Bone regeneration using injectable BMP-7 loaded chitosan microparticles in rat femoral defect

Abstract

Injectable chitosan microparticles were prepared using a simple coacervation method under physiologically friendly conditions by eliminating oil or toxic chemical, and employing low temperature and pressure for growth factor stability. Amount of 200 ng of bone morphogenetic protein-7 (BMP-7) was incorporated in the chitosan microparticles by two methods: encapsulating and coating techniques. These microparticles were tested in vivo to determine the biological response in a rat femoral bone defect at 6 and 12 weeks. Four groups (n=10) were tested which include two groups for BMP-7 incorporated microparticles (by two techniques), microparticles without BMP-7, and defect itself (negative control). Healthy bone formation was observed around the microparticles, which were only confined to the defect site and did not disperse. Histology indicated minor inflammatory response around the microparticles at 6 weeks, which reduced by 12 weeks. Micro-CT analysis of bone surface density and porosity was found to be significantly more (p<0.05) for microparticles containing groups, in comparison with controls, which suggests that the new bone formed in the presence of microparticles is more interconnected and porous. Collagen fibrils analysis conducted using multiphoton microscopy showed significant improvement in the formation of bundled collagen area (%) in microparticles containing groups in comparison with controls, indicating higher cross-linking between the fibrils. Microparticles were biocompatible and did not degrade in the 12 week implant period.

Keywords: Bone morphogenetic protein-7; Chitosan microparticles; Histology; In vivo; Micro-CT; Multiphoton microscopy.

Fig. 1

(a) The surgical procedure showing a 4–5 mm defect in diaphysial region of femur, which was implanted with chitosan-TPP microparticles (b) SEM image of chitosan-TPP microparticles.

Fig. 2

Microscopic images of transverse sections (H&E) of rat femur at 6 weeks at 100x and 200x in (a,b) controls (c,d) microparticles group (e,f) BMP-7 coated microparticles group (g,h) BMP-7 encapsulated microparticles group respectively. CT, connective tissue; FB, fibroblasts; MP, microparticle; NB, new bone; OB, osteoblasts; OC, osteocytes; OCL, osteoclasts; OS, osteoid; IR, inflammatory response; BV, blood vessel (Scale bar represents 100 µm).

Fig. 3

Microscopic images of transverse sections (H&E) of rat femur at 12 weeks at 100x and 200x in (a,b) controls (c,d) microparticles group (e,f) BMP-7 coated microparticles group (g,h) BMP-7 encapsulated microparticles group respectively. CT, connective tissue; FB, fibroblasts; MP, microparticle; NB, new bone; OB, osteoblasts; OC, osteocytes; OCL, osteoclasts; OS, osteoid; IR, inflammatory response; BV, blood vessel (Scale bar represents 100 µm).

Fig. 4

Box plot representation of a,b) Bone volume fraction (BV/TV); c,d) Porosity of new bone; e,f) Bone surface density (BS/BV); g,h) Number of new bone fragments; i,j)

Fig. 4

Box plot representation of a,b) Bone volume fraction (BV/TV); c,d) Porosity of new bone; e,f) Bone surface density (BS/BV); g,h) Number of new bone fragments; i,j)

Fig. 5

Representative week 6 post-surgery micro-CT images of femurs for controls (a,b) and treated groups- microparticles (c,d); BMP-7 coated microparticles (e,f); BMP-7 encapsulated microparticles (g,h). Scale bar = 1 mm

Fig. 6

Representative week 12 post-surgery micro-CT images of femurs for controls (a,b) and treated groups- microparticles (c,d); BMP-7 coated microparticles (e,f); BMP-7 encapsulated microparticles (g,h). Scale bar = 1 mm.

Fig. 7

Representative histological section (H&E) as observed by multiphoton confocal microscopy under bright field view and the corresponding view of collagen fibers in the newly formed bone region (ROI) at 6 weeks for (a,b) controls (c,d) microparticles group (e,f) BMP-7 coated microparticles group (g,h) BMP-7 encapsulated microparticles group respectively. (Scale bar represents 250 µm).

Fig. 8

Representative histological section (H&E) as observed by multiphoton confocal microscopy under bright field view and the corresponding view of collagen fibers in the newly formed bone region (ROI) at 12 weeks for (a,b) controls (c,d) microparticles group (e,f) BMP-7 coated microparticles group (g,h) BMP-7 encapsulated microparticles group respectively. (Scale bar represents 250 µm).

Fig. 9

Box plot representation of bundled collagen formed in the new bone region (ROI) obtained by analyzing the confocal multiphoton microscopy images (n=15) using Image J software at a) 6 weeks b) 12 weeks.