The Cortical-inductive Properties Of NELL1 In A Rat Femur Distraction Osteogenesis Model

Objective:NELL1 is a cranisynostosis-associated molecule directly regulated by Runx2, the master molecule in controlling osteoblastic differentiation. NELL1 has exhibited potent osteoinductive activity for bone regeneration in several animal models. However, its capacity for promoting repair of long-bone defects remains unknown, In this study, we investigated the osteogenic effects of NELL1 on femoral distraction osteogenesis using adenoviral gene delivery and multiple approaches of in vivo analysis. Materials and Methods:We subcloned a 2.3-kb full-length human NELL1 cDNA and inserted into the pAdxsi vector to package the recombinant adenovirus vector carrying NELL1 and GFP (Ad-GFP-NELL1). Rat bone-marrow-derived stromal cells (BMSCs) were infected with various doses of adenovirus to establish the optimal multiplicity of infection (MOI). NELL1 expression was verified by RT-PCR, and the co-expression of NELL1 with GFP was confirmed by fluoreScent immunocytochemistry. Femur distraction osteogenesis model were made on 30 Sprague-Dawley (SD) rats. After 7 days latency, the femurs were distracted at a speed of 0.25mm every 12 hours for 14 days. At the mid-distraction period (Day 14), each animal received a single dose of 0.2ml injection of either adenovirus Ad-GFP-NELL1 diluted in physiological saline, adenovirus Ad-GFP diluted in physiological saline, or physiological saline into the distraction site. After the distraction completed (Day 21), the steel external fixators were substituted by radio-transparent polymer splint material made fixators, and maintained for another 5 weeks for the distracted zone to consolidate. Healing was assessed with serial radiographs and micro-computed tomography (Micro-CT) of the living animals on Days 21,28,42,56. Animals were killed on Day 56 for biomechanical, histological, and histochemical analysis. Results: Recombinant adenoviral vector Ad-GFP-NELL1, which encodes a fusion protein of human NELL 1, was successfully constructed and amplified with titer of 1×1011 pfu/ml. An MOI of 200 pfu/cell produced optimal effects in transfer efficiency without excessive cell death in vitro. Exogenous NELL1 was expressed in the distracted gap for at least 14 days after Ad-GFP-NELL1 transfection. The bone union rate in the distracted gap was significantly higher with Ad-GFP-NELL1 than with Ad-GFP (9/9 vs. 4/9 rats) or saline alone (5/9 rats) at day 56. The serial 3-D micro-CT images and quantitation obtained with the development and application of radiolucent external fixators showed less callus but more mature cortical bones formed with Ad-GFP-NELL1 than with Ad-GFP transfection and saline administration during distraction osteogenesis. The biomechanical properties of femur samples with Ad-GFP-NELL1 transfection and unoperated femurs were similar. Histology revealed cartilaginous tissues in the middle of distraction gaps with Ad-GFP transfection and saline treatment but only bony bridges with Ad-GFP-NELL1 transfection at the final time point (day 56). Coincidently, the expression of Runx2, BMP2, and BMP7 did not differ among groups at day 56, whereas the expression of osteocalcin and osteopontin was slightly higher with Ad-GFP-NELL1 transfection. Conclusions:Recombinant adenovirus vector Ad-GFP-NELL1 can steady expressing both GFP and NELL1 protein after infected into rat BMSCs. It provided us a useful tool for real time trace the expression of NELL1 and investigate its'function in vitro and in vivo. We developed a method that could serially acquire Micro-CT data during rat femur distraction osteogenesis. By applying this method, we showed the increasing of bone volume began at the end of distraction, lasting at least 5 weeks, while the remodeling of new formed bone began 3 weeks after distraction ended. The increasing and remodeling of new formed bone was not concord. Sustained Ad-NELL1 protein delivery into a local area of a rat femoral distraction osteogenesis model remarkably improved regeneration of good-quality bones and accelerated bone union at high rate. Acquiring serial micro-CT data during rat femur distraction osteogenesis and regional adeno virus delivery of NELL1 may facilitate future in vivo studies.