Biomechanical analysis of engineered bone with anti-BMP2 antibody immobilized on different scaffolds

Abstract

Recently we have demonstrated the ability of monoclonal antibodies (mAb) specific for bone morphogenetic protein (BMP)-2 immobilized on different scaffolds to mediate bone formation, a process referred to as Antibody Mediated Osseous Regeneration (AMOR). One of the key properties of regenerated bone is its biomechanical strength, in particular in load-bearing areas. This study sought to test the hypothesis that the biomechanical strength of regenerated bone depends of the mode of regeneration, as well as the scaffold used. Four different scaffolds, namely titanium granules (Ti), alginate hydrogel, anorganic bovine bone mineral (ABBM), and absorbable collagen sponge (ACS) were functionalized with anti-BMP-2 or isotype control mAb and implanted into rat critical-size calvarial defects. The morphology, density and strength of the regenerated bone were evaluated after 8 weeks. Results demonstrated that scaffolds functionalized with anti-BMP-2 mAb exhibited varying degrees of bone volume and density. Ti and ABBM achieved the highest bone volume, density, and strength of bone. When anti-BMP-2 mAb was immobilized on Ti or ABBM, the strength of the regenerated bone were 80% and 77% of native bone respectively, compared with 60% of native bone in sites implanted with rh-BMP-2. Control interventions with isotype mAb did not promote considerable bone regeneration and exhibited significantly lower mechanical properties. SEM analysis showed specimens immobilized with anti-BMP-2 mAb formed new bone with organized structure bridging the crack areas. Altogether, the present data demonstrated that the morphological and mechanical properties of bone bioengineered through AMOR could approximate that of native bone, when appropriate scaffolds are used. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1465-1473, 2016.

Keywords: antibody-mediated bone regeneration; biomaterials; bone morphogenetic protein; mechanical properties; monoclonal antibody; tissue engineering.

Figure 1

Micro computed tomographic analysis of specimens at 8-weeks post- implantation of different biomaterials used as scaffolds for AMOR. (a) Representative 3-D rendered images following micro-CT scanning of calvarial defects implanted with biomaterials preloaded with chimeric anti-BMP-2 mAb or isotype control mAb. (b) Quantitative analysis of micro-CT data was carried out and expressed as bone volume fraction (BV/TV) for each group (N=4). (c) The bone mineral density (BMD) of each retrieved specimen revealed that sites implanted with anti-BMP-2 mAb immobilized on Ti granules or ABBM exhibited significantly higher density in comparison to sites implanted with anti-BMP-2 mAb immobilized on ACS or alginate. ACS scaffold immobilized with rhBMP2 (1.5 mg/ml) was used as the positive control intervention. *p<0.05,**p < 0.01. NS= not significant.

Figure 2

Antibody mediated bone regeneration in vivo histological analysis. (a) Histological analysis of rat calvarial defects implanted with chimeric anti-BMP-2 mAb immobilized on 4 different types of scaffolds. Scale bar = 1 mm for low magnification images and 50 μm for histomicrographs in high magnification. (b) Quantitative histomorphometric analysis of newly formed osteoid bone. ACS scaffold immobilized with rhBMP2 (1.5 mg/ml) was used as the positive control group. *p<0.05,**p < 0.01. NS= not significant.

Figure 3

Mechanical strength of the engineered bone depends on the type of the scaffold utilized. (a) Results of biomechanical evaluation of the regenerated bone using chimeric anti-BMP-2 mAb immobilized on different biomaterials, showing Ti granules and ABBM scaffolds regenerated the strongest new bone in comparison to ACS and alginate. (b) Results of biomechanical evaluation of the regenerated bone in comparison to native bone. ABBM, Ti, alginate and ACS achieved 77%, 80%, 40% and 28% of the biomechanical strength of native bone, respectively. rhBMP-2 used in conjunction with ACS, served as positive control, achieved 66% of the strength of native bone. (c) Stiffness (N/mm) of tested specimens 8 weeks post implantation. *p<0.05, NS= not significant.

Figure 4

(a) X-Y scatter plot illustrating the correlation between bone mineral density (BMD) and maximum load to fracture for each scaffold. (b) X-Y scatter plot showing the correlation between bone volume fraction (BV/TV) and maximum load to fracture of tested scaffolds. (c) X-Y scatter plot illustrating the correlation between percentage of osteoid bone area based on histomorphometric analysis and maximum load to fracture for each scaffold confirming the important role of the utilized biomaterial in the mechanical strength of the bioengineered bone via AMOR.

Figure 5

SEM analysis. Representative SEM photomicrographs of the fracture site following mechanical testing showing specimens immobilized with chimeric anti-BMP-2 mAb formed new bone with organized collagen fibrils bridging the crack areas while the negative control group did not promote any bone regeneration. Further analysis confirmed that in ACS and alginate specimens the fracture happened within the regenerated bone (white arrows) while, in the ABBM and Ti groups immobilized with anti-BMP-2 mAb, the fractures were observed closer to the junction of regenerated bone and the native bone (black arrows).