The osteoinductive properties of Nell-1 in a rat spinal fusion model

Recombinant growth factors bone morphogenetic protein-2 (BMP-2) and BMP-7 are currently approved for human use but are associated with various adverse effects including ectopic bone formation and local inflammatory reaction. The development of alternative growth factors may help minimize the adverse effects of current osteoinductive therapeutics. Nell-1 (Nel-like molecule-1; Nel [a protein strongly expressed in neural tissue encoding epidermal growth factor like domain]) is a novel secretory molecule that appears to act more specifically on osteoblasts than the BMPs, which can act on multiple cell types. From a molecular point of view, Nell-1 is directly regulated by runt-related transcription factor 2 (Runx2/Cbfa1), a master regulatory gene controlling bone formation. Previous studies showed that Nell-1 accelerates osteogenic differentiation in vitro and calvarial bone formation in vivo. We hypothesize that Nell-1 may also effectively form bone in spinal fusion.

Our primary aim was to assess if direct adenoviral gene delivery with Nell-1 in a demineralized bone matrix (DBM) carrier can improve spinal fusion in a rat model. Because adenoviral vectors allow for sustained growth factor delivery, they were used for initial feasibility testing before protein studies.

Two groups of 20 athymic rats underwent posterolateral intertransverse process spinal fusion at L4-L5 with implanted DBM carrier containing either adenovirus coding for Nell-1 (AdNell-1) or control, Lac Z (AdLacZ). No cells were implanted. The 20 rats were sacrificed at 6 weeks for evaluation of spinal fusion.

All animals underwent Faxitron radiographs at 2, 4, and 6 weeks, manual spine palpation at 6 weeks, and high-resolution micro computerized tomography (microCT) at 6 weeks. Spinal fusion rate was assessed by: 1) 6-week Faxitron images; 2) manual palpation by three independent observers; 3) microCT; and 4) histology. New bone formation was assessed by hematoxylin-eosin and Masson trichrome staining on decalcified, coronally sectioned spine segments.

All differences achieved statistical significance. After 6 weeks, direct application of adenoviral Nell-1 in a DBM carrier achieved significantly higher rates of spinal fusion over Lac Z controls: 60% Nell-1 versus 20% Lac Z by manual palpation and 70% Nell-1 versus 20% Lac Z by microCT and histology. Histological assessment of bone quality and maturity revealed more mature, higher quality bone in all the Nell-1 treated specimens relative to Lac Z at 6 weeks.

Spinal fusion is more accurately assessed by microCT and histology than manual palpation. Direct application of adenoviral Nell-1 in a DBM carrier achieved significantly higher rates of spinal fusion over Lac Z controls at 6 weeks. Direct application of adenoviral Nell-1 in a DBM carrier also achieved significantly higher rates of spinal fusion over other reports in the literature using direct adenoviral BMP application. Direct application of adenoviral BMP in an allograft carrier achieved 8% fusion for BMP-2 and 16% fusion for BMP-7 at 8 weeks. These results indicate that Nell-1 may be a potent osteoinductive molecule. In addition, the regulation of Nell-1 by the master bone regulatory gene, Runx2 suggests that Nell-1 may exert its effects more specifically in osteoblastic cells than BMPs which affect multiple cell types. Overall, Nell-1 may fulfil a current need for an osteoinductive factor.