Teriparatide (1-34 human PTH) regulation of osterix during fracture repair

Abstract

Based on remarkable success of PTH as an anabolic drug for osteoporosis, case reports of off-label use of teriparatide (1-34 PTH) in patients with complicated fractures and non-unions are emerging. We investigated the mechanisms underlying PTH accelerated fracture repair. Bone marrow cells from 7 days 40 microg/kg of teriparatide treated or saline control mice were cultured and Osx and osteoblast phenotypic gene expression assessed by real-time RT-PCR in these cells. Fractured animals injected daily with either saline or 40 microg/kg of teriparatide for up to 21 days were X-rayed and histological assessment performed, as well as immunohistochemical analyses of the Osx expression in the fracture callus. Osx, Runx2 and osteoblast or chondrocyte phenotypic gene expression was also assessed in fracture calluses. Our data shows that Osx and Runx2 are up-regulated in marrow-derived MSCs isolated from mice systemically treated with teriparatide. Furthermore, these MSCs undergo accelerated osteoblast maturation compared to saline injected controls. Systemic teriparatide treatments also accelerated fracture healing in these mice concomitantly with increased Osx expression in the PTH treated fracture calluses compared to controls. Collectively, these data suggest a mechanism for teriparatide mediated fracture healing possibly via Osx induction in MSCs.

Figure 1. Teriparatide induces bone marrow derived mesenchymal stem cell differentiation into osteoblasts

Bone marrow derived progenitor cells were isolated from femurs of mice previously injected daily with teriparatide or saline solution for 7 days. Cells were cultured for 7, 14, and 28 days for total RNA analysis. mRNA levels of Osx (A), Runx2 (B), type I collagen (C), alkaline phosphatase (D), and osteocalcin (E) were measured using real time RT-PCR. Values, which are normalized to β actin, are expressed as means ± SE (n=3). * denotes statistical significance from controls at each time point (p<0.05).

Figure 2. Teriparatide accelerates femur fracture repair

7–9 week old C57BL/6 mice underwent unilateral femur fractures and were subsequently injected with daily teriparatide (lower panels) or saline solution (upper panels) as described in the Methods section. X-rays (A), histology (B), and histomophometric analysis (C) were performed 7, 10, and 14 days post-fracture. Values are expressed as means ± SE (n=3). * denotes statistical significance from controls at each time point (p<0.05).

Figure 3. The effects of teriparatide on Osx protein expression during fracture repair

7–9 week old C57BL/6 mice underwent unilateral femur fractures and were subsequently injected daily with teriparatide (right panels; x10 and x40) or saline solution (left panels; x10 and x40) as described in the Methods section. Osx proteins were detected in the fracture callus at day 7, 10, 14, and day 21 post-fracture using immunohistochemical analysis. Of note is the strong immunostaining in the progenitor cells (arrows) as well as osteoblasts (*) and osteocytes (^@) seen in newly formed bone. In addition, there is a lack of staining in the hypertrophic chondrocytes (#).

Figure 3. The effects of teriparatide on Osx protein expression during fracture repair

7–9 week old C57BL/6 mice underwent unilateral femur fractures and were subsequently injected daily with teriparatide (right panels; x10 and x40) or saline solution (left panels; x10 and x40) as described in the Methods section. Osx proteins were detected in the fracture callus at day 7, 10, 14, and day 21 post-fracture using immunohistochemical analysis. Of note is the strong immunostaining in the progenitor cells (arrows) as well as osteoblasts (*) and osteocytes (^@) seen in newly formed bone. In addition, there is a lack of staining in the hypertrophic chondrocytes (#).

Figure 4. Teriparatide increases Osx transcript expression in mouse callus during fracture healing

7–9 week old C57BL/6 mice underwent unilateral femur fractures and received daily injection of teriparatide or saline solution for 7 to 21 days. Fracture calluses at various healing time points (7, 10, 14, and 21 days post fracture) were collected and total RNA extracted. mRNA levels of Osx (A), Runx2 (B), Sox9 (C), type II collagen (D), type 1 collagen (E), and osteocalcin (F) were measured using real time RT-PCR. Values are expressed as means ± SE (n=3), normalized to β-actin. * denotes statistical significance from saline controls at each time point (p<0.05).