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This study evaluated 3D printed polycaprolactone (PCL) composite scaffold and recombinant human bone morphogenetic protein-2 (rhBMP-2), loaded either onto a PCL composite scaffold or implant surface, for vertical bone augmentation with implant placement. Three-dimensional printed PCL frames were filled with powdered PCL, hydroxyapatite, and β-tricalcium phosphate. RhBMP-2 was loaded to the PCL composite scaffolds and implant surfaces, and rhBMP-2 release was quantified for 21 days. Experimental implants were placed bilaterally on 20 rabbit calvaria, and the PCL composite scaffolds were vertically augmented. The randomly allocated experimental groups were divided by carrier and rhBMP-2 dosage as no rhBMP-2 (control), 5 μg rhBMP-2 loaded to PCL composite (Scaffold/rhBMP-2[5 μg]), 5 μg rhBMP-2 loaded to implant (Implant/rhBMP-2[5 μg]), 30 μg rhBMP-2 loaded to PCL composite (Scaffold/rhBMP-2[30 μg]), and 30 μg rhBMP-2 loaded to implant (Implant/rhBMP-2[30 μg]). Histologic and histometric analyses were conducted after 8 weeks. In both scaffold-loading and implant-loading, rhBMP-2 released initially rapidly, then slowly and constantly. Released rhBMP-2 totaled 23.02 ± 1.03% and 24.69 ± 1.14% in the scaffold-loaded and implant-loaded groups, respectively. There were no significant differences in histologic bone-implant contact (%). Peri-implant bone density (%) was significantly higher in the Scaffold/rhBMP-2(30 μg) and Implant/rhBMP-2(30 μg) groups. Total bone density (%) was not significantly different between the Scaffold/rhBMP-2(5 μg), Implant/rhBMP-2(5 μg), and control groups, or between the Scaffold/rhBMP-2(30 μg) and Implant/rhBMP-2(30 μg) groups, but was significantly higher in the Scaffold/rhBMP-2(30 μg) and Implant/rhBMP-2(30 μg) groups than in the controls. Three-dimensional printed PCL composite scaffold with rhBMP-2 produced vertical osteogenesis and osseointegration, regardless of rhBMP-2 loading to the PCL composite scaffold or implant surface., (© 2021 Wiley Periodicals LLC.)

Objective: to investigate whether harmine has a promotive effect on human periodontal ligament cells (hPDLCs)-induced tissue regeneration., Materials and Methods: Various concentrations of harmine on hPDLCs proliferation were tested. Osteogenic and cementogenic characteristics were examined in hPDLC/rhBMP-2 and hPDLC/harmine by alizarin red S staining, real-time PCR, and Western blotting assay. The activity of harmine was investigated in an ectopic transplantation nude mouse model., Results: We determined that 10 μM of harmine was the threshold concentration. hPDLC/harmine showed similar mineralized nodule formation in alizarin S staining compared to hPDLC/rhBMP-2. In real-time PCR, the highest gene expression level was observed for Runx2 in hPDLC/harmine at all time points. The level of CEMP-1 in hPDLC/harmine was higher at 7 days than hPDLCs alone. Thicker band of Runx2 in hPDLC/harmine was observed than in hPDLC/rhBMP-2 at 7 days by Western blotting. The band for CEMP-1 in hPDLC/harmine was thicker than hPDLCs alone at both 7 and 14 days. In ectopic transplantation, hPDLCs with harmine showed a comparable amount of mineralized tissue formation compared to rhBMP-2. hPDLCs with harmine or rhBMP-2 formed both bone and cementum-like tissue with Sharpey's fiber-like collagen insertion., Conclusion: Harmine can be a potential candidate for promoting hPDLCs-induced tissue regeneration., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

This is the first report on the development of a covalently bone morphogenetic protein-2 (BMP2)-immobilized hydrogel that is suitable for osteogenic differentiation of human periodontal ligament stem cells (hPLSCs). O-propargyl-tyrosine (OpgY) was site-specifically incorporated into BMP2 to prepare BMP2-OpgY with an alkyne group. The engineered BMP2-OpgY exhibited osteogenic characteristics after in vitro osteogenic differentiation of hPLSCs, indicating the osteogenic ability of BMP2-OpgY. A methoxy polyethylene glycol-(polycaprolactone-(N 3 )) block copolymer (MC-N 3 ) was prepared as an injectable in situ-forming hydrogel. BMP2 covalently immobilized on an MC hydrogel (MC-BMP2) was prepared quantitatively by a simple biorthogonal reaction between alkyne groups on BMP2-OpgY and azide groups on MC-N 3 via a Cu(I)-catalyzed click reaction. The hPLSCs-loaded MC-BMP2 formed a hydrogel almost immediately upon injection into animals. In vivo osteogenic differentiation of hPLSCs in the MC-BMP2 formulation was confirmed by histological staining and gene expression analyses. Histological staining of hPLSC-loaded MC-BMP2 implants showed evidence of mineralized calcium deposits, whereas hPLSC-loaded MC-Cl or BMP2-OpgY mixed with MC-Cl, implants showed no mineral deposits. Additionally, MC-BMP2 induced higher levels of osteogenic gene expression in hPLSCs than in other groups. In conclusion, BMP2-OpgY covalently immobilized on MC-BMP2 induced osteogenic differentiation of hPLSCs as a noninvasive method for bone tissue engineering.

Human periodontal ligament stem cells (hPDLSCs) are considered potential cellular carriers for gene delivery in the field of tissue regeneration. This study tested the osseoregenerative potential of hPDLSCs transduced with replication-deficient recombinant adenovirus (rAd) containing the gene encoding bone morphogenetic protein-2 (BMP2; hPDLSCs/rAd-BMP2) in both in vivo and in vitro osteogenic environments. After the optimal condition for rAd-mediated transduction was determined, hPDLSCs were transduced to express BMP2. In vivo bone formation was evaluated in a critical-size rat calvarial bone defect model that more closely mimics the harsher in vivo milieu for bone regeneration than subcutaneous transplantation model. As support materials for bone regeneration, block-type biphasic calcium phosphate (BCP) scaffolds were combined with hPDLSCs and/or BMP2 and transplanted into critical-size bone defects in rats. Experimental groups were as follows: BCP scaffold control (group 1 [Gr1]), scaffold containing recombinant human BMP2 (rhBMP2; group 2 [Gr2]), scaffold loaded with normal hPDLSCs (group 3 [Gr3]), scaffold combined with both normal hPDLSCs and rhBMP2 (group 4 [Gr4]), and scaffold loaded with hPDLSCs transduced with rAd-BMP2 (hPDLSCs/rAd-BMP2; group 5 [Gr5]). Our data showed that new bone formation was highest in Gr2. Less mineralization was observed in Gr3, Gr4, and Gr5 in which hPDLSCs were transplanted. In vitro transwell assay demonstrated that hPDLSCs exert an inhibitory activity on BMP2-induced osteogenic differentiation. Our findings suggest that the in vivo bone regenerative potential of BMP2-overexpressing hPDLSCs could be compromised in a critical-size rat calvarial bone defect model. Thus, further investigations are required to elucidate the underlying mechanisms and to develop efficient techniques for improved tissue regeneration.

Aim: This study compared the effects of Escherichia-coli-produced recombinant human bone morphogenetic protein 2 (ErhBMP-2) with a biphasic calcium phosphate (BCP) carrier to those of deproteinized bovine bone in human maxillary sinus floor augmentation., Material and Methods: Screening for this clinical trial selected 56 sites that provided informed consent to participate, of which 46 were ultimately enrolled and 41 were finally included in the study. The sites were divided into two groups using a random-number table, and the material was applied. A trephine biopsy was performed after 24 weeks, and implants wider than the biopsy site were inserted. Computed tomography and plain panoramic images were obtained immediately and then again at 24 weeks after the surgery. Radiographic images were reconstructed to allow measurement of the linear and volumetric changes. The biopsy samples were processed for histologic and histometric analyzes., Results: All sites healed uneventfully with no complications. Radiographic analysis revealed a tendency for the volume to increase, but the difference was not statistically significant in either group. Comparison of volumetric changes between the two groups also revealed no significant difference. Moreover, none of the histometric parameters differed significantly between the groups, although different healing patterns were observed on histologic analysis., Conclusions and Clinical Implications: It can be concluded that sinus augmentation with ErhBMP-2 carrying BCP carrier did not enhance bone regeneration compared to the conventional treatment using deproteinized bovine bone at 24 weeks after the surgery., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Human periodontal ligament stem cells (hPDLSCs) are considered an appropriate cell source for therapeutic strategies. The aims of this study were to investigate the sustainability of bone morphogenetic protein 2 (BMP2) secretion and the bone regenerative capacity of hPDLSCs that had been genetically modified to express the gene encoding BMP2 (BMP2). hPDLSCs isolated from healthy third molars were transduced using replication-deficient recombinant adenovirus (rAd) encoding BMP2 (hPDLSCs/rAd-BMP2), and the cellular characteristics and osteogenic potentials of hPDLSCs/rAd-BMP2 were analyzed both in vitro and in vivo. hPDLSCs/rAd-BMP2 successfully secreted BMP2, formed colonies, and expressed immunophenotypes similar to their nontransduced counterparts. As to their osteogenic potential, hPDLSCs/rAd-BMP2 formed greater mineralized nodules and exhibited significantly higher levels of expression of BMP2 and the gene encoding alkaline phosphatase, and formed more and better quality bone than other hPDLSC-containing or recombinant human BMP2-treated groups, being localized at the initial site until 8 weeks. The findings of the present study demonstrate that hPDLSCs/rAd-BMP2 effectively promote osteogenesis not only in vitro but also in vivo. The findings also suggest that hPDLSCs can efficiently carry and deliver BMP2, and that hPDLSCs/rAd-BMP2 could be used in an attractive novel therapeutic approach for the regeneration of deteriorated bony defects.

Block-type biphasic calcium phosphate (BCP) carriers are more effective at delivering recombinant human bone morphogenetic protein-2 (rhBMP-2) in various clinical situations than are particle-type carriers, due to their potential for highly successful three-dimensional bone regeneration. The aim of this study was to confirm the bone-regenerative capabilities of three-dimensional BCP blocks with a low hydroxyapatite/β-tricalcium phosphate ratio (20/80) combined with collagen (10% wt) as an rhBMP-2 delivery system in a craniofacial vertical bone augmentation model. BCP blocks and BCP-collagen blocks (with average macropore sizes of 296 and 390 μm, respectively) with or without rhBMP-2 were fixed with osteosynthesis screws to the calvarial surface of rabbits. After 8 weeks, histologic and histomorphometric analyses were performed to evaluate the resulting new bone area, augmented area, bone density, and degree of integration. The area of new bone was significantly greater in specimens containing rhBMP-2 than in the control group (p < 0.05). Moreover, the area fractions of newly formed bone within the augmented area and a degree of integration between the regenerative bone and the calvarium were both significantly greater in the BCP-collagen/rhBMP-2 group than in the BCP/rhBMP-2 group (p < 0.05), whereas the two carrier systems exhibited similar rhBMP-2 release profiles, with sustained and linear release. The BCP and BCP/rhBMP-2 blocks exhibited excellent structural integrity, with large fragments of residual ceramic. In conclusion, the BCP-collagen composite block exhibited enhanced osteoinductive potential and could be a good candidate as a carrier of rhBMP-2 due to its characteristics of favorable volumetric stability, ease of handling, and excellent remodeling properties., (Copyright © 2012 Wiley Periodicals, Inc.)

Objectives: The purpose of this pilot study was to determine the osteoinductive effect of absorbable collagen sponge (ACS) loaded with Escherichia coli-expressed recombinant human bone morphogenetic protein 2 (ErhBMP-2) and evaluate structural stability of ACS in a standardized rabbit sinus model., Material and Methods: The maxillary sinuses were prepared bilaterally in six male white rabbits. The windows were prepared using a 6 mm trephine bur, and circular bony windows were carefully removed. Following reflection of the sinus membrane, a saline-soaked ACS alone and an ErhBMP-2-loaded ACS were inserted into the left and right maxillary sinuses, respectively. After a healing period of 8 weeks, sections of the augmented sinus and surrounding bone were made and analyzed by microcomputed tomography and histologically for signs of window closure and bone augmentation., Results: Radiographic analysis revealed new bone formation in both groups of augmented sinus (i.e., with and without ErhBMP-2). The maximum augmented height did not differ significantly between the groups; however, window closure was significantly more advanced in the ErhBMP-2 group than in the control group (P=0.02). The defect was significantly deeper in the control group than in the ErhBMP-2-treated group (P=0.02)., Conclusions: In conclusion, ErhBMP-2-loaded ACS showed enhanced osteoinductive potential, particularly with regard to bone closure of a sinus window and facilitated maturation of the newly formed bone within the rabbit sinus cavity. However, the structural durability of ACS was not sufficient to maintain the augmented volume in the sinus., (© 2011 John Wiley & Sons A/S.)

Objective: The aim of the current study was to determine whether a hydroxyapatite (HA)/beta-tricalcium phosphate (β-TCP) ratio of 20/80 impregnated with recombinant human bone morphogenetic protein (rhBMP-2) enhances new bone formation and to evaluate the dose-dependent response of rhBMP-2., Study Design: Critical-sized calvarial defects were made in rats, and biphasic calcium phosphate (BCP) with different rhBMP-2 doses was loaded into rat calvarial defects. The animals were allowed to heal for either 2 or 8 weeks., Results: The percentages of new bone after 2 and 8 weeks of healing were significantly greater in the rhBMP-2-treated groups (at all doses) than in the control groups. The percentage of remaining BCP was significantly lower at 8 weeks than at 2 weeks in all groups that included BCP., Conclusions: rhBMP-2 administered using a BCP carrier significantly induces new bone formation. A dose-dependent response was not shown in the present study., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background and Objective: The potential of the Escherichia coli-expressed recombinant human bone morphogenetic protein-2 (ErhBMP-2) to support new bone formation/maturation using a block-type of macroporous biphasic calcium phosphate (bMBCP) carrier was evaluated in an orthotopic and ectopic rat model., Material and Methods: Critical-size (Φ 8 mm) calvarial defects and subcutaneous pockets in 32 Sprague-Dawley rats received implants of rhBMP-2 (2.5 μg) in a bMBCP carrier or bMBCP alone (control). Implant sites were evaluated using histological and histometric analysis following 2- and 8-wk healing intervals (eight animals/group/interval)., Results:   ErhBMP-2/bMBCP supported significantly greater bone formation at 2 and 8 wk (10.8% and 25.4%, respectively) than the control at 2 and 8 wk (5.3% and 14.0%, respectively) in calvarial defects (p < 0.01). Bone formation was only observed for the ErhBMP-2/bMBCP ectopic sites and was significantly greater at 8 wk (7.5%) than at 2 wk (4.5%) (p < 0.01). Appositional and endochondral bone formation was usually associated with a significant increase in fatty marrow at 8 wk. The bMBCP carrier showed no evidence of bioresorption., Conclusion: ErhBMP-2/bMBCP induced significant bone formation in both calvarial and ectopic sites. Further study appears to be required to evaluate the relevance of the bMBCP carrier., (© 2011 John Wiley & Sons A/S.)

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., (Crown Copyright © 2011. Published by Mosby, Inc. All rights reserved.)

We investigated the ability of recombinant human bone morphogenetic protein-2, produced from Escherichia coli (ErhBMP-2), to form orthotopic and ectopic bone in rat models. BMP-2 was expressed in E. coli and extracted from the inclusion bodies. Critical-sized calvarial defects and subcutaneous pouches were created in rats, and an absorbable collagen sponge (ACS) was loaded with different doses of ErhBMP-2 for implantation. ACS alone and sham surgery controls were also included. Implant sites were evaluated by histological and/or histometric analyses following a 2- or 8-week healing interval. In the calvarial defect model, enhanced bone formation was observed with all doses of ErhBMP-2, while only limited amounts of new bone were found in controls. In the ectopic subcutaneous implant model, bone formation was clearly observed in all animals treated with ErhBMP-2 at 2 weeks. However, at 8 weeks, less new bone formation was detected than at 2 weeks. Nevertheless, the remaining new bone showed an advanced degree of bone remodeling and more maturity than that observed at 2 weeks. These results showed that ErhBMP-2 was osteoinductive under controlled in vivo conditions. Thus, ErhBMP-2 has definite potential as an alternative to rhBMP-2 produced in a eukaryotic system for clinical use., (Copyright 2010 Elsevier Ltd. All rights reserved.)