Evaluation of Maxillary Alveolar Reconstruction Using a Resorbable Collagen Sponge with Recombinant Human Bone Morphogenetic Protein-2 in Cleft Lip and Palate Patients

Introduction: A resorbable collagen matrix with recombinant human bone morphogenetic protein (rhBMP-2) was compared with traditional iliac crest bone graft for the closure of alveolar defects during secondary dental eruption. Methods: Sixteen patients with unilateral cleft lip and palate, aged 8 to 12 years, were selected and randomly assigned to group 1 (rhBMP-2) or group 2 (iliac crest bone graft). Computed tomography was performed to assess both groups preoperatively and at months 6 and 12 postoperatively. Bone height and defect volume were calculated through Osirix Dicom Viewer (Pixmeo, Apple Inc.). Overall morbidity was recorded. Results: Preoperative and follow-up examinations revealed progressive alveolar bone union in all patients. For group 1, final completion of the defect with a 65.0% mean bone height was detected 12 months postoperatively. For group 2, final completion of the defect with an 83.8% mean bone height was detected 6 months postoperatively. Dental eruption routinely occurred in both groups. Clinical complications included significant swelling in three group 1 patients (37.5%) and significant donor-site pain in seven group 2 patients (87.5%). Conclusions: For this select group of patients with immature skeleton, rhBMP-2 therapy resulted in satisfactory bone healing and reduced morbidity compared with traditional iliac crest bone grafting.

Effect of recombinant human bone morphogenetic protein-2 on bone formation in the acute distraction osteogenesis of rat mandibles

Background Distraction osteogenesis (DO) is a method of producing new bone directly from the osteotomy site by gradual traction of the divided bone fragments. Aim The purpose of the present study was to evaluate histomorphometrically whether acute DO would constitute a viable alternative to the conventional continuous distraction treatment and also to verify the capacity of a recombinant human BMP (rhBMP-2) associated with monoolein gel to stimulate bone formation in the acute distraction process. Materials and methods Forty-eight Wistar rats were assigned to three groups: Group 1, treated at a conventional continuous distraction rate (0.5 mm/day), Group 2, treated with acute distraction of 2.5 mm at the time of the surgical procedure, and Group 3, subjected to acute distraction associated with rhBMP-2. The animals from each experimental group were killed at the end of the second or fourth post-operative weeks and the volume fraction of newly formed bone trabeculae was estimated in histological images by a differential point-counting method. Results The results showed that after 2 and 4 weeks, bone volumes in the rhBMP-2 group were significantly higher than in the other groups (P < 0.05), but no significant difference was observed in the volume fraction of newly formed bone between the continuous and acute DO groups. Conclusion In conclusion, the study indicates that rhBMP-2 can enhance the bone formation at acute DO, which may potentially reduce the treatment period and complications related to the distraction procedure. To cite this article:Issa JPM, do Nascimento C, Lamano T, Iyomasa MM, Sebald W, de Albuquerque Jr RF. Effect of recombinant human bone morphogenetic protein-2 on bone formation in the acute distraction osteogenesis of rat mandibles.Clin. Oral Impl. Res. 20, 2009; 1286-1292.doi: 10.1111/j.1600-0501.2009.01799.x.

Immunolocalization of bone morphogenetic protein 2 during the early healing events after guided bone regeneration

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Immunolocalization of bone morphogenetic protein 2 during the early healing events after guided bone regeneration

Objective. The objective of this study was to evaluate the immunolocalization of bone morphogenetic protein 2 (BMP-2) after autogenous block grafting covered or not with an e-PTFE membrane. Study Design. Forty-eight rats were divided into 2 groups, autogenous block graft (B) and autogenous block graft + e-PTFE membrane (MB), and were evaluated by immunohistochemistry at baseline and 3, 7, 14, 21, and 45 days. Results. The largest number of positive cells in the recipient bed was observed after 3 days in both groups. At the graft border, the largest number of positive cells was seen after 7 days in group B and after 14 days in group MB. The highest proportion of staining in the graft was observed after 3 days in group B and after 21 days in group MB. Conclusions. High proportions of stain were related to intense revascularization and osteogenesis. Except for the interface, BMP-2 staining occurred later in group MB than in group B in all structures analyzed. (Oral Surg Oral Med Oral Pathol Oral Radiol 2012;113:533-541)

Cyclooxygenase-2 inhibition selectively attenuates bone morphogenetic protein-6 synthesis and bone formation during guided tissue regeneration in a rat model

OBJECTIVES: Bone formation during guided tissue regeneration is a tightly regulated process involving cells, extracellular matrix and growth factors. The aims of this study were (i) to examine the expression of cyclooxygenase-2 (COX-2) during bone regeneration and (ii) the effects of selective COX-2 inhibition on osseous regeneration and growth factor expression in the rodent femur model. MATERIAL AND METHODS: A standardized transcortical defect of 5 x 1.5 mm was prepared in the femur of 12 male rats and a closed half-cylindrical titanium chamber was placed over the defect. The expression of COX-2 and of platelet-derived growth factor-B (PDGF-B), bone morphogenetic protein-6 (BMP-6) and insulin-like growth factor-I/II (IGF-I/II) was analyzed at Days 3, 7, 21 and 28 semiquantitatively by reverse transcriptase-polymerase chain reaction and immunohistochemistry. The effects of COX-2 inhibition by intraperitoneal injection of NS-398 (3 mg/kg/day) were analyzed in five additional animals sacrificed at Day 14. RESULTS: Histomorphometry revealed that new bone formation occurred in the cortical defect area as well as in the supracortical region, i.e. region within the chamber by Day 7 and increased through Day 28. Immunohistochemical evidence of COX-2 and PDGF-B levels were observed early (i.e. Day 3) and decreased rapidly by Day 7. BMP-6 expression was maximal at Day 3 and slowly declined by Day 28. In contrast, IGF-I/II expression gradually increased during the 28-day period. Systemic administration NS-398 caused a statistically significant reduction (P<0.05) in new bone formation (25-30%) and was associated with a statistically significant reduction in BMP-6 protein and mRNA expression (50% and 65% at P<0.05 and P<0.01, respectively). PDGF-B mRNA or protein expression was not affected by NS-398 treatment. CONCLUSION: COX-2 inhibition resulted in reduced BMP-6 expression and impaired osseous regeneration suggesting an important role for COX-2-induced signaling in BMP synthesis and new bone formation.

Recombinant Human Bone Morphogenetic Protein 9 (rhBMP9) Induced Osteoblastic Behaviour on a Collagen Membrane Compared With rhBMP2

BACKGROUND Bone morphogenetic protein 9 (BMP9) has previously been characterized as one of the most osteogenic growth factors of the BMP-family, however, up until now, these experiments have only been demonstrated using adenovirus-transfection experiments (gene therapy). With the recent development of recombinant human (rh)BMP9, the aim of the present study was to investigate its osteopromotive potential versus rhBMP2 when loaded onto a collagen membrane. METHODS ST2 stromal bone marrow cells were seeded onto 1)control; 2)rhBMP2-low(10ng/ml); 3)rhBMP2-high(100ng/ml); 4)rhBMP9-low(10ng/ml); and 5)rhBMP9-high(100ng/ml) porcine collagen membranes. Groups were then compared for cell adhesion at 8 hours, cell proliferation at 1, 3 and 5 days real-time PCR at 3 and 14 days for genes encoding Runx2, alkaline phosphatase(ALP) and bone sialoprotein(BSP) at 3 and 14 days and alizarin red staining at 14 days. RESULTS While rhBMP2 and rhBMP9 demonstrated little effects on cell attachment and proliferation, pronounced increases were observed on osteoblast differentiation. It was found that all groups significantly induced ALP mRNA levels at 3 days and BSP levels at 14 days, however rhBMP9-high demonstrated significantly higher values when compared to all other groups for ALP levels (5-fold increase at 3 days and 2-fold increase at 14 days). Alizarin red staining further revealed that both concentrations of rhBMP9 induced up to 3-fold more staining when compared to rhBMP2. CONCLUSION These results indicate that the combination of collagen membranes with rhBMP9 significantly induced significantly higher ALP mRNA expression and alizarin red staining when compared to rhBMP2. These findings suggest that rhBMP9 may be a suitable growth factor for future regenerative procedures in bone biology.

Effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) on the viability, proliferation and differentiation of osteoblast-like cells cultured on a chemically modified titanium surface

Aim: The aim of the present study was to assess the influence of the chemical characteristics and roughness of titanium surfaces on the viability, proliferation and differentiation of osteoblast-like cells cultured in a medium supplemented with recombinant human bone morphogenetic protein-7 (rhBMP-7). Material and methods: Osteo-1 cells were grown on titanium disks presenting with the following surfaces: (1) machined, (2) coarse grit-blasted and acid-attacked (SLA) and (3) chemically modified SLA (SLAmod) in the absence or presence of 20 ng/ml rhBMP-7 in culture medium. The viability and number of osteo-1 cells were evaluated after 24 h. Analyses of total protein content (TP) and alkaline phosphatase (AP) activity at 7, 14 and 21 days, collagen content at 7 and 21 days and mineralized matrix formation at 21 days were performed. Results: Cell viability (P=0.5516), cell number (P=0.3485), collagen content (P=0.1165) and mineralized matrix formation (P=0.5319) were not affected by the different surface configurations or by the addition of rhBMP-7 to the medium. Osteo-1 cells cultured on SLA surfaces showed a significant increase in TP at 21 days. The ALPase/TP ratio (P=0.00001) was affected by treatment and time. Conclusion: The results suggest that the addition of rhBMP-7 to the culture medium did not exert any effect on the viability, proliferation or differentiation of osteoblast-like cells grown on the different surfaces tested. All titanium surfaces analyzed allowed the complete expression of the osteoblast phenotype such as matrix mineralization by osteo-1 cells.

Osteogenesis induced in goat bone marrow progenitor cells by recombinant adenovirus coexpressing bone morphogenetic protein 2 and basic fibroblast growth factor

Bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) have been shown to exhibit a synergistic effect to promote bone repair and healing. In this study, we constructed a novel adenovirus with high coexpression of BMP2 and bFGF and evaluated its effect on osteogenic differentiation of goat bone marrow progenitor cells (BMPCs). Recombinant adenovirus Ad-BMP2-bFGF was constructed by using the T2A sequence. BMPCs were isolated from goats by density gradient centrifugation and adherent cell culture, and were then infected with Ad-BMP2-bFGF or Ad-BMP2. Expression of BMP2 and bFGF was detected by ELISA, and alkaline phosphatase (ALP) activity was detected by an ALP assay kit. In addition, von Kossa staining and immunocytochemical staining of collagen II were performed on BMPCs 21 days after infection. There was a high coexpression of BMP2 and bFGF in BMPCs infected with Ad-BMP2-bFGF. Twenty-one days after infection, ALP activity was significantly higher in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. Larger and more mineralized calcium nodules, as well as stronger collagen II staining, were observed in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. In summary, we developed a novel adenovirus vector Ad-BMP2-bFGF for simultaneous high coexpression of BMP2 and bFGF, which could induce BMPCs to differentiate efficiently into osteoblasts.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2. © 2013 Society for Reproduction and Fertility.

A new heterologous fibrin sealant as scaffold to recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins for the repair of tibial bone defects

Tissue engineering has special interest in bone tissue aiming at future medical applications Studies have focused on recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins due to the osteogenic properties of rhBMP-2 and the angiogenic characteristic of fraction 1 protein (P-1) extracted from the rubber tree Hevea brasiliensis. Furthermore, heterologous fibrin sealant (FS) has been shown as a promising alternative in regenerative therapies. The aim of this study was to evaluate these substances for the repair of bone defects in rats. A bone defect measuring 3 mm in diameter was created in the proximal metaphysis of the left tibia of 60 rats and was implanted with rhBMP-2 or P-1 in combination with a new heterologous FS derived from snake venom. The animals were divided into six groups: control (unfilled bone defect), rhBMP-2 (defect filled with 5 mu g rhBMP-2), P-1 (defect filled with 5 mu g P-1), FS (defect filled with 8 mu g FS), FS/rhBMP-2 (defect filled with 8 mu g FS and 5 mu g rhBMP-2), FS/P-1 (defect filled with 8 mu g FS and 5 mu g P-1). The animals were sacrificed 2 and 6 weeks after surgery. The newly formed bone projected from the margins of the original bone and exhibited trabecular morphology and a disorganized arrangement of osteocyte lacunae. Immunohistochemical analysis showed intense expression of osteocalcin in all groups. Histometric analysis revealed a significant difference in all groups after 2 weeks (p < 0.05), except for the rhBMP-2 and FS/rhBMP-2 groups (p > 0.05). A statistically significant difference (p < 0.05) was observed in all groups after 6 weeks in relation to the volume of newly formed bone in the surgical area. In conclusion, the new heterologous fibrin sealant was found to be biocompatible and the combination with rhBMP-2 showed the highest osteogenic and osteoconductive capacity for bone healing. These findings suggest a promising application of this combination in the regeneration surgery.

Human Bone Morphogenetic Protein-2 Use for Maxillary Reconstruction in Cleft Lip and Palate Patients

Background: The conventional methods of maxillary alveolar reconstruction in patient with cleft are the periosteoplasty and autologous bone grafting. As an important alternative of bone substitution, there is the recombinant human bone morphogenetic protein-2 (rhBMP-2). This study compares the rhBMP-2 with periosteoplasty and autologous bone grafting. Methods: Patients with cleft and alveolar defect were divided into 3 groups of 6 patients who underwent to autologous iliac crest bone grafting, resorbable collagen sponge with rhBMP2, and periosteoplasty, respectively. The analysis was performed through computed tomographic scan preoperatively and at months 3, 6, and 12 postoperatively. The variables analyzed were the alveolar defect volume, formed bone volume, bone formation rate, maxillary height repair rate, and the formed bone density mean. Results: The formed bone volume was similar comparing the bone graft and BMP groups at 1-year postoperative analysis (P = 0.58). Both of them had the formed bone volume significantly larger than the periosteoplasty group at 3 and 6 months postoperatively. In this last group, the 1-year follow-up was canceled because the bone formation was insufficient. The bone formation rate, the maxillary height repair rate, and the mean of density of the formed bone were similar in the bone graft and BMP groups at 1-year follow-up with P values of 0.93, 0.90, and 0.81, respectively. Conclusions: The amount of formed bone in the periosteoplasty group was insufficient. There was no difference among the bone graft and rhBMP-2 therapy considering the parameters analyzed.

Bone morphogenetic protein 2 incorporated into biomimetic coatings retains its biological activity

We have previously shown that proteins can be incorporated into the latticework of calcium phosphate layers when biomimetically coprecipitated with the inorganic components, upon the surfaces of titanium-alloy implants. In the present study, we wished to ascertain whether recombinant human bone morphogenetic protein 2 (rhBMP-2) thus incorporated retained its bioactivity as an osteoinductive agent. Titanium alloy implants were coated biomimetically with a layer of calcium phosphate in the presence of different concentrations of rhBMP-2 (0.1-10 microg/mL). rhBMP-2 was successfully incorporated into the crystal latticework, as revealed by protein blot staining. rhBMP-2 was taken up by the calcium phosphate coatings in a dose-dependent manner, as determined by ELISA. Rat bone marrow stromal cells were grown directly on these coatings for 8 days. Their osteogenicity was then assessed quantitatively by monitoring alkaline phosphatase activity. This parameter increased as a function of rhBMP-2 concentrations within the coating medium. rhBMP-2 incorporated into calcium phosphate coatings was more potent in stimulating the alkaline phosphatase activity of the adhering cell layer than was the freely suspended drug in stimulating that of cell layers grown on a plastic substratum. This system may be of osteoinductive value in orthopedic and dental implant surgery.

Characterization of a shorter recombinant polypeptide chain of bone morphogenetic protein 2 on osteoblast behaviour

BACKGROUND Recombinant bone morphogenetic protein two (rhBMP2) has been utilised for a variety of clinical applications in orthopaedic surgery and dental procedures. Despite its widespread use, concerns have been raised regarding its short half-life and transient bioactivity in vivo. Recent investigation aimed at developing rhBMP2 synthesized from a shorter polypeptide chain (108 amino acids) has been undertaken. METHODS The osteopromotive properties of BMP2 were investigated on cell behaviour. Five concentrations of rhBMP2_108 including 10, 50, 100, 200 and 500 ng/ml were compared to a commercially available rhBMP2 (100 ng/ml). Each of the working concentrations of rhBMP2_108 were investigated on MC3T3-E1 osteoblasts for their ability to induce osteoblast recruitment, proliferation and differentiation as assessed by alkaline phosphatase (ALP) staining, alizarin red staining, and real-time PCR for genes encoding ALP, osteocalcin (OCN), collagen-1 (COL-1) and Runx2. RESULTS The results demonstrate that all concentrations of rhBMP2_108 significantly improved cell recruitment and proliferation of osteoblasts at 5 days post seeding. Furthermore, rhBMP2_108 had the most pronounced effects on osteoblast differentiation. It was found that rhBMP2_108 had over a four fold significant increase in ALP activity at seven and 14 days post-seeding and the concentrations ranging from 50 to 200 ng/ml demonstrated the most pronounced effects. Analysis of real-time PCR for genes encoding ALP, OCN, COL-1 and Runx2 further confirmed dose-dependant increases at 14 days post-seeding. Furthermore, alizarin red staining demonstrated a concentration dependant increase in staining at 14 days. CONCLUSION The results from the present study demonstrate that this shorter polypeptide chain of rhBMP2_108 is equally as bioactive as commercially available rhBMP2 for the recruitment of progenitor cells by facilitating their differentiation towards the osteoblast lineage. Future in vivo study are necessary to investigate its bioactivity.

Effect of bone morphogenetic protein-7 (BMP-7) on in vitro survival of caprine preantral follicles

This study was conducted in order to verify the effect of different concentrations of BMP-7 in the in vitro survival and development of caprine preantral follicles. Fragments of caprine ovarian cortical tissue were cultured for 1 or 7 days in Minimum Essential Medium (MEM+) supplemented with different concentrations of BMP-7 (1, 10, 50 or 100ng/ml). Non-cultured fragments or those cultured for 1 or 7 days were processed for classical histology and transmission electron microscopy (TEM). Parameters such as follicular survival, activation and growth were evaluated. The results showed that, after 1 or 7 days of culture, the percentage of morphologically normal follicles was significantly reduced in all treatments when compared with fresh control, except at 1ng/ml of BMP-7 for 1 day. In addition, the concentration of 10ng/ml of BMP-7 significantly increases follicular diameter from day 1 to 7 of culture. There was no influence of the other concentrations of BMP-7 regarding to the follicular and oocyte diameter. Ultrastructure studies confirmed follicular integrity after 7 days of culture in 1ng/ml BMP-7. In conclusion, small concentrations of BMP-7 can improve the survival and growth of caprine preantral follicles during in vitro culture.

Bone Morphogenetic Protein-6 (BMP-6) induces atresia in goat primordial follicles cultured in vitro

This study investigated the effects of bone morphogenetic protein 6 (BMP-6) on in vitro primordial follicle development in goats. Samples of goat ovarian cortex were cultured in vitro for 1 or 7 days in Minimum Essential Medium (control medium) supplemented with different concentrations of BMP-6. Follicle survival, activation and growth were evaluated through histology and transmission electron microscopy (TEM). After 7 days of culture, histological analysis demonstrated that BMP-6 enhanced the percentages of atretic primordial follicles when compared to fresh control (day 0). Nevertheless, BMP-6 increased follicular and oocyte diameter during both culture periods. As the culture period progressed from day 1 to day 7, a significant increase in follicle diameter was observed with 1 or 50ng/ml BMP-6. However, on the contrary to that observed with the control medium TEM revealed that follicles cultured for up to 7 days with 1 or 50ng/ml BMP-6 had evident signs of atresia. In conclusion, this study demonstrated that BMP-6 negatively affects the survival and ultrastructure of goat primordial follicles.