Sclerostin deficient mice rapidly heal bone defects by activating β-catenin and increasing intramembranous ossification

Authors

Meghan E. McGee-Lawrence, Zachary C. Ryan, Lomeli R. Carpioc, Sanjeev Kakar, Jennifer J. Westendorf, Rajiv Kumar

Abstract

We investigated the influence of the osteocyte protein, sclerostin, on fracture healing by examining the dynamics and mechanisms of repair of single-cortex, stabilized femoral defects in sclerostin knockout (Sost−/−; KO) and sclerostin wild-type (Sost+/+; WT) mice. Fourteen days following generation of bone defects, Sost KO mice had significantly more bone in the healing defect than WT mice. The increase in regenerating bone was due to an increase in the thickness of trabecularized spicules, osteoblast numbers and surfaces within the defect. Enhanced healing of bone defects in Sost KO mice was associated with significantly more activated β-catenin expression than observed in WT mice. The findings were similar to those observed in Axin2−/− mice, in which β-catenin signaling is known to be enhanced to facilitate bone regeneration. Taken together, these data indicate that enhanced β-catenin signaling is present in Sost−/− mice that demonstrate accelerated healing of bone defects, suggesting that modulation of β-catenin signaling in bone could be used to promote fracture repair.

Link to Article

http://dx.doi.org/10.1016/j.bbrc.2013.10.155

Evaluation of the effects of systemic treatment with a sclerostin neutralizing antibody on bone repair in a rat femoral defect model

Authors

Farhang Alaee, Mandeep S. Virk, Hezhen Tang, Osamu Sugiyama, Douglas J. Adams, Marina Stolina, Denise Dwyer, Michael S. Ominsky, Hua Zhu Ke, Jay R. Lieberman

Abstract

Systemic administration of a sclerostin neutralizing antibody (Scl-Ab) has been shown to enhance fracture callus density and strength in several animal models. In order to further evaluate the potential of Scl-Ab to improve healing in a bone defect model, we evaluated Scl-Ab in a 3 mm femoral defect in young male outbred rats. Scl-Ab was given either continuously for 6 or 12 weeks after surgery or with 2 weeks of delay for 10 weeks. Bone formation was assessed by radiographs, µ-CT, and histology. Complete bony union was achieved in only a few defects after 12 weeks of healing (Scl-Ab treated 5/30, vehicle treated 1/15). µ-CT evaluation demonstrated a significant increase in the BV/TV in the defect in the delayed treatment group (65%, p < 0.05), but a non-significant increase in the continuous group (35%, p = 0.11) compared to control. However, both regimens induced an anabolic response in the bone proximal and distal to the defect and in the un-operated femurs. We demonstrate that treatment with Scl-Ab can enhance bone repair in a bone defect and in the surrounding host bone, but lacks the osteoinductive activity to heal it. This agent seems to be most effective in bone repair scenarios where there is cortical integrity.

Link to Article

http://onlinelibrary.wiley.com/doi/10.1002/jor.22498/abstract

Fracture healing with alendronate treatment in the Brtl/+ mouse model of osteogenesis imperfecta

Authors

J.A. Meganck, D.L. Begun, J.D. McElderry, A. Swick, K.M. Kozloff, S.A. Goldstein, M.D. Morris, J.C. Marini, M.S. Caird

Abstract

Osteogenesis imperfecta (OI) is a heritable bone dysplasia characterized by increased skeletal fragility. Patients are often treated with bisphosphonates to attempt to reduce fracture risk. However, bisphosphonates reside in the skeleton for many years and long-term administration may impact bone material quality. Acutely, there is concern about risk of non-union of fractures that occur near the time of bisphosphonate administration. This study investigated the effect of alendronate, a potent aminobisphosphonate, on fracture healing. Using the Brtl/+ murine model of type IV OI, tibial fractures were generated in 8-week-old mice that were untreated, treated with alendronate before fracture, or treated before and after fracture. After 2, 3, or 5 weeks of healing, tibiae were assessed using microcomputed tomography (μCT), torsion testing, quantitative histomorphometry, and Raman microspectroscopy. There were no morphologic, biomechanical or histomorphometric differences in callus between untreated mice and mice that received alendronate before fracture. Alendronate treatment before fracture did not cause a significant increase in cartilage retention in fracture callus. Both Brtl/+ and WT mice that received alendronate before and after fracture had increases in the callus volume, bone volume fraction and torque at failure after 5 weeks of healing. Raman microspectroscopy results did not show any effects of alendronate in wild-type mice, but calluses from Brtl/+ mice treated with alendronate during healing had a decreased mineral-to-matrix ratio, decreased crystallinity and an increased carbonate-to-phosphate ratio. Treatment with alendronate altered the dynamics of healing by preventing callus volume decreases later in the healing process. Fracture healing in Brtl/+ untreated animals was not significantly different from animals in which alendronate was halted at the time of fracture.

Link to Article

http://dx.doi.org/10.1016/j.bone.2013.06.003

Neuropeptide Y modulates fracture healing through Y1 receptor signaling

Authors

Daniela M. Sousa, Michelle M. McDonald, Kathy Mikulec, Lauren Peacock, Herbert Herzog, Meriem Lamghari, David G. Little, Paul A. Baldock

Abstract

Neuropeptide Y acting via it's Y1 receptor represents a powerful pathway in the control of bone mass. The global or osteoblast-specific Y1 receptor deletion induces pronounced bone anabolic effects in mice. However, the contribution of Y1 receptor deletion in bone repair/healing remained to be clarified. Therefore, in this study we characterized the role of Y1 receptor deletion in fracture healing. Closed tibial fractures were generated in germline (Y1−/−) and osteoblastic-specific Y1 receptor knockout mice. The progression of tibial repair monitored from 1- until 6-weeks post-fracture demonstrated that in Y1−/− mice there is a delay in fracture repair, as seen by a decrease in bone callus volume and callus strength. Moreover, the histological features included elevated avascular and cartilage area and consequently delayed cartilage removal, and hence impaired union. Interestingly, this delay in bone repair was not related directly to Y1 receptors expressed by mature osteoblasts. These findings suggest that the global absence of the Y1 receptor delays fracture healing, through impairing the early phases of fracture repair to achieve bony union. The data acquired on the role of Y1 receptor signaling disruption in bone regeneration is critical for the design of future therapeutic strategies.

Link to Article

http://dx.doi.org/10.1002/jor.22400

Mice Lacking Pten in Osteoblasts Have Improved Intramembranous and Late Endochondral Fracture Healing

Authors

Travis A. Burgers, Martin F. Hoffmann, Caitlyn J. Collins, Juraj Zahatnansky, Martin A. Alvarado, Michael R. Morris, Debra L. Sietsema, James J. Mason, Clifford B. Jones, Heidi L. Ploeg, Bart O. Williams

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-cretg/+;Ptenflox/flox). Mid-diaphyseal femoral fractures induced in wild-type and Ocn-cretg/+;Ptenflox/flox mice were studied via micro-computed tomography (µCT) scans, biomechanical testing, histological and histomorphometric analysis, and protein expression analysis. Ocn-cretg/+;Ptenflox/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.

Link to Article

http://dx.doi.org/10.1371/journal.pone.0063857

Angiogenesis is required for stress fracture healing in rats

Authors

Ryan E. Tomlinson, Jennifer A. McKenzie, Anne H. Schmieder, Gregory R. Wohl, Gregory M. Lanza, Matthew J. Silva

Abstract

Although angiogenesis and osteogenesis are critically linked, the importance of angiogenesis for stress fracture healing is unknown. In this study, mechanical loading was used to create a non-displaced stress fracture in the adult rat forelimb. Fumagillin, an anti-angiogenic agent, was used as the water soluble analogue TNP-470 (25 mg/kg) as well as incorporated into lipid-encapsulated αvβ3 integrin targeted nanoparticles (0.25 mg/kg). In the first experiment, TNP-470 was administered daily for 5 days following mechanical loading, and changes in gene expression, vascularity, and woven bone formation were quantified. Although no changes in vascularity were detected 3 days after loading, treatment-related downregulation of angiogenic (Pecam1) and osteogenic (Bsp, Osx) genes was observed at this early time point. On day 7, microCT imaging of loaded limbs revealed diminished woven bone formation in treated limbs compared to vehicle treated limbs. In the second experiment, αvβ3 integrin targeted fumagillin nanoparticles were administered as before, albeit with a 100-fold lower dose, and changes in vascularity and woven bone formation were determined. There were no treatment-related changes in vessel count or volume 3 days after loading, although fewer angiogenic (CD105 positive) blood vessels were present in treated limbs compared to vehicle treated limbs. This result manifested on day 7 as a reduction in total vascularity, as measured by histology (vessel count) and microCT (vessel volume). Similar to the first experiment, treated limbs had diminished woven bone formation on day 7 compared to vehicle treated limbs. These results indicate that angiogenesis is required for stress fracture healing, and may have implications for inducing rapid repair of stress fractures.

Link to Article

http://dx.doi.org/10.1016/j.bone.2012.09.035