Discussion: Combining Smoothened Agonist and NEL-Like Protein-1 Enhances Bone Healing
The hedgehog signaling family includes three mammalian ligands, Sonic hedgehog, Indian hedgehog, and Desert hedgehog, each with well-characterized importance in diverse developmental processes.2 The activity of the hedgehog pathway was first identified in Drosophila, and its expression was later found in all vertebrates. All of the hedgehog homologues undergo the same highly conserved hedgehog signaling pathway.3 In vertebrates, Smoothened leads to the transcription of target genes downstream through interaction with the glioblastoma gene products (Gli) family of transcription factors (Gli1, Gli2, and Gli3). Nel-like protein-1 (NELL-1) is a unique secreted protein of 810 amino acids first studied in the context of human craniofacial skeletal development, where NELL-1 was noted to be osteoinductive and its overexpression associated with human craniosynostosis.4 Since that time, transgenic Nell-1–overexpressing mice have been observed to recapitulate a craniosynostosis-like phenotype.5 Conversely, Nell-1–deficient mice (as developed by N-ethyl-N-nitrosourea–induced mutagenesis) exhibit cranial bone defects with undermineralization.6 Mechanistically, NELL-1 binds to the cell surface receptor integrin β1 and regulates activity of the master osteogenic transcription factor, Runt-related transcription factor-2 (Runx2).7
The authors previously observed evidence of a combinatorial effect between hedgehog signaling and Nell-1 signaling in driving osteogenic differentiation of adipose-derived stromal cells in vitro.8 To evaluate the potential efficacy of these findings in vivo, the authors sought to manipulate NELL-1 and hedgehog signaling pathways in a mouse critical-size calvarial bone defect model. In their article, the authors examined whether use of an acellular scaffold treated with small molecule Smoothened agonist (SAG) and recombinant NELL-1 protein could aid in repair of critical-size (4 mm in diameter defects in parietal bone) calvarial bone defects in mice. After 8 weeks, the combination of SAG plus NELL-1 treatment resulted in significantly greater trabecular bone volume and bone mineral density compared with NELL-1 or SAG alone. Bone thickness distribution maps obtained from micro–computed tomographic imaging revealed a significant increase in bone thickness of the newly formed regenerate after SAG plus NELL-1 treatment compared to NELL-1 or SAG alone. Radiographic findings were confirmed using qualitative histologic and quantitative histomorphometric analyses. In addition to parameters of bone formation, histomorphometric analyses showed an increase in blood vessel number and density in response to each treatment group compared with the control group.
The elevated healing rate of calvarial defects achieved using combinatorial delivery of SAG plus NELL-1 demonstrates the efficacy of a novel molecular therapy for healing critical-size bone defects. This approach would circumvent complications associated with autologous bone graft procedures and use of alloplastic materials, such as donor-site morbidity, biocompatibility, and infection. Moreover, these molecular compounds have not been associated with the side effects reported with recombinant human bone morphogenetic protein-2 use.