Regeneration of calvarial defects with Escherichia coli -derived rhBMP-2 adsorbed in PLGA membrane.

OBJECTIVE: Escherichia coli-derived recombinant human bone morphogenetic protein-2 (E-BMP-2) has been shown to be as effective as mammalian cell-derived BMP-2. However, several in vitro and in vivo experiments are still necessary to validate the effectiveness of E-BMP-2 due to the difference in synthesis process, mainly related to protein nonglycosylation. The objective of this study was to investigate whether biodegradable polylactide-co-glycolide (PLGA) membrane is a suitable carrier for E-BMP-2 delivery for bone regeneration of critical-sized defects in rat calvaria. MATERIALS AND METHODS: First, the osteoinductive effect of E-BMP-2 was confirmed in vitro in mouse bone marrow stromal cells by analysis of osteocalcin mRNA levels, and calcium deposition was detected by alizarin red staining. Before in vivo experiments, the release profile of E-BMP-2 from PLGA membranes was determined by ELISA. E-BMP-2 (0, 1, 5 and 10 µg/µl) was applied for ectopic and orthotopic bone formation and was analyzed by X-ray, micro-CT and histology. RESULTS: Release-profile testing showed that PLGA membrane could retain 94% of the initially applied E-BMP-2. Ectopic bone formation assay revealed that combination of E-BMP-2/PLGA membrane strongly induced bone formation. Stronger osteoinductivity with complete repair of critical-sized defects was observed only with PLGA membranes adsorbed with 5 and 10 µg/µl of E-BMP-2, whereas no bone formation was observed in the groups that received no membrane or 0-µg/µl dose of E-BMP-2. CONCLUSION: PLGA membrane was shown to be a suitable carrier for sustained release of E-BMP-2, and the E-BMP-2/PLGA membrane combination was demonstrated to be efficient in bone regeneration in a model of critical-sized defects.

Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model.

OBJECTIVE: The aim of this work was to analyse qualitatively and quantitatively the newly formed bone after insertion of rhBMP-2 and protein extracted from Hevea brasiliensis (P-1), associated or not with a carrier in critical bone defects created in Wistar rat calvarial bone, using histological and histomorphometrical analyses. MATERIALS AND METHODS: Eighty-four male Wistar rats were used, divided into two groups, according to the period of time until the sacrifice (2 and 6 weeks). Each one of these groups was subdivided into six groups with seven animals each, according to the treatments: (1) 5 µg of pure rhBMP-2, (2) 5 µg of rhBMP-2/monoolein gel, (3) pure monoolein gel, (4) 5 µg of pure P-1, (5) 5 µg of P-1/monoolein gel and (6) critical bone defect controls. The animals were euthanised and the calvarial bone tissue removed for histological and histomorphometrical analyses. RESULT AND CONCLUSION: The results showed an improvement in the bone healing process using the rhBMP-2 protein, associated or not with a material carrier in relation to the other groups, and this process demonstrated to be time dependent.

Site-specific PEGylation of bone morphogenetic protein-2 cysteine analogues.

Three cysteine analogues of bone morphogenetic protein (BMP)-2, BMP2A2C, BMP2N56C, and BMP2E96C, were generated in order to enable the attachment of SH-reactive poly(ethylene glycol) (PEG) at specific sites. Three different approaches (Ap) were used for SH-specific PEGylation: (Ap1) reaction of glutathione activated proteins with thiol PEG; (Ap2) reaction of DTT reduced proteins with orthopyridyl disulfide PEG; (Ap3) reaction of DTT reduced proteins with maleimide PEG. Non-, mono-, and di-PEGylated BMP-2 analogues could be separated by RP-HPLC. Trypsin digestion of PEGylated proteins and Trypsin and GluC double-digestion of N-ethylmaleimide-labeled proteins confirmed that the modifications were site-specific. Surface plasmon resonance analysis of type I and type II receptor binding of the PEGylated BMP-2 analogues revealed that all three PEGylation approaches were equivalent. PEGylation at positions 2 and 96 caused a similar decrease in receptor affinity. PEGylation at position 56 resulted in a larger decrease in affinity for both types of receptors. Mono-PEGylated BMP-2 analogues exhibited intermediate affinities in comparison with unmodified and di-PEGylated proteins. However, the biological activity of the PEGylated BMP-2 analogues as measured in alkaline phosphatase assay was higher than BMP-2 wild-type for the PEGylated BMP2A2C, slightly reduced for the BMP2N56C, and strongly reduced for the BMP2E96C. These results taken together indicate that specific attachment of PEG at engineered sites of BMP-2 is possible and that the attachment site is critical for biological activity. Furthermore, the biological activity of PEGylated BMP-2 analogues in cell culture seems to be determined not only by receptor affinity, but also by other factors such as protein solubility and stability. It is also discussed that the attached PEG interferes with the binding of BMP-2 to modulator proteins, co-receptors, or heparinic sites of proteoglycans in the extracellular matrix.

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.

Bone healing process in critical-sized defects by rhBMP-2 using poloxamer gel and collagen sponge as carriers.

The bone morphogenetic protein type 2, is an osteoinduction protein, but new carriers to improve its actions are being studied in recent researches. The objective of this study was to evaluate the poloxamer gel as potential carrier of the rhBMP-2 during the bone healing process and verify if the collagen sponge can improve this process. Fifty-six male Wistar rats were used, which were divided into two groups, considering two periods of time. Thus, each one of these groups was divided in four groups with seven animals each, according to the treatment and the defect filled by 4 microg rhBMP-2 in aqueous solution, 4 microg rhBMP-2 in aqueous solution+collagen sponge, 4 microg rhBMP-2 combined with poloxamer gel, and 4 microg rhBMP-2 combined with poloxamer gel+collagen sponge. After 2 and 4 weeks, respectively, animals were perfused and the hemi-mandibles removed for histological analysis. Statistical analysis of the data showed significant difference between all analyzed groups (p<0.01), and between the periods of time (p=0.0118). There was no interaction between the applied treatment and considered periods of time (p=0.642). Results showed that the rhBMP-2 used in this study was able to induce bone regeneration and had its action optimized when combined to the used carriers, being the bone repair time-dependent.

Bone repair in rat mandible by rhBMP-2 associated with two carriers.

This study evaluated the quantity and quality of newly formed bone, stimulated by rhBMP-2 in combination with monoolein or chitosan gel as carriers, in critical bone defects created in 36 Wistar rat mandibles. Two weeks after surgery, the animals were anesthetized with 37.5% urethane submitted to perfusion and the hemi-mandibles removed for histological and histomorphometrical analysis. The results showed that there was a statistical difference between groups of animals receiving or not rhBMP-2 (p<0.05). Newly formed bone was more intense in the occlusal region, followed by the basal and middle regions, respectively. Both carriers, monoolein and chitosan gels were adequate for defect filling and control of protein release.

RGD-grafted thermoreversible polymers to facilitate attachment of BMP-2 responsive C2C12 cells.

The purpose of this study was to design thermoreversible biomaterials for enhanced adhesion of bone morphogenetic protein-2 (BMP-2)-responsive cells. Peptides containing the arginine-glycine-aspartic acid (RGD) sequence were conjugated to N-isopropylacrylamide (NiPAM) polymers via amine-reactive N-acryloxysuccinimide (NASI) groups. In monolayer cultures, the adhesion of BMP-2-responsive C2C12 cells to RGD-grafted NiPAM/NASI surfaces was significantly higher than adhesion on ungrafted NiPAM/NASI surfaces. Although the morphology of cells adhered to RGD-grafted NiPAM/NASI surfaces was comparable to cells adhered on tissue culture polystyrene (TCPS), long-term cell growth was limited on the NiPAM/NASI surfaces, even for RGD-grafted surfaces. Treatment of C2C12 cells with recombinant BMP-2 induced dose-dependent osteoblastic differentiation as assessed by alkaline phosphatase (ALP) activity. In the absence of BMP-2, cells cultured on NiPAM/NASI polymers (either grafted with RGD peptide or not) expressed significantly higher levels of ALP activity than the cells cultured on TCPS, indicating that the polymer surfaces induced some osteoblastic activity in C2C12 cells without the need for BMP-2. We conclude that NiPAM-based thermoreversible biomaterials, despite their limited ability to support cell growth, allowed an enhanced expression of the chosen osteogenic marker (ALP) by C2C12 cells in vitro.

Human osteoprogenitor bone formation using encapsulated bone morphogenetic protein 2 in porous polymer scaffolds.

The ability to deliver, over time, biologically active osteogenic growth factors by means of designed scaffolds to sites of tissue regeneration offers tremendous therapeutic opportunities in a variety of musculoskeletal diseases. The aims of this study were to generate porous biodegradable scaffolds encapsulating an osteogenic protein, bone morphogenetic protein 2 (BMP-2), and to examine the ability of the scaffolds to promote human osteoprogenitor differentiation and bone formation in vitro and in vivo. BMP-2-encapsulated poly(DL-lactic acid) (PLA) scaffolds were generated by an innovative supercritical fluid process developed for solvent-sensitive and thermolabile growth factors. BMP-2 released from encapsulated constructs promoted adhesion, migration, expansion, and differentiation of human osteoprogenitor cells on three-dimensional scaffolds. Enhanced matrix synthesis and cell differentiation on growth factor-encapsulated scaffolds was observed after culture in an ex vivo model of bone formation developed on the basis of the chick chorioallantoic membrane model. BMP-2-encapsulated polymer scaffolds showed morphologic evidence of new bone matrix and cartilage formation after subcutaneous implantation and within diffusion chambers implanted into athymic mice as assessed by X-ray analysis and immunocytochemistry. The generation of three-dimensional biomimetic structures incorporating osteoinductive factors such as BMP-2 indicates their potential for de novo bone formation that exploits cell-matrix interactions and, significantly, realistic delivery protocols for growth factors in musculoskeletal tissue engineering.

Bone repair investigation using rhBMP-2 and angiogenic protein extracted from latex.

BACKGROUND: The aim of this work was to study the new bone tissue formation after bone morphogenetic protein type 2 (rhBMP-2) and P-1 application, using 5 and 10 µg of each, combined to a material carrier, in critical bone defects. METHODS: It was used 70 Wistar rats (male, ∼250 g) that were divided in 10 groups with seven animals on each. Groups are the following: critical bone defect only, pure monoolein gel, 5 µg of pure P-1, 5 µg of pure rhBMP-2, 5 µg of P-1/monoolein gel, 5 µg of rhBMP-2/monoolein gel, 10 µg of pure P-1, 10 µg of pure rhBMP-2, 10 µg of P-1/monoolein gel, 10 µg of rhBMP-2/monoolein gel. Animals were sacrificed after 4 weeks of the surgical procedure and the bone samples were submitted to histological, histomorphometrical, and immunohistochemical evaluations. RESULTS: Animals treated with pure P-1 protein, in both situations with 5 µg and 10 µg, had no significant difference (P > 0.05) for new bone formation; other groups treated with 10 µg were statistically significant (P < 0.05) among themselves and when compared with groups in which it was inserted the monoolein gel or critical bone defect only (P < 0.05). In the group involving the 10 µg rhBMP-2/monoolein gel association, it was observed an extensive bone formation, even when compared with the same treatment without the gel carrier. CONCLUSION: Using this experimental animal model, more new bone tissue was found when it was inserted the rhBMP-2, especially when this protein was combined to the vehicle, and this process seems to be dose dependent.

N-terminal specificity of PEGylation of human bone morphogenetic protein-2 at acidic pH.

Site-specific PEGylation offers the possibility to modify a therapeutic protein without interfering with its biological activity. Previously, a preferential N-terminal PEGylation has been reported for several proteins when the reaction was performed at acidic pH. In the present study it was explored if acidic pH favors N-terminal PEGylation of bone morphogenetic protein-2 (BMP-2). PEGylation by poly(ethylene glycol) aldehyde (PEG-AL) or poly(ethylene glycol) carboxymethyl succinimidyl ester (PEG-NHS) was performed at moderate acidic pH of 4. Comparing with PEG-NHS, PEG-AL converted more BMP-2 mainly to mono- or di-PEGylated derivatives at much less molar excess and shorter duration. Analysis of Tryptic fragments of the PEGylated derivatives indicated a partial N-terminal PEGylation specificity. PEG-AL exhibited higher specificity than PEG-NHS. UV spectrometry proved that PEGylation improved the solubility of BMP-2 in PBS. Surface plasmon resonance showed that PEGylation decreased the binding of BMP-2 proteins to a type II receptor. Remarkably, mono-PEGylated BMP-2 with PEG-AL showed higher cellular bioactivity than unmodified protein. Higher N-terminal PEGylation specificity correlates with higher receptor binding affinity and cellular activity. In summary, PEGylation of BMP-2 by PEG-AL and PEG-NHS at acidic pH exhibits a partial N-terminal specificity which however might be sufficient for an efficient site-specific PEGylation process.

Polyethylenimine-PEG coated albumin nanoparticles for BMP-2 delivery.

Bone Morphogenetic Protein-2 (BMP-2) plays an important role in stimulating new bone formation, and has been utilized in clinical bone repair by implantation. In this study, we report a nanoparticulate (NP) system for BMP-2 delivery based on bovine serum albumin (BSA) NPs stabilized with a poly(ethylene glycol) modified polyethylenimine (PEI-PEG) coating. PEI-PEG with different PEG substitutions were synthesized, and the cell viability assay showed PEG substitution greatly reduced the cytotoxicity of the native PEI. Furthermore, PEI-PEG coated BSA NPs demonstrated smaller size and decreased zeta potential compared to PEI-coated NPs. The bioactivity of the encapsulated BMP-2 and the toxicity of PEI-PEG coated NPs were examined by the alkaline phosphatase (ALP) induction assay and the MTT assay, respectively, using human C2C12 cells. The results indicated that BMP-2 remained bioactive in NPs and PEI-PEG coating was advantageous in reducing the NP toxicity as compared to PEI. A 7-day pharmacokinetics study showed the BMP-2 retention in PEI-PEG coated NPs was similar to the uncoated NPs, but lower than that of the PEI-coated NPs. The osteoinductivity of BMP-2 delivered in NPs was determined by subcutaneous implantation in rats, and the results revealed that PEI-PEG coated BSA NPs induced significant de novo bone formation after implantation, while PEI-coated NPs demonstrated much less bone formation. We conclude that BMP-2 delivered by PEGylated PEI-coated BSA NPs displays favorable biocompatibility and promotes new bone formation after implantation.

Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds.

The aim of this study was to determine the feasibility of adenoviral gene transfer into primary human bone marrow osteoprogenitor cells in combination with biodegradeable scaffolds to tissue-engineer bone. Osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene. Alkaline phosphatase activity was induced in C2C12 cells following culture with conditioned media from BMP-2 expressing cells, confirming successful secretion of active BMP-2. Expression of alkaline phosphatase activity, type I collagen and mineralisation confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks on PLGA porous scaffolds. In vivo implantation of adenoviral osteoprogenitor constructs on PLGA biodegradeable scaffolds, using diffusion chambers, also demonstrated bone cell differentiation and production of bone tissue. The maintenance of the osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs indicate the potential of such bone tissue engineering approaches in bone repair.