[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cell commitment to an osteoprogenitor lineage requires the activity of Runx2, a molecule implicated in the etiopathology of multiple congenital craniofacial anomalies. Through promoter analyses, we have recently identified a new direct transcriptional target of Runx2, Nell-1, a craniosynostosis (CS)-associated molecule with potent osteogenic properties. This study investigated the mechanistic and functional relationship between Nell-1 and Runx2 in regulating osteoblast differentiation. The results showed that spatiotemporal distribution and expression levels of Nell-1 correlated closely with those of endogenous Runx2 during craniofacial development. Phenotypically, cross-mating Nell-1 overexpression transgenic (CMV-Nell-1) mice with Runx2 haploinsufficient (Runx2(+/-)) mice partially rescued the calvarial defects in the cleidocranial dysplasia (CCD)-like phenotype of Runx2(+/-) mice, whereas Nell-1 protein induced mineralization and bone formation in Runx2(+/-) but not Runx2(-/-) calvarial explants. Runx2-mediated osteoblastic gene expression and/or mineralization was severely reduced by Nell-1 siRNA oligos transfection into Runx2(+/+) newborn mouse calvarial cells (NMCCs) or in N-ethyl-N-nitrosourea (ENU)-induced Nell-1(-/-) NMCCs. Meanwhile, Nell-1 overexpression partially rescued osteoblastic gene expression but not mineralization in Runx2 null (Runx2(-/-)) NMCCs. Mechanistically, irrespective of Runx2 genotype, Nell-1 signaling activates ERK1/2 and JNK1 mitogen-activated protein kinase (MAPK) pathways in NMCCs and enhances Runx2 phosphorylation and activity when Runx2 is present. Collectively, these data demonstrate that Nell-1 is a critical downstream Runx2 functional mediator insofar as Runx2-regulated Nell-1 promotes osteoblastic differentiation through, in part, activation of MAPK and enhanced phosphorylation of Runx2, and Runx2 activity is significantly reduced when Nell-1 is blocked or absent.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 04/2011; 26(4):777-91. DOI:10.1002/jbmr.267 · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: Nell-1 has previously displayed promising osteoinductive capabilities in multiple models. The objective of the current study was to evaluate the effects of several microenvironments on Nell-1's osteogenic role using a 3D in vitro system and a rat model.
Methods: The PLGA scaffolds were fabricated by solvent casting and coated with collagen I (CoI) at 2.5mg/ml, hydroxyapatite spheres (HAS) at 45% (w/v), and chitosan (CS) at 0.1% before loading 100ng rhNell-1. MC3T3-E1 cells were seeded onto each scaffold for evaluating (1) cell attachment and proliferation by environmental SEM and MTT, (2) osteogenic differentiation with RT-PCR and immunocytochemistry, and (3) mineralization by Micro-CT. Furthermore, a calvarial defect model was performed and bone regeneration was monitored using live optical imaging and Micro-CT analysis.
Results: All three coatings significantly increased cell attachment upon loading the cells onto scaffolds, but had no effect on proliferation during 9 days culture. Compared to samples with no coating, the gene expression of OCN was remarkably upregulated at week 1 while BSP was reduced at week 4 in samples with rhNell-1 and either HAS or CS coatings. Both OCN and BSP exhibited stronger immunostaining in samples with rhNell-1 plus any of coatings as compared to their relevant controls at week 4. Micro-CT analysis revealed two-fold higher mineralized volume in samples with rhNell-1 plus HAS or CS over relevant controls. Surprisingly, the most significant improvement of bone regeneration was identified in rats implanted with rhNell-1 plus CoI, although HAS coating appeared to induce better mineralization in 3D system. Notably, the local retention of rhNell-1 lasted significantly longer on CoI over HAS coated implants.
Conclusion: Nell-1 exerts an osteoinductive effect in a microenvironment-dependent manner. The optimal microenvironment affecting Nell-1's function in vivo may not be readily drawn from in vitro optimization, even using 3D system.
NIH R21 DE0177711-01