Enhancing Calvarial Regeneration through Inhibition of TGF-β1 Signaling

Abstract

INTRODUCTION: Given the significant biomedical burden posed by calvarial defects and the limitations of contemporary craniofacial skeletal reconstructive approaches, a need exists to better understand the biology of calvarial osteoblasts and how to direct them towards endogenous bone regeneration. The mammalian skull vault is the result of a tightly regulated evolutionary process which components of disparate embryonic origin are integrated with frontal bones arising from neural crest cells and parietal bones arising from paraxial mesoderm. Frontal neural crest-derived osteoblasts possess greater osteogenic potential relative to parietal bone derived cells. This is, in part, due to increased TGF-β1 signaling in parietal cells, which is known to promote apoptosis in a number of different cell types. We therefore investigated the potential of enhancing calvarial regeneration through modulation of TGF-β1 signaling.1–5 METHODS: 2mm calvarial defects were created in both the frontal and parietal bones of wild-type CD-1 mice. A collagen sponge was then used to deliver either human recombinant TGF-β1 (800ng) or the TGF-β signaling pathway inhibitor SB431542 (26 mM) into defects. Micro-Computed Tomography was employed to evaluate bone regeneration and percent osseous healing was calculated using the GE Microview program. Immunohistochemistry was performed on coronal sections through treated calvarial defects for Phospho-Smad 2 and 3, the downstream effectors for TGFβ signaling to assess endogenous activation of this pathway and whether our treatment with SB431542 was effectively inhibiting this pathway in vivo. RESULTS: Inhibition of TGF-β1 signaling using SB431542, a specific small molecule inhibitor of TGF-β1 mediated signaling, resulted in significantly enhanced bone regeneration in parietal defects (*p<0.05) and engendered a more ‘frontal like’ healing capacity (Figure 1). In contrast, delivery of TGF-β1 significantly reduced healing relative to non-treated defects (*p<0.05). Immunohistochemistry for downstream effectors of TGF-β1 signaling, Phospho-Smad 2 and 3, established that this pathway was being effectively inhibited by SB431542 in vivo.Figure 1: Calvarial Healing with SB431542 or DMSO control treatment.CONCLUSION: Increased TGF-β1 signaling impairs calvarial healing whereas inhibition of this pathway promotes bone regeneration, potentially through inhibition of apoptotic activity. This study provides an insight into the potential use of specific small molecule inhibitors of TGF-β signaling as a novel approach that could be used in conjunction with currently available treatments and may allow treatment of larger, more complex craniofacial skeletal defects.