Parathyroid hormone stimulates bone regeneration in an atrophic non-union model in aged mice

Abstract

Background: Non-union formation still represents a major burden in trauma and orthopedic surgery. Moreover, aged patients are at an increased risk for bone healing failure. Parathyroid hormone (PTH) has been shown to accelerate fracture healing in young adult animals. However, there is no information whether PTH also stimulates bone regeneration in atrophic non-unions in the aged. Therefore, the aim of the present study was to analyze the effect of PTH on bone regeneration in an atrophic non-union model in aged CD-1 mice.

Methods: After creation of a 1.8 mm segmental defect, mice femora were stabilized by pin-clip fixation. The animals were treated daily with either 200 µg/kg body weight PTH 1-34 (n = 17) or saline (control; n = 17) subcutaneously. Bone regeneration was analyzed by means of X-ray, biomechanics, micro-computed tomography (µCT) imaging as well as histological, immunohistochemical and Western blot analyses.

Results: In PTH-treated animals bone formation was markedly improved when compared to controls. This was associated with an increased bending stiffness as well as a higher number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and CD31-positive microvessels within the callus tissue. Furthermore, PTH-treated aged animals showed a decreased inflammatory response, characterized by a lower number of MPO-positive granulocytes and CD68-positive macrophages within the bone defects when compared to controls. Additional Western blot analyses demonstrated a significantly higher expression of cyclooxygenase (COX)-2 and phosphoinositide 3-kinase (PI3K) in PTH-treated mice.

Conclusion: Taken together, these findings indicate that PTH is an effective pharmacological compound for the treatment of non-union formation in aged animals.

Keywords: Aging; Angiogenesis; Bone regeneration; Fracture healing; Inflammation; Mice; Non-union; Parathyroid hormone; Segmental defect.

Fig. 1

A–D Representative X-rays of femora of controls (A, B) and PTH-treated mice (C, D) at 2 (A, C) and 10 weeks (B, D) after surgery. Scale bars: 1 mm. Representative µCT-3D reconstructions (E, G, I, K) and transversal µCT images (F, H, J, L) of controls (E–H) and PTH-treated animals (I–L) at 2 (E, F, I, J) and 10 weeks (G, H, K, L) after surgery. Scale bars: 0.5 mm. M, N Poorly (PM) and highly mineralized (HM) bone volume of the callus tissue of controls (white bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) and PTH-treated mice (black bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) at 2 (M) and 10 weeks (N) after surgery, as assessed by µCT analyses. Mean ± SEM; *p < 0.05 vs. control

Fig. 2

A–E BV/TV (%) (A), bone surface density (1/mm) (B), trabecular thickness (mm) (C), trabecular separation (mm) (D) and trabecular number (1/mm) (E) of the callus tissue of controls (white bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) and PTH-treated mice (black bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) at 2 and 10 weeks after surgery, as assessed by µCT analyses. F Bending stiffness (N/mm) of femora of controls (white bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) and PTH-treated mice (black bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) at 2 and 10 weeks after surgery, as assessed by biomechanical analysis. Mean ± SEM; *p < 0.05 vs. control

Fig. 3

A–D Representative histological images of Safranin-O-stained femora of controls (A, B) and PTH-treated mice (C, D) at 2 (A, C) and 10 weeks (B, D) after surgery. Fibrous tissue (ft), cartilaginous tissue (ct), woven bone (wb) and cortical bone (cb) are indicated. Scale bars: 0.5 mm. E, F Callus composition (%), including fibrous tissue (white), cartilaginous tissue (gray) and osseous tissue (black), of the callus of controls (n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) and PTH-treated mice (n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) at 2 (E) and 10 (F) weeks after surgery, as assessed by histomorphometric analysis. G Representative histological images of TRAP-positive osteoclasts (arrowheads) within the callus tissue of controls and PTH-treated mice at 2 and 10 weeks after surgery. Scale bars: 25 μm. H TRAP-positive osteoclasts/HPF within the callus tissue of controls (white bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) and PTH-treated mice (black bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) at 2 and 10 weeks after surgery, as assessed by histological analysis. Mean ± SEM; *p < 0.05 vs. control

Fig. 4

A Representative immunohistochemical images of CD31-positive microvessels (arrowheads) within the callus tissue of controls and PTH-treated mice at 2 and 10 weeks after surgery. Scale bars: 25 μm. B CD31-positive microvessels/HPF within the callus tissue of controls (white bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) and PTH-treated mice (black bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) at 2 and 10 weeks after surgery, as assessed by immunohistochemical analysis. Mean ± SEM; *p < 0.05 vs. control. C Representative immunohistochemical images of MPO-positive cells (arrowheads) within the callus tissue of controls and PTH-treated mice at 2 and 10 weeks after surgery. Scale bars: 25 μm. D MPO-positive cells/HPF within the callus tissue of controls (white bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) and PTH-treated mice (black bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) at 2 and 10 weeks after surgery, as assessed by immunohistochemical analysis. Mean ± SEM; *p < 0.05 vs. control. E Representative immunohistochemical images of CD68-positive cells (arrowheads) within the callus tissue of controls and PTH-treated mice at 2 and 10 weeks after surgery. Scale bars: 25 μm. F CD68-positive cells/HPF within the callus tissue of controls (white bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) and PTH-treated mice (black bars, n = 5 at 2 weeks after surgery, n = 9 at 10 weeks after surgery) at 2 and 10 weeks after surgery, as assessed by immunohistochemical analysis. Mean ± SEM; *p < 0.05 vs. control

Fig. 5

A Representative Western blots of VEGF, COX-2, PI3K and β-actin expression within the callus tissue of controls and PTH-treated mice at 2 weeks after surgery. B–D Expression of VEGF (pixel intensity × 10⁴) (B), COX-2 (pixel intensity × 10⁴) (C) and PI3K (pixel intensity × 10⁴) (D) within the callus tissue of controls (white bars, n = 3) and PTH-treated mice (black bars, n = 3) at 2 weeks after surgery, as assessed by Western blot analysis. Mean ± SEM; *p < 0.05 vs. control