Sequential dual-drug delivery of BMP-2 and alendronate from hydroxyapatite-collagen scaffolds for enhanced bone regeneration

Abstract

The clinical use of bioactive molecules in bone regeneration has been known to have side effects, which result from uncontrolled and supraphysiological doses. In this study, we demonstrated the synergistic effect of two bioactive molecules, bone morphogenic protein-2 (BMP-2) and alendronate (ALN), by releasing them in a sequential manner. Collagen-hydroxyapatite composite scaffolds functionalized using BMP-2 are loaded with biodegradable microspheres where ALN is encapsulated. The results indicate an initial release of BMP-2 for a few days, followed by the sequential release of ALN after two weeks. The composite scaffolds significantly increase osteogenic activity owing to the synergistic effect of BMP-2 and ALN. Enhanced bone regeneration was identified at eight weeks post-implantation in the rat 8-mm critical-sized defect. Our findings suggest that the sequential delivery of BMP-2 and ALN from the scaffolds results in a synergistic effect on bone regeneration, which is unprecedented. Therefore, such a system exhibits potential for the application of cell-free tissue engineering.

Figure 1

Schematic illustration of sequential dual-drug delivery using BMP-2 and ALN.

Figure 2

Morphological analysis of CHAS. SEM images of (A) PLGA microspheres; (B) hydroxyapatite; (C) collagen scaffold; (D) CHAS; (E) µ-CT images of CHAS (green threshold level at 147).

Figure 3

Initial or delayed drug release of BMP-2 (A) and alendronate (B) depending on the presence of PLGA microparticles in CHAS.

Figure 4

(A) Cell viability evaluation of CHAS with drugs (i.e., BMP-2 and/or ALN) using CCK-8 assay on osteoblasts. (B) ALP activity of osteoblasts in CHAS depending on the drugs (i.e., BMP-2 and/or ALN).

Figure 5

Micro-computed tomography-reconstructed images at 2, 4, and 8 weeks postoperatively. µ-CT radiographic images of rat calvarial bone formed at 2, 4, and 8 weeks postoperatively in different groups, showing degree and extent of mineral deposition at the defect site.

Figure 6

Quantitative analysis of new bone volume at 2, 4 and 8 weeks postoperatively. µ-CT based histomorphometry analysis of the defect site showing the amount of highly mineralized volume and BV/TV percentage at the defect site at each time point (2, 4, and 8 weeks). (*p < 0.05; **p < 0.01; ***p < 0.001 vs. control group; one-way ANOVA test).

Figure 7

Histological evaluation of bone regeneration in rat calvarial defects at 4 and 8 weeks postoperatively: (A) Image for hematoxylin and eosin staining and Masson’s trichrome staining. Yellow background shows where defects were created. Semi-quantitative analysis of red-stained area ratio (%) of Masson’s trichrome staining in (B) 4 weeks and (C) 8 weeks using ImageJ software (V1.8.0_172, NIH, USA) (https://imagej.nih.gov/ij/). CB, cortical bone; LB, lamellar bone; WB, woven bone; F, fibrous tissue; IC, inflammation cell; S, scaffold; BM, bone marrow; DI, diploe; V: vessels. (×40 and ×100 magnification, scale bar = 200 μm. *p < 0.05; ***p < 0.001 versus control group; Two-way krANOVA test).