The effect of NELL1 and bone morphogenetic protein-2 on calvarial bone regeneration

Abstract

Purpose: Most craniofacial birth defects contain skeletal components that require bone grafting. Although many growth factors have shown potential for use in bone regeneration, bone morphogenetic proteins (BMPs) are the most osteoinductive. However, supraphysiologic doses, high cost, and potential adverse effects stimulate clinicians and researchers to identify complementary molecules that allow a reduction in dose of BMP-2. Because NELL1 plays a key role as a regulator of craniofacial skeletal morphogenesis, especially in committed chondrogenic and osteogenic differentiation, and a previous synergistic mechanism has been identified, NELL1 is an ideal molecule for combination with BMP-2 in calvarial defect regeneration. We investigated the effect of NELL1 and BMP-2 on bone regeneration in vivo.

Materials and methods: BMP-2 doses of 589 and 1,178 ng were grafted into 5-mm critical-sized rat calvarial defects, as compared with 589 ng of NELL1 plus 589 ng of BMP-2 and 1,178 ng of NELL1 plus 1,178 ng of BMP-2, and bone regeneration was analyzed.

Results: Live micro-computed tomography data showed increased bone formation throughout 4 to 8 weeks in all groups but a significant improvement when the lower doses of each molecule were combined. High-resolution micro-computed tomography and histology showed more mature and complete defect healing when the combination of NELL1 plus BMP-2 was compared with BMP-2 alone at lower doses.

Conclusion: The observed potential synergy has significant value in the future treatment of patients with craniofacial defects requiring extensive bone grafting that would normally entail extraoral autogenous bone grafts or doses of BMP-2 in milligrams.

FIGURE 1

A, Unilateral critical-sized rat calvarial defect. B, Saline solution or growth factor adsorbed onto PLGA scaffold. C, PLGA scaffold implanted into calvarial defect.

FIGURE 2

Live microCT analysis of bone formation over time. A, Qualitative live microCT analysis in rat calvarial defect 4, 6, and 8 weeks after implantation of PLGA scaffolds coated with control (saline solution), 589 ng of BMP-2, 1,178 ng of BMP-2, or 589 ng of NELL1 plus BMP-2. B, Quantitative measurements of percent bone regeneration after implantation of scaffolds alone or with 589 ng of BMP-2, 1,178 ng of BMP-2, or 589 ng of NELL1 plus BMP-2 (N+B). Asterisk, Significant increase compared with control (P < .05); pound sign, significant increase compared with both doses of BMP-2 alone (P < .05).

FIGURE 3

High-resolution microCT and quantification of regenerated bone volume. A, Three-dimensionally reconstructed high-resolution microCT images of defects implanted with control, 589 ng of BMP-2, 1,178 ng of BMP-2, 589 ng of NELL1 plus BMP-2, and 1,178 ng of NELL1 plus BMP-2 after 12 weeks of healing. B, Quantification of percent volume bone regeneration in calvarial defects after 12 weeks of healing compared with uninjured calvaria. All groups produced significantly more regenerated bone than control samples (P < .05).

FIGURE 4

Histologic analysis of bone regeneration in calvarial defects after 12 weeks of healing. H&E staining of calvarial defects (arrows represent defect margins) shows fibrous tissue in control samples, as compared with more woven bone formation in groups with BMP-2 alone and more lamellar bone formation in groups with NELL1 plus BMP-2.

FIGURE 5

Masson trichrome staining of bone healing in calvarial defects. After 12 weeks of healing, the maturity of the regenerated bone was evaluated. Combined NELL1 and BMP-2 show areas of osteoid (blue) and mature lamellar (red) bone formation, with an increased thickness in the regenerated bone when the 2 factors are used in combination.