Inhibin A enhances bone formation during distraction osteogenesis

Abstract

Given the aging population and the increased incidence of fracture in the elderly population, the need exists for agents that can enhance bone healing, particularly in situations of delayed fracture healing and/or non-union. Our previous studies demonstrated that overexpression of the gonadal peptide, human inhibin A (hInhA), in transgenic mice enhances bone formation and strength via increased osteoblast activity. We tested the hypothesis that hInhA can also exert anabolic effects in a murine model of distraction osteogenesis (DO), using both transgenic hInhA overexpression and administration of normal physiological levels of hInhA in adult male Swiss-Webster mice. Tibial osteotomies and external ring fixation were performed, followed by a 3-day latency period, 14-day distraction, and sacrifice on day 18. Supraphysiological levels of hInhA in transgenic mice, but not normal physiological levels of hInhA, significantly increased endosteal bone formation and mineralized bone area in the distraction gap, as determined by radiographic and µCT analysis. Significantly, increased PCNA and osteocalcin expression in the primary matrix front suggested that hInhA increased osteoblast proliferation. This mechanism is consistent with the effects of other agents and pathologies that modulate bone formation during DO, and demonstrates the potential of hInhA to enhance bone repair and regeneration.

Figure 1. Serum hInhA levels are increased in vivo

(A) Normal Swiss-Webster mice treated with vehicle or hInhA (40.8+/−7.2 pg/ml) for 18 days. (B) Glvp/InhA bigenic mice were implanted with Vehicle or MFP to stimulate the systemic overexpression of hInhA. Human InhA was undetectable in both Swiss Webster (A) and Glvp/InhA mice treated with Vehicle (B). Data shown are mean +/− SD.;*=p<0.05.

Figure 2. Supraphysiological concentrations of hInh A enhance new bone formation during DO

(Top) Percent total new bone area in the DO gap of (A) Swiss-Webster mouse tibiae treated with vehicle or physiological concentrations of hInhA by Alzet minipump for the 18 day duration of the experiment or (B) Glvp/InhA mice treated with Vehicle or MFP to overexpress InhA. Data shown are mean +/− SD; *=p<0.05. (Bottom) Representative X-ray images from each treatment are shown under the corresponding bars in the top panels.

Figure 3. hInhA overexpression enhanced new microcolumn formation but not the area of new matrix formation during DO

Photomicrographs of the proximal half of a decalcified paraffin embedded histological sections of the DO gap in Glvp/InhA mice treated with Vehicle during DO (A), or overexpressing hInhA during the time of DO (B) and stained with H&E depicting the major biological zones of the distraction gap. Analytically, the zone of microcolumn formation (MCF) is referred to as Endosteal New Bone (ENB). BM, Bone Marrow; C, Cortex; FIZ, Fibrous Interzone; PMF, Primary Matrix Front. Bars = 500µm. (C) The formation of new bone matrix in the endosteal distraction gap of Vehicle-treated Glvp/InhA mice and Glvp/InhA mice treated with MFP was assessed in central sections of decalcified DO gaps stained with H&E. (D) The osteocyte density in the ENB was enumerated per bone area in central sections of decalcified DO gaps stained with H&E. No significant differences in cellularity were observed. Data shown are mean +/− SD.

Figure 4. hInhA overexpression increases endosteal new bone formation during DO

(A) MicroCT reconstruction of mouse tibiae. (Top) Swiss-Webster treated with vehicle or hInhA for 18 days. (Bottom) Glvp/InhA mice treated with Vehicle or MFP to overexpress hInhA. Mice were sacrificed 18 days post-operatively and the DO gap scanned. (B) MicroCT translucent reconstructions illustrate hInhA effects on endosteal bone volume. 3-Dimensional reconstruction of endosteal new bone in Vehicle treated (yellow) and InhA overexpressing (gray) Glvp/InhA mice. (C) Total volume and bone volume of the endosteal distraction gap were calculated directly from the voxel volumes in the reconstruction. Data shown are mean +/− SD; *=P<0.05.

Figure 5. hInhA overexpression increases osteoblast proliferation in the primary matrix front (PMF) during DO

Paraffin embedded decalcified DO gaps containing ENB from Vehicle-treated Glvp/InhA mice and Glvp/InhA mice treated with MFP to stimulate hInhA overexpression (Glvp/InhA), stained for expression of proliferating cell nuclear antigen (PCNA, brown stain). (A) Vehicle treated, (B, C) hInhA overexpressing samples. (C) Negative control (no primary anti-PCNA antibody). Bars=500µm. (D) PCNA+ cells adjacent to the ENB were quantified in four 20× visual fields in the proximal PMF. Data are mean +/− SD; (*=P<0.05).

Figure 6. Osteocalcin expression identifies osteoblastic cells in the PMF during DO

Paraffin embedded decalcified DO gaps containing ENB from Vehicle-treated Glvp/InhA mice and Glvp/InhA mice treated with MFP to stimulate hInhA overexpression (Glvp/InhA), and stained for osteocalcin. (A) Vehicle treated, (B, C) InhA overexpressing samples. (C) Negative control (no primary anti-osteocalcin antibody) Bar=500µm.