Topical co-administration of zoledronate with recombinant human bone morphogenetic protein-2 can induce and maintain bone formation in the bone marrow environment

Abstract

Background: Bone morphogenetic proteins (BMPs) induce osteogenesis in various environments. However, when BMPs are used alone in the bone marrow environment, the maintenance of new bone formation is difficult owing to vigorous bone resorption. This is because BMPs stimulate the differentiation of not only osteoblast precursor cells but also osteoclast precursor cells. The present study aimed to induce and maintain new bone formation using the topical co-administration of recombinant human BMP-2 (rh-BMP-2) and zoledronate (ZOL) on beta-tricalcium phosphate (β-TCP) composite.

Methods: β-TCP columns were impregnated with both rh-BMP-2 (30 µg) and ZOL (5 µg), rh-BMP-2 alone, or ZOL alone, and implanted into the left femur canal of New Zealand white rabbits (n = 56). The implanted β-TCP columns were harvested and evaluated at 3 and 6 weeks after implantation. These harvested β-TCP columns were evaluated radiologically using plane radiograph, and histologically using haematoxylin/eosin (H&E) and Masson's trichrome (MT) staining. In addition, micro-computed tomography (CT) was performed for qualitative analysis of bone formation in each group (n = 7).

Results: Tissue sections stained with H&E and MT dyes revealed that new bone formation inside the β-TCP composite was significantly greater in those impregnated with both rh-BMP-2 and ZOL than in those from the other experimental groups at 3 and 6 weeks after implantations (p < 0.05). Micro-CT data also demonstrated that the bone volume and the bone mineral density inside the β-TCP columns were significantly greater in those impregnated with both rh-BMP-2 and ZOL than in those from the other experimental groups at 3 and 6 weeks after implantations (p < 0.05).

Conclusions: The topical co-administration of both rh-BMP-2 and ZOL on β-TCP composite promoted and maintained newly formed bone structure in the bone marrow environment.

Keywords: Bone morphogenetic proteins; Histology; Rabbit; micro computed tomography; β-tricalcium phosphate.

Fig. 1

Representative photos of the left distal femurs of rabbits cut in the sagittal plane at 3 and 6 weeks after implantation. In the images, the left side is distal side of femur and the upper side is dorsal side of femur. Arrows point to a section of the edge of the implanted β-TCP. The gross appearances of implanted β-TCP columns gradually disappeared (a). The gross appearances of β-TCP columns in groups containing ZOL (f and h) were comparatively more recognizable than in the other groups without ZOL (b and d) at 6 weeks after implantation

Fig. 2

Representative X-ray images of the left distal femurs of rabbits from each group at 0, 3, and 6 weeks after implantation. In the images, the left side is distal side of femur and the upper side is dorsal side of femur. The radiolucencies at the area of implanted β-TCP columns gradually increased. The radiolucencies of β-TCP columns in groups containing ZOL (i and l) were comparatively more recognizable than in the other groups without ZOL (c and f) at 6 weeks after implantation

Fig. 3

Representative H&E and Masson’s Trichrome stained sections of the left distal femurs of rabbits cut in the sagittal plane in each group at 3 and 6 weeks after implantation. In each image, the proximal section is displayed on the right and the dorsal section is displayed on the upper parts of the figure. The dotted box in the low-powered view (2×) indicates the range of high-powered view. The high-powered views (20×) were captured randomly from inside the implanted β-TCP areas for quantitative evaluation. The uniformly-stained tissue area, pointed by arrows, indicate newly formed trabecular bone structure. At 3 weeks after implantation, stained tissue areas were recognized as new bone area was significantly increased in groups containing rh-BMP-2 (f, h,n, and p). New bone area only remained in groups treated with both rh-BMP-2 and ZOL (d’ and f’) at 6 weeks after implantation. Note: H&E, Hematoxylin-Eosin; rh-BMP-2, recombinant human bone morphogenetic protein 2; ZOL, zoledronate; β-TCP, β-tricalcium phosphate

Fig. 4

Quantitative evaluation of H&E sections and Masson’s Trichrome sections of the of the left distal femurs of rabbits in each group at 3 and 6 weeks after implantation. The columns and bars represent the means and standard deviations (n = 7), respectively. At 3 weeks after implantation, the groups containing rh-BMP-2 (Group 2 or 4) showed greater areas of new bone formation than the other groups (P < 0.05). However, at 6 weeks after implantation, only the group (Group 4) that involved the combination usage of both rh-BMP-2 and ZOL still showed areas of newly formed bone (P < 0.05). *: P < 0.05. Statistical differences between groups were determined using a one-way analysis of variance with Bonferroni’s multiple comparison test. Note: H&E, Hematoxylin-Eosin; MT, Masson Trichrome; rh-BMP-2, recombinant human bone morphogenetic protein 2; ZOL, zoledronate

Fig. 5

μ-CT evaluation of BV/TV and BMD in retrieved β-TCP implants at 3 and 6 weeks after implantation. The columns and bars represent the means and standard deviations (n = 7), respectively. *: P < 0.05. Statistical differences between groups were determined with the one-way ANOVA and post-hoc Bonferroni test. Note: BV/TV, Bone volume/ Total tissue volume; BMD, Bone mineral density