Enhanced bone morphogenetic protein-2-induced ectopic and orthotopic bone formation by intermittent parathyroid hormone (1-34) administration

Abstract

Bone morphogenetic proteins (BMPs) play a central role in local bone regeneration strategies, whereas the anabolic features of parathyroid hormone (PTH) are particularly appealing for the systemic treatment of generalized bone loss. The aim of the current study was to investigate whether local BMP-2-induced bone regeneration could be enhanced by systemic administration of PTH (1-34). Empty or BMP-2-loaded poly(lactic-co glycolic acid)/poly(propylene fumarate)/gelatin composites were implanted subcutaneously and in femoral defects in rats (n = 9). For the orthotopic site, empty defects were also tested. Each of the conditions was investigated in combination with daily administered subcutaneous PTH (1-34) injections in the neck. After 8 weeks of implantation, bone mineral density (BMD) and bone volume were analyzed using microcomputed tomography and histology. Ectopic bone formation and almost complete healing of the femoral defect were only seen in rats that received BMP-2-loaded composites. Additional treatment of the rats with PTH (1-34) resulted in significantly (p < 0.05) enhanced BMD and bone volume in the BMP-2 composites at both implantation sites. Despite its effect on BMD in the humerus and vertebra, PTH (1-34) treatment had no significant effect on BMD and bone volume in the empty femoral defects and the ectopically or orthotopically implanted empty composites. Histological analysis showed that the newly formed bone had a normal woven and trabecular appearance. Overall, this study suggests that intermittent administration of a low PTH dose alone has limited potential to enhance local bone regeneration in a critical-sized defect in rats. However, when combined with local BMP-2-releasing scaffolds, PTH administration significantly enhanced osteogenesis in both ectopic and orthotopic sites.

FIG. 1.

Bone mineral density of the ectopic implants, orthotopic implants, vertebra L1, and humerus after 8 weeks of follow-up. The symbols indicate significant differences relative to (*) ectopic bone morphogenetic proteins (BMP)-2-loaded implants, (#) unfilled and empty scaffold filled orthotopic defects, (+) all other groups, or (x) nontreated vertebrae or humeri (p < 0.05). PTH, parathyroid hormone.

FIG. 2.

Three-dimensional microcomputed tomography reconstructions (A) and bone volumes (B) of subcutaneously formed bone after 8 weeks of implantation in rats. The asterisk (*) indicates significant difference relative to BMP-2-loaded implants (p = 0.02).

FIG. 3.

Three-dimensional microcomputed tomography reconstructions (A) and bone volumes (B) of newly formed bone in the 5-mm femoral defects after 8 weeks of follow-up. The symbols indicate significant differences relative to (#) unfilled defects, defects filled with empty scaffolds, or defects filled with empty scaffolds treated with parathyroid hormone (PTH) injections and (+) all defects (p < 0.05).

FIG. 4.

Histological sections of subcutaneously implanted empty (A) or BMP-2 (B–D) scaffolds in rats that were treated with (A, C, D) or without (B) PTH. Hematoxylin/eosin-stained section (A) showing the interconnected porous polymer network and fibrous tissue (f) in the scaffold. Goldner-stained sections (B–D) showing bone formation (b) and osteoid deposition (arrows) at the surface and in the pores of BMP-2 scaffolds (s). Scale bars represent 500 μm (A, D), 200 μm (B), and 50 μm (C). Color images available online at www.liebertonline.com/ten.

FIG. 5.

Histological sections of an empty femoral defect (A) and defects filled with empty (B) or BMP-2 scaffolds (C–F) in rats treated without (A, C) or with (B, D–F) PTH. Methylene blue/basic fuchsin-stained sections (A–E) showing interpositioning of muscles (m), fibrous tissue formation (f), and bone formation (b) extending from the original femur cortices (c) into the defect and the pores of the scaffold (s). The vascular network in the bone and fibrous tissue is observed by the blue Microfil^® agent (arrows). Endochondral ossification was seen in the center of the defects of BMP-2-containing implants (E). Fluorochrome analysis of unstained sections (F) showed calceine green and alizarin red deposition in newly formed bone. Scale bars represent 500 μm (A–D), 50 μm (D, E), and 500 μm (F). Color images available online at www.liebertonline.com/ten.