The Osteogenic Properties Of Ad-GFP-NELL1in Polyethylene Particles Induced Calvarial Osteolysis

Objective: NELL1as a craniosynostosis-associated molecule, has exhibitedpotent osteoinductive activity for bone regeneration in several animal models.However, its capacity for promoting repair of osteolysis remains unknown. In thisstudy, we investigated the osteogenic effects of NELL1on polyethylene (PE)-particledebris induced calvarial osteolysis model using adenoviral gene delivery andmultiple approaches of in vivo analysis. Materials and Methods: We constructed therecombinant adenovirus vector carrying NELL1and GFP (Ad-GFP-NELL1), and theco-expression of NELL1with GFP was confirmed by fluoreScentimmunocytochemistry by infecting Rat bone-marrow-derived stromal cells (BMSCs)with Ad-GFP-NELL1. Then the mice osteoblasts（MC3T3E1）were infected withAd-GFP-NELL1to observe the recombinant adenovirus’ toxic and osteogenic effectsby NELL1. In vivo,76female Balb/c mice were used for calvarial osteolysis model.They were randomly assigned to4groups for treatment1day post-operation: SHAM(injected with0.1ml saline without particles implantation); PE control (injected with0.1ml saline after implantation of particles); PE+(Ad-GFP-NELL1)(injected with0.1ml Ad-GFP-NELL1in saline after implantation of particles); and PE+(Ad-GFP)group (injected with0.1ml Ad-GFP in saline after implantation of particles). GFPand NELL1delivery in vivo after injection were validated by optical imaging at10day post-op, and then all mice were sacrificed for analysis by3-D micro-computed tomography (micro-CT), real-time PCR, histology and biomechanical testing. Results:Recombinant adenoviral vector Ad-GFP-NELL1, which encodes a fusion protein ofhuman NELL1, was successfully constructed. At the best MOI(Multiplicity ofInfection=200pfu/cell), Cellular proliferation was not effected by Ad-GFP-NELL1Calcium nodules were much more in NELL1group（72） compared to（21）（P<0.05）. The NELL1stimulated expression of OCN (3.7folds) and OPN(5.1folds)significantly（P<0.05）. Exogenous NELL1and GFP were expressed in the head for atleast9days after Ad-GFP-NELL1injection. Serial3-D micro-CT images and testingof bone volume, bone mineral density, trabecular thickness, bone surface density andConnectivity density revealed the new bone promoted with Ad-GFP-NELL1injectioncould almost compensate the PE-induced osteolysis and regenerate significantly betterthan with Ad-GFP treatment. The expression of osteopontin (OPN) was significantlyhigher with Ad-GFP-NELL1transduction among all of samples with or without PEimplantation. Real-time PCR examination confrmed the augmented expression ofOPN, Runx-2and receptor activator of nuclear factor-kappa B ligand (RANKL). TheElastic modulus was significantly greater with Ad-GFP-NELL1than with PE and/orAd-GFP group (p<0.01). Conclusions: Recombinant adenovirus vectorAd-GFP-NELL1can steady expressing both GFP and NELL1protein.It provided usa useful tool for real time trace the expression of NELL1and investigate its’ functionin vitro and in vivo. Ad-GFP-NELL1can still promote osteoblasts differentiation andincreasing osteogenic effect. Ad-GFP-NELL1gene transfer effectively reversed thecalvarial osteolysis and could be a new treatment for osteolysis through promoting bone regeneration.