Mice lacking pten in osteoblasts have improved intramembranous and late endochondral fracture healing

Abstract

The failure of an osseous fracture to heal (development of a non-union) is a common and debilitating clinical problem. Mice lacking the tumor suppressor Pten in osteoblasts have dramatic and progressive increases in bone volume and density throughout life. Since fracture healing is a recapitulation of bone development, we investigated the process of fracture healing in mice lacking Pten in osteoblasts (Ocn-cre(tg/+;)Pten(flox/flox) ). Mid-diaphyseal femoral fractures induced in wild-type and Ocn-cre(tg/+;)Pten(flox/flox) mice were studied via micro-computed tomography (µCT) scans, biomechanical testing, histological and histomorphometric analysis, and protein expression analysis. Ocn-cre(tg/+;)Pten(flox/flox) mice had significantly stiffer and stronger intact bones relative to controls in all cohorts. They also had significantly stiffer healing bones at day 28 post-fracture (PF) and significantly stronger healing bones at days 14, 21, and 28 PF. At day 7 PF, the proximal and distal ends of the Pten mutant calluses were more ossified. By day 28 PF, Pten mutants had larger and more mineralized calluses. Pten mutants had improved intramembranous bone formation during healing originating from the periosteum. They also had improved endochondral bone formation later in the healing process, after mature osteoblasts are present in the callus. Our results indicate that the inhibition of Pten can improve fracture healing and that the local or short-term use of commercially available Pten-inhibiting agents may have clinical application for enhancing fracture healing.

Figure 1. Fracture femoral (A) stiffness and (B) maximum strength.

Pten mutants had significantly higher stiffness at 28 d PF and maximum strength at 14, 21, and 28 d PF in fractured femurs. The stiffness and strength increased for both the wild-type and mutant groups during the healing process. (*p<0.05, **p<0.01, and ***p<0.001 WT to Mut at the time point.).

Figure 2. Representative longitudinal µCT sections of the fracture.

The Pten mutants had more bone formation at the proximal and distal ends of the fracture callus at each time point.

Figure 3. Representative µCT cross sections of bone formation from the periosteum.

Sections were taken an average of 2.7 mm away from the fracture. A black line was drawn around the existing bone to indicate the transition between it and newly formed bone. The Pten mutants had more ossification, especially around the existing bone, at each time point.

Figure 4. BV/TV µCT callus analysis: (A) BV/TV in proximal and distal callus ends, (B) BV/TV in center of callus.

Pten mutants had higher BV/TV at each time point in ends and center. (*p<0.05 WT to Mut at the time point.).

Figure 5. µCT callus analysis: (A) callus mineral content, (B) callus volume, (C) callus mineral density.

Pten mutants had more callus mineral content, volume and density at each time point. (*p<0.05 WT to Mut at the time point.).

Figure 6. p-Akt and Pten protein expression during healing.

Pten mutants had increased p-Akt expression at days 21 and 28 PF.

Figure 7. H&E of fracture calluses at day 14 PF.

(A) 4× magnification of wild-type callus; (B) 10× magnification of the box from (A); (C) 4× magnification of Pten mutant callus; and (D) 10× magnification of box from (C). Woven bone was spread throughout the callus and was surrounded hypertrophic chondrocytes in both cases.

Figure 8. TRAP stain of fracture calluses at day 14 PF.

(A) 10× magnification of wild-type callus; (B) 20× magnification of box from (A); (C) 10× magnification of Pten mutant callus; (D) 20× magnification of box from (C). TRAP staining was more intense in the mutant group.