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

doi:10.1016/j.bbrc.2013.10.155

Biochemical and Biophysical Research Communications

Volume 441, Issue 4, 29 November 2013, Pages 886-890

https://doi.org/10.1016/j.bbrc.2013.10.155 Get rights and content

Highlights

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Repair of cortical bone defects is enhanced in sclerostin-deficient (Sost^−/−) mice.
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Osteoblasts and β-catenin are increased in cortical bone defects of Sost^−/− mice.
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Enhanced β-catenin expression and bone repair occur in Sost^−/− and Axin2^−/− mice.

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.

Introduction

Fractures are among the most common injuries in people of all ages. The lifetime risk of sustaining a traumatic fracture that must be referred for orthopedic treatment exceeds that of stroke, type 2 diabetes, and either breast or prostate cancer [1]. Fractures reduce mobility, productivity and quality of life. Therefore, interventions that accelerate fracture repair will be of great therapeutic value.

The innate healing capacity of bone tissue is widely recognized. Wnts are potent morphogens and mitogens essential for normal bone formation and fracture healing [2], [3]. The canonical Wnt pathway is well recognized as a crucial pathway for proper skeletal development, maintenance of bone mass, and bone regeneration. This pathway dictates osteoblast specification from osteo/chondro-progenitors, inhibits early stages of chondrogenesis, stimulates osteoblast proliferation, enhances osteoblast and osteocyte survival, and transmits mechanical loading signals to bone lining cells [2], [4], [5], [6], [7]. A simplified description of “canonical” Wnt signaling begins with Wnt ligands binding to receptor complexes consisting of Lrp5/6 and Frizzled (Fzd) proteins [8]. This inactivates a destruction complex composed of Axin1/2, Gsk3β, Disheveled (Dsh), APC and other proteins, leading to β-catenin stabilization and its translocation to the nucleus where it displaces co-repressors from Tcf7 and Lef1 transcription factors and regulates the expression of genes involved in cell proliferation (e.g., cyclin D1) [9], negative feedback (e.g., Axin2) [10], osteoblast differentiation (e.g., osteocalcin) [11] and other activities (e.g., Wisp1, Wisp2). Several secreted proteins (e.g., sclerostin, Dkk1, and Sfrps) prevent Lrp5/6- and/or Wnt-mediated signaling.

Sclerostin is encoded by the SOST gene, which is mutated in several genetic disorders of high bone mass and expressed at high levels by osteocytes [12], [13]. The seminal discoveries that mutations in SOST and LRP5 alter bone density in humans sparked immense interest in these and other Wnt pathway components [12], [14], [15], [16]. Canonical Wnt signaling positively regulates proliferation of mesenchymal stem cells that act as the progenitor cell population for the osteoblast lineage [22]. Several studies indicate that sclerostin plays important roles in fracture healing. Sclerostin levels are increased near healing human fracture callus [17]. Genetic deletion of Sost [18] or administration of sclerostin-neutralizing antibodies [19] to WT mice accelerated the healing of transverse fractures through endochondral ossification. Anti-sclerostin antibodies also improved bone regeneration in cases of biologically impaired fracture healing, such as type 2 diabetes [20]. Previous studies emphasized the role of sclerostin as an inhibitor of osteogenic canonical Wnt signaling, and as such it might be inferred that β-catenin is required for the anabolic effect of sclerostin inhibition on bone formation. However, the involvement of this intracellular signaling mediator has not been tested or proven in vivo.

We recently described a new Sost-KO mouse line that has high bone mass due to increased bone mineral accretion and elevated osteoblast numbers [21]. In the current study, we examined the rate and mechanism of defect repair in a fracture model that heals by intramembranous ossification and differentiation of mesenchymal progenitors directly into osteoblast lineage cells. Our studies reveal that Sost-KO mice rapidly heal bone defects by activating β-catenin and increasing osteoblast numbers.

Section snippets

Materials and methods

All animal research was conducted according to guidelines provided by the National Institutes of Health and the Institute of Laboratory Animal Resources, National Research Council. The Mayo Clinic Institutional Animal Care and Use Committee approved all animal studies. Male WT (Sost^+/+) and sclerostin-deficient (Sost^−/−) mice [21] were 13 weeks-old when bone defects were introduced. Male mice deficient in Axin2 (Axin2^−/−), an intracellular negative feedback inhibitor of canonical

Results and discussion

As previously reported, Sost^−/− animals presented with noticeably enhanced bone mass as compared to WT (Sost^+/+) controls at 13 weeks of age (Fig. 1; note differences in cortical bone thickness between Sost^+/+ and Sost^−/− animals) [21]. Surgical defects were successfully induced in mice from both groups (Fig. 1A). Fourteen days after surgery, Sost^−/− animals had significantly more bone in the healing defect as compared to Sost^+/+ controls (Fig. 1A and B). MicroCT analysis confirmed an increase

Acknowledgments

The NIH (R01 DE020194, AR60869, T32 AR056950, F32 AR60140) a grant from the Dr. Ralph and Marion Falk Foundation and the Mayo Clinic Center for Regenerative Medicine supported this work. The authors thank David Razidlo and Bridget Stensgard for assistance with surgical procedures and mouse colony maintenance, and the Mayo Clinic Biomaterials and Quantitative Histomorphometry Core Laboratory for assistance with histological specimen preparation.

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Citation Excerpt :
For all the other conditions, bone formation was observed at both the edge and the center of the defects. As expected [11,74], increased BV/TV was systemically found in Sost KO mice when compared to their WT counterparts and the addition of mDPSC, either WT or KO, did not improve bone formation in KO animals (Fig. 1a). In contrast, in WT mice, the addition of mDPSC significantly improved bone formation compared to acellular hydrogels.
Scaffolds associated with different types of mesenchymal stromal stem cells (MSC) are extensively studied for the development of novel therapies for large bone defects. Moreover, monoclonal antibodies have been recently introduced for the treatment of cancer-associated bone loss and other skeletal pathologies. In particular, antibodies against sclerostin, a key player in bone remodeling regulation, have demonstrated a real benefit for treating osteoporosis but their contribution to bone tissue-engineering remains uncharted. Here, we show that combining implantation of dense collagen hydrogels hosting wild-type (WT) murine dental pulp stem cells (mDPSC) with weekly systemic injections of a sclerostin antibody (Scl-Ab) leads to increased bone regeneration within critical size calvarial defects performed in WT mice. Furthermore, we show that bone formation is equivalent in calvarial defects in WT mice implanted with Sost knock-out (KO) mDPSC and in Sost KO mice, suggesting that the implantation of sclerostin-deficient MSC similarly promotes new bone formation than complete sclerostin deficiency. Altogether, our data demonstrate that an antibody-based therapy can potentialize tissue-engineering strategies for large craniofacial bone defects and urges the need to conduct research for antibody-enabled local inhibition of sclerostin.
The use of monoclonal antibodies is nowadays broadly spread for the treatment of several conditions including skeletal bone diseases. However, their use to potentialize tissue engineering constructs for bone repair remains unmet. Here, we demonstrate that the neutralization of sclerostin, through either a systemic inhibition by a monoclonal antibody or the implantation of sclerostin-deficient mesenchymal stromal stem cells (MSC) directly within the defect, improves the outcome of a tissue engineering approach, combining dense collagen hydrogels and MSC derived from the dental pulp, for the treatment of large craniofacial bone defects.
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The process of bone healing largely recapitulates bone development in the embryo and ideally achieves complete restoration of bone shape and structure. However, because successful fracture healing requires tight interactions of numerous cell types and signaling molecules, any disruption of this highly coordinated processes can result in delayed healing or even non-union formation. The rate of fracture healing complications in orthopedic patients is reported to be 5–20%. Therefore, there is a need for new therapeutic strategies to improve fracture healing in patients with healing complications. One treatment strategy would include the easy and safe application of a pharmacological agent inducing osteoanabolic effects during fracture healing. One potential promising molecular target is the osteoanabolic WNT signaling pathway. This pathway plays an important role during embryonic bone development, homeostasis, mechanotransduction, development of osteoporosis and bone regeneration. This review focuses on preclinical studies targeting WNT signaling molecules to accelerate fracture healing. The three main investigated antagonists of the WNT signaling pathway, which can be blocked experimentally by antibodies, are Sclerostin, Dickkopf-1 and Midkine. Treating animals with antibodies against these proteins enhanced bone formation in the fracture callus. This indicates a therapeutic potential for these antibodies to accelerate fracture healing in patients with orthopedic complications.

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

Highlights

Abstract

Introduction

Section snippets

Materials and methods

Results and discussion

Acknowledgments

Gene

Bone

Gene

J. Biol. Chem.

Cell

Am. J. Hum. Genet.

Bone

Dev. Biol.

J. Biol. Chem.

Bone

Bone

The incidence of fractures and dislocations referred for orthopaedic services in a capitated population

J. Bone Joint Surg. Am.

Wnt signaling during fracture repair

Curr. Osteoporos. Rep.

Building bone to reverse osteoporosis and repair fractures

J. Clin. Invest.

Where Wnts went: the exploding field of Lrp5 and Lrp6 signaling in bone

J. Bone Miner. Res.