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.

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- β 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.

Remodeling of cortical and corticocancellous fresh-frozen allogeneic block bone grafts--a radiographic and histomorphometric comparison to autologous bone grafts.

OBJECTIVES: To compare cortical (AL-C) and corticocancellous (AL-CC) fresh-frozen block bone allografts to cortical block bone autografts (AT) used for lateral ridge augmentation in terms of radiographic dimensional maintenance and histomorphometrical graft remodeling. MATERIALS AND METHODS: Twenty-four patients, requiring ridge augmentation in the anterior maxilla prior to implant placement, were treated with AT, AL-C or AL-CC bone blocks (eight patients per graft type). Patients were examined with CBCT prior to, 14 days, and 6-8 months after grafting. Amount of augmentation and dimensional block graft maintenance over time was evaluated by comparing planimetric measurements of the alveolar ridge made on CBCT sections of the augmentation area. During implant installation surgery, 6-8 months after grafting, cylindrical biopsies were harvested perpendicularly to the lateral aspect of the augmented alveolar ridge. The relative volumes of vital and necrotic bone and soft tissues were histomorphometrically estimated. Comparisons among groups and observation times were performed using Friedman test followed by Dunn's post-hoc test. RESULTS: Radiographic evaluation showed that the three types of grafts resulted in a significant increase in alveolar ridge width, with no significant differences among the groups in terms of ridge dimensions at the various observation times. However, significant graft resorption (P = 0.03) was observed in the AL-CC group over time (-8.3 ± 7.1%) compared with the AT and AL-C groups, where a slight increase was observed, on average (1.5 ± 20.6% and 1.3 ± 14.9%, respectively). Histomorphometrical analysis showed that larger amounts of vital bone were found in the biopsies from the AT augmented sites (25.1 ± 11.2%) compared with AL-CC and AL-C augmented sites (9.3 ± 3.8% and 3.9 ± 4.6%, respectively; P ≤ 0.01). AL-CC and AT biopsies had the smallest amount of necrotic bone (38.2 ± 12.1% and 56.7 ± 26.0, respectively) compared with AL-C (83.7 ± 10.8%, P < 0.01) biopsies. AL-CC biopsies showed the largest amount of soft tissues (52.5 ± 11.7%) compared with those from AT (18.1 ± 17.1%, P = 0.03) and AL-C (12.3 ± 8.5%, P < 0.01) sites. CONCLUSIONS: AL block bone graft architecture influences significantly its dimensional incorporation and remodeling. Compared with AT bone graft, a small portion of the AL block consists of vital bone 6-8 months after grafting. Cortical AL blocks seem to show the least amounts of vital bone, while corticocancellous AL blocks seem to undergo more resorption over time.

Antenatal steroid and tracheal occlusion restore vascular endothelial growth factor receptors in congenital diaphragmatic hernia rat model.

OBJECTIVE: Investigate the effects of antenatal steroids and tracheal occlusion on pulmonary expression of vascular endothelial growth factor receptors in rats with nitrofen-induced congenital diaphragmatic hernia. STUDY DESIGN: Fetuses were exposed to nitrofen at embryonic day 9.5. Subgroups received dexamethasone or were operated on for tracheal occlusion, or received combined treatment. Morphologic variables were recorded. To analyze vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 expression, we performed Western blotting and immunohistochemistry. Morphologic variables were analyzed by analysis of variance and immunohistochemistry by Kruskal-Wallis test. RESULTS: Congenital diaphragmatic hernia decreased body weight, total lung weight, and lung-to-body weight ratio. Tracheal occlusion increased total lung weight and lung-to-body weight ratio (P < .05). Fetuses with congenital diaphragmatic hernia had reduced vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 expression, whereas steroids and tracheal occlusion increased their expression. Combined treatment increased expression of receptors, but had no additive effect. CONCLUSION: Vascular endothelial growth factor signaling disruption may be associated with pulmonary hypertension in congenital diaphragmatic hernia. Tracheal occlusion and steroids provide a pathway for restoring expression of vascular endothelial growth factor receptors.