Combining Smoothened Agonist and NEL-Like Protein-1 Enhances Bone Healing

Abstract

Background: Nonhealing bone defects represent an immense biomedical burden. Despite recent advances in protein-based bone regeneration, safety concerns over bone morphogenetic protein-2 have prompted the search for alternative factors. Previously, the authors examined the additive/synergistic effects of hedgehog and Nel-like protein-1 (NELL-1) on the osteogenic differentiation of mesenchymal stem cells in vitro. In this study, the authors sought to leverage their previous findings by applying the combination of Smoothened agonist (SAG), hedgehog signal activator, and NELL-1 to an in vivo critical-size bone defect model.

Methods: A 4-mm parietal bone defect was created in mixed-gender CD-1 mice. Treatment groups included control (n = 6), SAG (n = 7), NELL-1 (n = 7), and SAG plus NELL-1 (n = 7). A custom fabricated poly(lactic-co-glycolic acid) disk with hydroxyapatite coating was used as an osteoinductive scaffold.

Results: Results at 4 and 8 weeks showed increased bone formation by micro-computed tomographic analyses with either stimulus alone (SAG or NELL-1), but significantly greater bone formation with both components combined (SAG plus NELL-1). This included greater bone healing scores and increased bone volume and bone thickness. Histologic analyses confirmed a significant increase in new bone formation with the combination therapy SAG plus NELL-1, accompanied by increased defect vascularization.

Conclusions: In summary, the authors' results suggest that combining the hedgehog signaling agonist SAG and NELL-1 has potential as a novel therapeutic strategy for the healing of critical-size bone defects. Future directions will include optimization of dosage and delivery strategy for an SAG and NELL-1 combination product.

Fig. 1. Micro-CT imaging and analysis

(A) Reconstructed micro-CT images taken at eight weeks postoperatively. Greatest new bone formation within the defect site was seen in SAG + NELL-1 treatment group. (B–D) Quantitative analysis of bone formation at defect site at four and eight weeks postoperatively, including (B) BMD, (C) BV/TV, and (D) bone healing score. The SAG + NELL-1 treatment group showed higher bone healing in general. *P < 0.05 and **P < 0.01 in comparison to control; +P < 0.05 and ++P < 0.01 in comparison to SAG alone; #P < 0.05 in comparison to NELL-1 alone. BMD: Bone mineral density, BV/TV: Bone volume/Tissue volume. Boxplot showing the median along with the first and third quartiles. Error bar means data range from minimum to maximum.

Fig. 2. Bone thickness analysis

(A) Color maps of bone thickness distribution. Thickest bone density shown as blue and thinnest as red. (B) Mean bone thickness was calculated for each treatment. SAG + NELL-1 demonstrated higher bone thickness compared to either SAG or NELL-1 alone. *P < 0.05 and **P < 0.01 in comparison to control; +P < 0.05 in comparison to SAG alone; #P < 0.05 in comparison to NELL-1 alone. Boxplot showing the median along with the first and third quartiles. Error bar means data range from minimum to maximum.

Fig. 3. Histologic analysis

Representative histologic appearance of the defect site under control (A–C), SAG alone (D–F), NELL-1 alone (G–I) or SAG + NELL-1 treatment conditions (J–L). Images taken at eight weeks postoperative, and appear in coronal cross section at 4x, 10x and 40x magnifications using routine H & E staining. Black arrowhead: foreign body giant cell; asterisk: residual scaffold material; white arrowheads: blood vessels. Black dashed lines indicated the bone margin in relation to fibrous tissue. Black scale bar: 200 um; White scale bar: 50 um.

Fig. 4. Histomorphometric analysis

Analyses were performed at the study endpoint (8 weeks). Analyses included (left) B.Ar per HPF (pixel number per HPF), (right) Fractional Bone Area (B.Ar/T.Ar). *P < 0.05 and **P < 0.01 in comparison to control; ++P < 0.01 in comparison to SAG alone; ##P < 0.01 in comparison to NELL-1 alone. B.Ar: Bone Area, HPF: High power field, B.Ar/T.Ar: Bone Area/Tissue Area. Boxplot showing the median along with the first and third quartiles. Error bar means data range from minimum to maximum.