Evaluation of the optimal dosage of BMP-9 through the comparison of bone regeneration induced by BMP-9 versus BMP-2 using an injectable microparticle embedded thermosensitive polymeric carrier in a rat cranial defect model

Abstract

Bone morphogenetic proteins (BMPs) are well known as enhancers and facilitators of osteogenesis during bone regeneration. The use of recombinant BMP-2 (rhBMP-2) in bone defect healing has drawbacks, which has driven the scouting for alternatives, such as recombinant BMP-9 (rhBMP-9), to provide comparable new bone formation. However, the dosage of rhBMP-9 is quintessential for the facilitation of adequate bone defect healing. Therefore, this study has been designed to evaluate the optimal dosage of BMP-9 by comparing the bone defect healing induced by rhBMP-9 over rhBMP-2. The chitosan (CS) microparticles (MPs), coated with BMPs, were embedded in a thermoresponsive methylcellulose (MC) and calcium alginate (Alg) based injectable delivery system containing a dosage of either 0.5 μg or 1.5 μg of the respective rhBMP per bone defect. A 5 mm critical-sized cranial defect rat model has been used in this study, and bone tissues were harvested at eight weeks post-surgery. The standard tools for comparing the new bone regeneration included micro computerized tomography (micro-CT) and histological analysis. A novel perspective of analyzing the new bone quality and crystallinity was employed by using Raman spectroscopy, along with its elastic modulus quantified through Atomic Force Microscopy (AFM). Results showed that the rhBMP-9 administered at a dosage of 1.5 μg per bone defect, using this delivery system, can adequately facilitate the bone void filling with ample new bone mineralization and crystallinity as compared to rhBMP-2, thus approving the hypothesis for a viable rhBMP-2 alternative.

Keywords: Atomic force microscopy; Bone morphogenetic protein; Bone regeneration; Microparticles; Raman spectroscopy.

Figure 1.

Schematic Representation of the Experimental Design for the In Vivo Study for the Injectable MPs-Gel System Bone Tissue Scaffold.

Figure 2.

MicroCT evaluation of cranial bone formation. (A) 3-D reconstructed images showing the new bone formation on the defects after eight weeks. (B) Quantification of bone volume normalized to the size of the defect after eight weeks, n=7. * and ** represents statistical significance at p < 0.05 and p < 0.001 respectively.

Figure 2.

MicroCT evaluation of cranial bone formation. (A) 3-D reconstructed images showing the new bone formation on the defects after eight weeks. (B) Quantification of bone volume normalized to the size of the defect after eight weeks, n=7. * and ** represents statistical significance at p < 0.05 and p < 0.001 respectively.

Figure 3.

Histological sections of bone samples harvested from the defect site stained with hematoxylin and eosin for different groups. FT: fibrous tissue, MP: microparticles, NB: new bone. Black arrows: new blood vessels, green arrows: osteocytes.

Figure 3.

Histological sections of bone samples harvested from the defect site stained with hematoxylin and eosin for different groups. FT: fibrous tissue, MP: microparticles, NB: new bone. Black arrows: new blood vessels, green arrows: osteocytes.

Figure 4.

(A) Schematic representation of Raman spectroscopy for the bone samples with 785 nm laser; (B) Raman spectra of rat pristine cranial bone (without defects). Raman spectrum normalized to 959 cm⁻¹.

Figure 5.

Raman spectra for all bone sample groups. y-axis: 100% → 20,000 intensity counts.

Figure 6.

MMR for the bone groups. Pristine bone has been designated as bone Only. (A) Phosphate MMR of the bone sample groups with significant difference observed between the groups, especially between BMP-9H and the other BMP-9 groups (including Gel Only and Void); (B) Carbonate MMR of the bone sample groups; similar trend compared to the Phosphate MMR with significantly higher BMP-9H carbonate MMR over the other BMP-9 groups has been observed; (C) Reciprocal of the full width at half maximum for the Phosphate band obtained for the bone sample groups; BMP-9H group shows significantly higher mineral crystallinity over BMP-9L and BMP-9L + VEGF groups; (D) BMP 9H group microscope image using 10x objective of the Raman microscope; scale =100 μm (E) BMP 2L sample microscope image with 10x objective of the Raman microscope; scale =100 μm. Statistical significance between groups was calculated at a 95% confidence interval; * represents a p-value < 0.05. n=39.

Figure 7.

(A) Schematic representation of the PFQNM mode of AFM used for measuring elastic modulus of the bone samples; (B) Atomic force microscopy measurements for all bone sample groups; p-value < 0.05 was used to establish statistical significance between groups. n=21. Pristine bone has been designated as bone Only.