Elsevier

Bone

Volume 49, Issue 6, December 2011, Pages 1178-1185
Bone

Original Full Length Article
Increased callus mass and enhanced strength during fracture healing in mice lacking the sclerostin gene,☆☆

https://doi.org/10.1016/j.bone.2011.08.012Get rights and content

Abstract

Humans with inherited sclerostin deficiency have high bone mass. Targeted deletion of the sclerostin gene in mice (SOST-KO) causes increases in bone formation, bone mass and bone strength. Inhibition of sclerostin by a monoclonal antibody increases bone formation and enhances fracture healing in rodent and primate models. In this study, we describe the temporal progression of femoral fracture healing in SOST-KO mice compared with wild type (WT) control mice to further characterize the role of sclerostin in fracture healing.

Sixty-seven male 9–10 week-old SOST-KO (N = 37) and WT (N = 30) mice underwent a closed femoral fracture. Weekly radiography was used to monitor the progress of healing. Histologic sections were used to characterize callus composition, evaluate callus bridging, and quantify lamellar bone formation on days 14 and 28. Densitometry and biomechanical testing were utilized to characterize bone mass and strength at the fractured and contralateral femurs on day 45.

A significant improvement in time to radiographic healing (no discernible fracture line) was observed in SOST-KO mice, which corresponded to an increase in histologic bony bridging at 14 days (38% versus 0% in WT). Both genotypes appeared to be nearly fully bridged at 28 days post-fracture. The increased bridging at 14 days was associated with 97% greater bone area and 40% lower cartilage area in the callus of SOST-KO mice as compared to WT mice. Bone formation-related endpoints were higher in SOST-KO mice at both 14 and 28 days. At 45 days post-fracture, peak load and bone mass were significantly greater in the fractured femurs of SOST-KO mice as compared to WT mice.

In conclusion, fractures in mice lacking sclerostin showed accelerated bridging, greater callus maturation, and increased bone formation and strength in the callus.

Highlights

► We examined differences in fracture healing in sclerostin KO and wild type mice. ► Closed femoral fracture model. ► Accelerated healing process in KO. Increased callus bone formation in KO. ► Increased fractured bone BMD in KO. ► Increased fractured bone strength in KO.

Introduction

Sclerostin, an antagonist of Wnt/beta-catenin signaling, negatively regulates osteoblast differentiation and function, thereby acting as an inhibitor of bone formation. Humans with inherited sclerostin deficiency (sclerosteosis and van Buchem disease) show increased bone mass [1], [2], [3]. Sclerostin knockout (SOST-KO) mice not only exhibit high bone mass and bone strength associated with increased bone formation [4], but also experience less bone loss during skeletal unloading [5].

Recently, it has been shown that Wnt/beta-catenin signaling plays an important role in fracture healing [6]. Treatment with lithium, an agonist of Wnt/beta-catenin signaling, improved fracture healing; whereas treatment with Dickkopf-1, an antagonist of this pathway, inhibited healing [6].

Similarly, systemic inhibition of sclerostin by administration of a sclerostin-neutralizing antibody (sclerostin-Ab) increased bone formation and bone mass in an estrogen-deficient, osteopenic rat model [7], in female non-human primates [8], and in healthy men and postmenopausal women [9]. Furthermore, we have reported that sclerostin-Ab increased bone area and strength of the fracture callus in both rats and non-human primates [10], [11]. Sclerostin-Ab was also shown to improve bone integration and pull-out strength in a cancellous bone healing model, in which a screw was inserted in the proximal tibia in rats [12].

The purpose of the current study was to determine if knockout of sclerostin would affect the fracture healing process in mice. In this study, we conducted a closed mid-femoral fracture in SOST-KO and WT mice. We then assessed the progress of fracture healing by serial radiography; bone formation, callus formation, and callus remodeling by histomorphometry; bone mass by DXA and pQCT; and fractured bone strength by biomechanical testing.

Section snippets

Sclerostin knockout (SOST-KO) mice

SOST-KO mice were maintained on a 129/SvJ/Black Swiss background [4]. Sixty-seven 9- to 10-week old male mice entered the study. One set of SOST-KO (N = 23) and wild type (WT) (N = 18) mice was used to conduct histomorphometric analysis at 14 (N = 12 and N = 9) and 28 (N = 11 and N = 9) days post fracture. A second set of SOST-KO (N = 14) and WT (N = 12) mice was used for densitometry and biomechanical testing at 45 days post fracture. Fluorochrome labels (10 mg/kg calcein) were injected subcutaneously 6 and 2 

Serial radiographic imaging

Weekly in vivo radiographic examination revealed that SOST-KO had a larger and denser callus by 14 days post fracture that persisted for the rest of the experiment (Fig. 1). Peak callus size appeared to have occurred at 14 days post fracture, with reductions in callus size at subsequent time points. At 14 days post fracture, radiographic healing (no discernible fracture line) was observed in 52% (17 of 33) of SOST-KO animals and 24% (5 of 21) of WT animals. At 28 days post fracture, 90% (22 of 24)

Discussion

In the United States, over 7.9 million adults per year experience fractures, of which approximately 5–20% results in nonunion or delayed union [17], [18], [19]. Thus, a therapeutic intervention that improved fracture outcomes could have important clinical benefits. Sclerostin has been identified as an inhibitor of bone formation. Inhibition of sclerostin by systemic administration of a sclerostin antibody has been confirmed as a potential novel bone anabolic approach to combat osteoporosis and

Acknowledgments

The authors thank all the members of the Amgen and UCB sclerostin teams for their support of these studies, Dr. Don Kimmel for his help on editing of this manuscript and Dr. Michael Eschenberg for his help on statistic analysis of X-ray imaging scoring.

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    Accumulating evidence revealed that this paracrine interaction controls cell behavior, tissue formation and bone modeling/remodeling. These observations led to extensive preclinical investigations [7–13], and to the development of several neutralizing antibodies raised against sclerostin. Their evaluation in several randomized clinical trials conducted in women with osteoporosis (romosozumab and blosozumab) [14–18], or in patients with osteogenesis imperfecta (setrusumab-BPS-804) [19,20], showed that the systemic delivery of sclerostin antibodies significantly increased bone mass density through promoting osteoblast differentiation while inhibiting osteoclast formation [6,21,22].

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    More bone tissue and less cartilage were seen in the fracture callus of knock-out mice. After 45 days callus bone mass was significantly greater in knock-out mice compared to wildtype mice [67]. Sclerostin antibodies (Scl-Ab) were developed in the 2000s and could prove to increase bone formation, bone mass and bone strength in osteoporotic and healthy animal trials [68–70].

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Parts of the manuscript were presented at the 56th Annual Meeting of the Orthopedic Research Society, New Orleans, USA, March 6–9, 2010.

☆☆

All authors are employees of Amgen and have received stock and stock options from Amgen.

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