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. 2019 Apr 19;49(2):114-126.
doi: 10.5051/jpis.2019.49.2.114. eCollection 2019 Apr.

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

Jae-Ho Hwang  1 , Seunghan Oh  2 , Sungtae Kim  1
Affiliations

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

Jae-Ho Hwang et al. J Periodontal Implant Sci. .

Abstract

Purpose: The 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).

Methods: The 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.

Results: The 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).

Conclusions: ErhBMP-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.

Keywords: Biphasic calcium phosphate; Bone morphogenetic protein 2; Bone substitute; epigallocatechin-3-gallate.

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Conflict of interest statement

Conflict of Interest: No potential conflict of interest relevant to this article was reported.

Figures

Figure 1
Figure 1. Schematic diagram of preparation of BCP coated with low-concentration ErhBMP-2 and EGCG 1. APTES treatment; 2. Bifunctional cross-linker (SMP) connection; 3. Immobilized ErhBMP-2 and EGCG grafting.
HA: hydroxyapatite, BCP: biphasic calcium phosphate, ErhBMP-2: Escherichia coli-expressed recombinant human bone morphogenetic protein 2, EGCG: epigallocatechin-3-gallate, APTES: 3-aminopropyltriethoxysilane, SMP: succinimidyl-3-maleimidopropionate.
Figure 2
Figure 2. Radiographic analysis: micro-CT. (A) Reconstructed image of micro-CT. (B) Region of interest: new bone volume was calculated as the remaining graft volume subtracted from the total augmented volume.
CT: computed tomography.
Figure 3
Figure 3. (A) Results of fluorescein diacetate staining at 2 and 24 hours. (B) Results of the MTT assay at 24 and 48 hours.
BCP: biphasic calcium phosphate, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Figure 4
Figure 4. Histological findings of the 4-week control group. (A) Area of interest (H&E). (B) Defect margin area (H&E, scale bar: 100 μm): immature osteocytes in the lacunae (arrowhead) were observed, which was a distinctive feature of the control group. (C) Near the Schneiderian membrane (H&E, scale bar: 100 μm). (D) Center of the augmented area (H&E, scale bar: 100 μm): the original curvature of the nasal bone was not restored, and loose connective tissue and adipose tissue were seen (black asterisk). Matured lamellar bone (white asterisk) can be hardly seen, either at the defect margin or in between graft materials.
H&E: hematoxylin and eosin.
Figure 5
Figure 5. Histological findings of the 4-week study group (A) Area of interest (H&E). (B) Defect margin area (H&E, scale bar: 100 μm): the number of immature osteocytes (arrowhead) was dramatically reduced. (C) Near the Schneiderian membrane (H&E, scale bar: 100 μm): black arrows indicate the shape of sharp, knife-edged bone graft materials, which were not greatly different from what was observed in the control group. (D) Center of the augmented area (H&E, scale bar: 100 μm): more vascularization and the presence of newly formed blood vessels (white asterisk) were identified. Black asterisks indicate that the majority of new bone formation occurred in a confined area, especially at the lateral side of the surgically created window.
H&E: hematoxylin and eosin.
Figure 6
Figure 6. Histological findings of the 8-week study group (A) Area of interest (H&E). (B) Defect margin area (H&E, scale bar: 100 μm). (C) Area near the Schneiderian membrane (H&E, scale bar: 100 μm): shallow, round, and ovoid-shaped graft materials were identified, indicating resorption of graft material particles. Intensely stained, well-lined patterns of compact lamellar bone (black asterisk) were prominent between graft material particles close to the Schneiderian membrane, showing very similar characteristics to the pre-existing bone. (D) Middle area (H&E, scale bar: 100 μm): surgically created window closure was markedly advanced and mature lamellar bone seemed to be predominant.
H&E: hematoxylin and eosin.
See this image and copyright information in PMC

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