Escherichia Coli-derived Recombinant Human Bone Morphogenetic Protein-2 Research Articles

Dose Effects of Slow-Released Bone Morphogenetic Protein-2 Functionalized β-Tricalcium Phosphate in Repairing Critical-Sized Bone Defects

The aim of this study is to investigate the efficacy and the effect of the dosage of the slow-released Escherichia coli-derived recombinant human bone morphogenetic protein 2 (ErhBMP-2) functionalized β-tricalcium phosphate (β-TCP) in repairing critical-sized bone defects. The functionalization was implemented by modifying the surface of β-TCP with biomimetic calcium phosphate coating with or without ErhBMP-2. Critical-sized calvarial defects were created in rats and filled with ErhBMP-2 functionalized β-TCP loaded with gradient doses of ErhBMP-2 (0, 50, 100, 150, 200, and 300 μg/g). The blank control group and the autologous bone group were also included. The systemic toxicity was evaluated using routine blood and histopathological examination. The efficiency in bone healing was evaluated using microcomputed tomography, histology, and histometric analyses. Neither local nor systemic adverse effects were found following the implantation of the ErhBMP-2 functionalized β-TCP. The new bone formation was significantly increased in the ErhBMP-2 functionalized β-TCP groups compared with the blank group and the β-TCP with coating only group. The efficacy of the ErhBMP-2 functionalized β-TCP in bone healing was comparable to that of the autologous bone. There was no significant difference in bone formation among all concentrations of ErhBMP-2 (0-300 μg/g). Increased bone maturation was found in the higher concentration groups (150, 200, and 300 μg/g) when compared with the lower concentration groups (50, and 100 μg/g). Our results demonstrated that the ErhBMP-2 functionalized β-TCP could significantly promote bone repairing in critical-sized defects, and it could clinically be a promising substitute for autologous bone. Besides, our results demonstrated that there was a dosage-dependent effect of ErhBMP-2 functionalized β-TCP on bone maturation rather than bone formation. The optimized concentrations of ErhBMP-2 recommended for this kind of model should be in the range of 150-300 μg/g. Impact Statement Bone substitutes functionalized by mammalian-derived recombinant human bone morphogenetic protein 2 (rhBMP-2) have demonstrated to be comparable to the autologous bone in repairing the critical-sized bone defects. To develop a commercial product with more effective cost, Escherichia coli-derived rhBMP-2 (ErhBMP-2) has been produced and evaluated as an alternative to the mammalian-derived rhBMP-2. In this study, we prepared gradient ErhBMP-2 functionalized β-tricalcium phosphate (β-TCP) with biomimetic calcium phosphate coating and investigate their efficacy and dose effects. We revealed the dose effects of the slow-released ErhBMP-2 and demonstrated that ErhBMP-2 functionalized β-TCP could be a promising bone substitute for bone regeneration in clinical settings.

Improvement of the osteogenic potential of ErhBMP-2-/EGCG-coated biphasic calcium phosphate bone substitute: in vitro and in vivo activity.

PurposeThe aim of this study was to evaluate the enhancement of osteogenic potential of biphasic calcium phosphate (BCP) bone substitute coated with Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2) and epigallocatechin-3-gallate (EGCG).MethodsThe cell viability, differentiation, and mineralization of osteoblasts was tested with ErhBMP-2-/EGCG solution. Coated BCP surfaces were also investigated. Standardized, 6-mm diameter defects were created bilaterally on the maxillary sinus of 10 male New Zealand white rabbits. After removal of the bony windows and elevation of sinus membranes, ErhBMP-2-/EGCG-coated BCP was applied on one defect in the test group. BCP was applied on the other defect to form the control group. The animals were sacrificed at 4 or 8 weeks after surgery. Histologic and histometric analyses of the augmented graft and surrounding tissue were performed.ResultsThe 4-week and 8-week test groups showed more new bone (%) than the corresponding control groups (P<0.05). The 8-week test group showed more new bone (%) than the 4-week test group (P<0.05).ConclusionsErhBMP-2-/EGCG-coated BCP was effective as a bone graft material, showing enhanced osteogenic potential and minimal side effects in a rabbit sinus augmentation model.

Bone Regenerative Efficacy of Limited-Dose Escherichia Coli-Derived rhBMP-2 With Biphasic Calcium Phosphate Carrier in Rabbit Calvarial Defect Model.

The aim of this study was to characterize the healing in rabbit calvarial bone defects after delivery of limited-dose (1.5 μg) Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2), and evaluate biphasic calcium phosphate (BCP) as a carrier. Four 8-mm diameter circular calvarial bone defects were made in 16 rabbits and filled with one of the following: (1) nothing, (2) BCP alone, (3) ErhBMP-2-loaded absorbable collagen sponge (ACS), or (4) ErhBMP-2-loaded BCP. The animals were allowed to heal for either 2 or 8 weeks and were evaluated in clinical, microcomputed tomographic, histological, and histomorphometric analyses. Microcomputed tomography revealed extensive new bone formation in both of the limited-dose ErhBMP-2-loaded groups. However, bony collapse of the upper defect borders was found in the ErhBMP-2-loaded ACS group. Histomorphometric examination revealed significantly greater new bone formation at 8 weeks than at 2 weeks in all 4 groups (P < 0.05). Both new bone formation and the size of the augmented area differed significantly between the ErhBMP-2-loaded BCP group (6.88 ± 0.74 and 19.62 ± 0.77) and the ErhBMP-2-loaded ACS group (3.04 ± 0.27 and 5.41 ± 0.43) at 8 weeks of healing. ErhBMP-2 promotes bone regeneration in rabbit calvarial defects, even at a limited dose (1.5 μg). The results of this study suggest that BCP is the more efficient carrier for this protein than ACS.

Four-week histologic evaluation of grafted calvarial defects with adjunctive hyperbaric oxygen therapy in rats.

PurposeThe aim of this study was to characterize the healing in the grafted calvarial defects of rats after adjunctive hyperbaric oxygen therapy.MethodsTwenty-eight male Sprague-Dawley rats (body weight, 250–300 g) were randomly divided into two treatment groups: with hyperbaric oxygen therapy (HBO; n=14) and without HBO (NHBO; n=14). Each group was further subdivided according to the bone substitute applied: biphasic calcium phosphate (BCP; n=7) and surface-modified BCP (mBCP; n=7). The mBCP comprised BCP coated with Escherichia-coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2) and epigallocatechin-3-gallate (EGCG). Two symmetrical circular defects (6-mm diameter) were created in the right and left parietal bones of each animal. One defect was assigned as a control defect and received no bone substitute, while the other defect was filled with either BCP or mBCP. The animals were allowed to heal for 4 weeks, during which those in the HBO group underwent 5 sessions of HBO. At 4 weeks, the animals were sacrificed, and the defects were harvested for histologic and histomorphometric analysis.ResultsWell-maintained space was found in the grafted groups. Woven bone connected to and away from the defect margin was formed. More angiogenesis was found with HBO and EGCG/BMP-2 (P<0.05). None of the defects achieved complete defect closure. Increased new bone formation with HBO or EGCG/BMP-2 was evident in histologic evaluation, but it did not reach statistical significance in histometric analysis. A synergic effect between HBO and EGCG/BMP-2 was not found.ConclusionsWithin the limitations of this study, the present findings indicate that adjunctive HBO and EGCG/BMP-2 could be beneficial for new bone formation in rat calvarial defects.

Rapid bone regeneration by Escherichia coli-derived recombinant human bone morphogenetic protein-2 loaded on a hydroxyapatite carrier in the rabbit calvarial defect model.

BackgroundThe aim of this study was to determine the osteoconductivity of hydroxyapatite particles (HAP) as a carrier for Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2). Two 8-mm diameter bicortical calvarial defects were created in each of 20 rabbits. One of each pair of defects was randomly assigned to be filled with HAP only (HAP group) or ErhBMP-2 loaded HAP (ErhBMP-2/HAP group), while the other defect was left untreated (control group). The animals were killed after either 2 weeks (n = 10) or 8 weeks (n = 10) of healing, and histological, histomorphometric, and tomographic analyses were performed.ResultsAll experimental sites showed uneventful healing during the postoperative healing period. In both histomorphometric and tomographic analyses, the new bone area or volume of the ErhBMP-2/HAP group was significantly greater than that of the HAP and control groups at 2 weeks (p < 0.05). However, at 8 weeks, no significant difference in new bone area or volume was observed between the ErhBMP-2/HAP and HAP groups. The total augmented area or volume was not significantly different between the ErhBMP-2/HAP and HAP groups at 2 and 8 weeks.ConclusionsCombining ErhBMP-2 with HAP could significantly promote rapid initial new bone formation. Moreover, HAP graft could increase new bone formation and space maintenance, therefore it might be one of the effective carriers of ErhBMP-2.

Bone Healing Properties of Autoclaved Autogenous Bone Grafts Incorporating Recombinant Human Bone Morphogenetic Protein-2 and Comparison of Two Delivery Systems in a Segmental Rabbit Radius Defect

Purpose:This study aims to validate the effect of autoclaved autogenous bone (AAB), incorporating Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2), on critical-sized, segmental radius defects in rabbits. Delivery systems using absorbable collagen sponge (ACS) and fibrin glue (FG) were also evaluated.Methods:Radius defects were made in 12 New Zealand white rabbits. After autoclaving, the resected bone was reinserted and fixed. The animals were classified into three groups: only AAB reinserted (group 1, control), and AAB and ErhBMP-2 inserted using an ACS (group 2) or FG (group 3) as a carrier. Animals were sacrificed six or 12 weeks after surgery. Specimens were evaluated using radiology and histology.Results:Micro-computed tomography images showed the best bony union in group 2 at six and 12 weeks after operation. Quantitative analysis showed all indices except trabecular thickness were the highest in group 2 and the lowest in group 1 at twelve weeks. Histologic results showed the greatest bony union between AAB and radial bone at twelve weeks, indicating the highest degree of engraftment.Conclusion:ErhBMP-2 increases bony healing when applied on AAB graft sites. In addition, the ACS was reconfirmed as a useful delivery system for ErhBMP-2.

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.

Bone formation of middle ear cavity using biphasic calcium phosphate lyophilized with Escherichia coli-derived recombinant human bone morphogenetic protein 2 using animal model

ObjectiveThe aim of the study was to analyze the value of Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2) coated biphasic calcium phosphate (BCP) for the obliteration of middle ear bone defect after mastoid surgery. MethodsTwenty-four specific pathogen-free Sprague-Dawley rats were randomly assigned to the BCP group (n=12) and BCP-ErhBMP-2 group (n=12; in which BCP scaffold of the granular type was coated with ErhBMP-2). In both groups, BCP scaffold was used to surgically fill the middle ear bulla. New bone formation was evaluated by measuring bone density (%) after 4 and 8 weeks in all rats in both groups. ResultsAt 4 weeks, new bone was visible at the periphery and center of the middle ear cavity in both groups. In the BCP group, a moderate amount of fibrous tissue had infiltrated into the interspace of the scaffolds. New bone almost totally filled the interspace in the BCP-ErhBMP-2 group. At 8 weeks, copious new bone formation had occurred. Histometric measurements showed that bone density in the BCP group was smaller than in the BCP-ErhBMP-2 group at 4 weeks (25.10% and 38.43%, respectively; p<0.05) and 8 weeks (25.54% and 34.18%, respectively; p<0.05). ConclusionsNew bone formation was greater in the presence of BCP-ErhBMP-2 scaffolds. ErhBMP-2 coated BCP scaffolds is a potentially useful material for middle ear obliteration after mastoidectomy.

The effects of ErhBMP‐2‐/EGCG‐coated BCP bone substitute on dehiscence around dental implants in dogs

The purpose was to evaluate the effect of Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2)-/epigallocatechin-3-gallate (EGCG)-coated biphasic calcium phosphate (BCP) and titanium barrier membrane on dehiscence defects in dogs. In five mongrel dogs, the dehiscence bony defects around dental implants were surgically created and in total three implants were placed at edentulous ridge of which teeth had been extracted 12weeks before. For the control group, BCP was applied to the dehiscence defect. For experimental groups, ErhBMP-2-coated BCP and ErhBMP-2-/EGCG-coated BCP were applied. The newly designed titanium barrier membrane was used to apply all the defects. The defects were evaluated histologically and histometrically after 12weeks. The comparative statistics of the groups were obtained through Kruskal-Wallis test. In bone-to-implant contact (BIC), bone density (BD), bone regeneration height (BRH), and bone mineralization apposition rate (BMAR), differences among groups were not found. ErhBMP-2/EGCG group appeared to have higher value. In fluorescence analysis, bone remodeling around graft material was more active in the ErhBMP-2/EGCG group. Within the limit of this study, it is reasonable to assume that BMP-2-/EGCG-coated biphasic BCP and the newly designed titanium membrane were more beneficial in dehiscence defect healing with increased bone remodeling.

Evaluation of the efficacy of Escherichia coli-derived recombinant human bone morphogenetic protein-2 in a mini-pig spinal anterior interbody fusion model

We evaluated the efficacy of Escherichia coli-derived recombinant human bone morphogenetic protein-2 (E-BMP-2) in a mini-pig model of spinal anterior interbody fusion. A total of 14 male mini-pigs underwent three-level anterior lumbar interbody fusion using polyether etherketone (PEEK) cages containing porous hydroxyapatite (HA). Four groups of cages were prepared: 1) control (n = 10 segments); 2) 50 μg E-BMP-2 (n = 9); 3) 200 μg E-BMP-2 (n = 10); and 4) 800 μg E-BMP-2 (n = 9). At eight weeks after surgery the mini-pigs were killed and the specimens were evaluated by gross inspection and manual palpation, radiological evaluation including plain radiographs and micro-CT scans, and histological analysis. Rates of fusion within PEEK cages and overall union rates were calculated, and bone formation outside vertebrae was evaluated. One animal died post-operatively and was excluded, and one section was lost and also excluded, leaving 38 sites for assessment. This rate of fusion within cages was 30.0% (three of ten) in the control group, 44.4% (four of nine) in the 50 μg E-BMP-2 group, 60.0% (six of ten) in the 200 μg E-BMP-2 group, and 77.8% (seven of nine) in the 800 μg E-BMP-2 group. Fusion rate was significantly increased by the addition of E-BMP-2 and with increasing E-BMP-2 dose (p = 0.046). In a mini-pig spinal anterior interbody fusion model using porous HA as a carrier, the implantation of E-BMP-2-loaded PEEK cages improved the fusion rate compared with PEEK cages alone, an effect that was significantly increased with increasing E-BMP-2 dosage.

Enhancement of osteogenesis by poly(lactide‐co‐glycolide) sponges loaded with surface‐embedded hydroxyapatite particles and rhBMP‐2

The bone mesenchymal stem cells (BMSCs) were seeded on [poly(lactide-co-glycolide) scaffolds with hydroxyapatite (HA) coating, and "s" stands for surface] (PLGA/HA-S), PLGA/HA-M (containing the same HA amount in the matrix as that of the PLGA/HA-S and "m" stands for matrix), and PLGA scaffolds, which were then cultured in a medium-containing Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2). In vitro culture of rat BMSCs found no different cell morphology in all the scaffolds, but the alkaline phosphatase activity and osteogenic gene expression of type I collagen (COL I) and osteocalcin (OCN) in the PLGA/HA-S scaffolds were always highest and were significantly improved in comparison with those in the PLGA scaffolds. In a rat calvarial defect model, new bone formation was enhanced in the PLGA/HA-S/ErhBMP-2 implants at 4 and 8 weeks after implantation too. Therefore, the PLGA/HA-S scaffold can better enhance the ErhBMP-2-induced osteogenic differentiation of BMSCs in vitro and osteogenesis in vivo.

Bone formation of block and particulated biphasic calcium phosphate lyophilized with Escherichia coli–derived recombinant human bone morphogenetic protein 2 in rat calvarial defects

The objective of this study was to evaluate bone formation in rat calvarial defects after surgical implantation of block or particulated biphasic calcium phosphate (BCP) lyophilized with Escherichia coli-derived recombinant human bone morphogenetic protein 2 (ErhBMP-2). Critical-size calvarial osteotomy defects were created in 5 groups of Sprague-Dawley rats. Each group received one of the following: 1) sham surgery control; 2) biphasic calcium phosphate particles (CPP); 3) biphasic calcium phosphate block (CPB); 4) ErhBMP-2-coated CPP; or 5) ErhBMP-2-coated CPB. ErhBMP was coated on BCP by a stepwise lyophilizing protocol. The new bone formation was significantly greater in ErhBMP-2-treated groups compared with the untreated group. In particular, the ErhBMP-2/CPB group showed stability of augmented areas during the period of healing, due to relevant space-providing capacity. Thus, it can be concluded that CPP and CPB lyophilized with ErhBMP-2 enhance the formation of new bone, and CPB appears to be a suitable carrier for ErhBMP-2 in which a 3-dimensional structural integrity is an important consideration factor.

Alveolar ridge augmentation using anodized implants coated with Escherichia coli–derived recombinant human bone morphogenetic protein 2

The aim of this study was to examine the effect of Escherichia coli-derived recombinant human bone morphogenetic protein 2 (ErhBMP-2) coated onto anodized implant to stimulate local bone formation, including osseointegration and the vertical augmentation of the alveolar ridge. Six young male adult beagle dogs were used. A crestal area was leveled on both sides of each test subject by removing minimal cortical bone using a round bur and without exposing cancellous bone. After a 2-month healing period, 3 anodized implants (length 8 mm, diameter 4 mm; Cowellmedi, Busan, Korea) were placed 5 mm into the mandibular alveolar ridge in either side. Each animal received 6 implants that were either coated with ErhBMP-2 (0.75 or 1.5 mg/mL concentration; Cowellmedi) or uncoated. This was performed using a randomized split-mouth design. A total of 36 implants were used for this study. Twelve noncoated implants were used as control, and 24 BMP-coated implants were used as our experimental group, which was further divided into 2 groups of 12 implants each with different BMP concentration of 0.75 and 1.5 mg/mL. Radiologic examinations were performed immediately after implant placement and 4 and 8 weeks after implant placement. The amount of bone augmentation was evaluated by measuring the distance from the uppermost point of the cover screw to the marginal bone. Implant stability quotient (ISQ) values were measured immediately after surgery and 8 weeks after implant placement. Statistical analysis was performed using one-way analysis of variance (SPSS version 17.0) and multiple-comparison tests. Statistical significance was established at the 95% confidence level. Implants coated with ErhBMP-2 at 0.75 mg/mL (BMP 0.75 group) and 1.5 mg/mL (BMP 1.5 group) exhibited significant vertical bone formation compared with the control group (mean ± SD): 0.88 ± 0.94 versus 0.60 ± 0.64 versus -0.52 ± 0.64 mm, respectively; P < .05. There was a significant difference between the 3 groups in bone level change (P < .05). The BMP 0.75 and BMP 1.5 groups exhibited significant changes in ISQ compared with the control group: 8.17 ± 8.31 versus 11.50 ± 9.02 versus 2.17 ± 7.61, respectively; P < .05. Within the limits of this study, the ErhBMP-2 coating on an anodized implant may stimulate vertical bone augmentation, which significantly increases implant stability on completely healed alveolar ridges.

アテロコラーゲンスポンジ（ＴＥＲＵＰＬＵＧ）を担体とする遺伝子組み換え ヒト骨形成因子の骨誘導に関する実験的研究―アテロペプチドコラーゲン（Ｃｅｌｌｍａｔｒｉｘ ＬＡ）を担体とした場合との比較―

We evaluated the usefulness of atelo-collagen sponge (ACS)(TERUPLUG®, TERMO CORPORATION, Tokyo) as a carrier for Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2) as compared with that of atelo-peptide collagen (CL)(Cellmatrix LA, Nitta Gelatin Inc., Osaka, Japan), which we previously demonstrated to be useful for bone induction. Five micrograms of ErhBMP- 2 with either 3 mg of ACS (ACS group) or 3 mg of CL (CL group) was implanted into the calf muscle of 13 10-week-old Wistar rats. The implanted material was sampled on days 7, 14, and 21 after implantation (5 specimens each per time, group). Radiographic evaluation, histological analysis, and biochemical examinations were performed. On radiographic evaluation, no opaque shadows were observed 7 days after implantation in either group. Oval shadows were observed 14 days after implantation. Twentyone days after implantation, the area of the shadows had increased. On histological analysis, much new cartilage was observed 7 days after implantation in both groups, but immature endochondral ossification was observed in only the ACS group. Endochondral ossification was found on the outermost edge of the implant after 14 days in both groups. After 21 days, mature bone had formed. On biochemical examinations on days 7 to 14 after implantation, alkaline phosphatase (ALP) activity and the calcium (Ca) content had increased markedly in both groups. On days 14 to 21 after implantation, ALP activity and Ca content increased gradually. These findings were similar in both groups.Our results suggest that, similar to CL, ACS is useful as a carrier for ErhBMP-2 designed to promote new bone formation.