstrated that is possible to change the pattern of cell differentiation via epigenetic mechanisms. The proposal of this study was to investigate whether 5-aza-2'-deoxycytidine (5-aza-dC) treatment would stimulate the osteoblast/cementoblast differentiation of periodontal ligament mesenchymal progenitor cells (PDL-CD105+ enriched cells), characterized as low osteoblast potential, through bone morphogenetic protein-2 (BMP-2) modulation. PDL-CD105+ cells from a single donor were cloned and characterized in two populations as high osteoblast/cementoblast potential (HOP) and low osteoblast/cementoblast potential (LOP) by mineralization in vitro and expression of osteogenic gene markers, such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), bone morphogenetic protein 2 (BMP-2) and asporin (ASPN). Next, two LOP clones (L1 and L2) were pretreated with 5-aza-dC (10 µM) for 48 h, cultured under osteogenic condition and evaluated for mineralized matrix in vitro, transcription modulation of osteogenic gene markers, methylated and hydroxymethylated DNA levels of BMP-2 and ASPN and intracellular/extracellular expression of BMP-2 protein. LOP clones showed high expression of ASPN transcripts associated with low mRNA levels of BMP-2, RUNX2, ALP, and OCN. 5-aza-dC treatment raised hydroxymethylated DNA levels of BMP-2 and increased the expression of BMP-2 transcripts in both LOP clones. However, BMP-2 protein (intracellular and secreted forms) was detected only in L1 cell clones, in which it was observed an increased expression of osteoblast/cementoblast markers (RUNX2, ALP, OCN) associated with higher mineralization in vitro. In L2 cell clones, 5-aza-dC increased gene expression of ASPN, with no great change in for osteoblast/cementoblast differentiation potential. These data show that 5-aza-dC improves osteoblast/cementoblast differentiation of PDL-CD105+ cells via BMP-2 secretion, and this effect depends on low levels of ASPN expression.

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

Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing.

Human BMP-2, a homodimeric protein that belongs to the TGF- ß family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Effect of rhBMP-2 applied with a 3D-printed titanium implant on new bone formation in rabbit calvarium.

OBJECTIVE: This study sought to compare the biocompatibility of a three-dimensional (3D)-printed titanium implant with a conventional machined titanium product, as well as the effect of such implant applied with recombinant human Bone Morphogenetic Protein Type 2 (rhBMP-2) for guided bone regeneration. METHODOLOGY: Disk-shaped titanium specimens fabricated either by the conventional machining technique or by the 3D-printing technique were compared by MC3T3-E1 cells cytotoxicity assay. New bone formation was evaluated using a rapid prototype titanium cap applied to the calvaria of 10 rabbits, which were divided into two groups: one including an atelopeptide collagen plug on one side of the cap (group I) and the other including a plug with rhBMP-2 on the other side (group II). At six and 12 weeks after euthanasia, rabbits calvaria underwent morphometric analysis through radiological and histological examination. RESULTS: Through the cytotoxicity assay, we identified a significantly higher number of MC3T3-E1 cells in the 3D-printed specimen when compared to the machined specimen after 48 hours of culture. Moreover, morphometric analysis indicated significantly greater bone formation at week 12 on the side where rhBMP-2 was applied when evaluating the upper portion immediately below the cap. CONCLUSION: The results suggest that 3D-printed titanium implant applied with rhBMP-2 enables new bone formation.