R. Siu, X. Zhang, T. Ko, B. Wu, K. Ting, C. Culiat, C. Soo

Nell-1 (a protein strongly expressed in neural tissue encoding EGF-like domain; novel epidermal growth factor (EGF)-like protein) is a potent osteochondrogenic growth factor. Since our first identification of Nell-1 overexpression in pathological suture specimens from human craniosynostosis patients, we have shown that Nell-1 (1) induces significant in vivo bone formation in animal models, (2) contains functional osteoblast-specific cis-acting element 2 (OSE2) response elements for the master osteogenic transcription factor runt-related transcription factor 2 (Runx2/Cbfa1), and (3) accelerates terminal osteoblast and chondrocyte differentiation. An N-ethyl-N-nitrosourea-induced nonsense point mutation in the mouse Nell-1 gene causes severe Nell-1 deficiency. Homozygous Nell-1 deficient (ND) mice are perinatal lethals with previously described alterations in cranial, vertebral, and ribcage morphology. The objective of this study is to elucidate the effect of Nell-1 deficiency on endochondral bone formation in the spine and long bones.

The vertebrae and long bones of newborn wildtype (WT) and ND mice were isolated and fixed in 4% paraformaldehyde. Quantitative and qualitative bone morphometric analysis was performed using microCT-based bone volume and density measurements as well as H&E and Masson's Trichrome histology. Chondrocyte maturation was assessed by type X collagen (ColX) immunohistochemistry.

CT images and histology showed normal cortical bone architecture with more densely packed lamellar bone in WT mice, while ND mice exhibited a discontiguous, highly trabeculated, and irregularly branched cortical architecture consistent with less mature bone (Figure 1). Bone marrow cavity area was also significantly decreased in ND mice (Figure 2). microCT measurements revealed reduced mineralization in ND compared to WT mice. Immunohistochemistry revealed dysregulated expression of ColX in ND mice (Figure 3). These results indicate that Nell-1 is important not only for proper terminal osteoblast and chondrocyte differentiation, but that Nell-1 is critical for normal endochondral ossification and the formation of mature compact/cortical bone during fetal development.

Disclosures: B. Wu, Bone Biologics, Inc.: Ownership or Partnership. C. Soo, Bone Biologics, Inc.: Ownership or Partnership. X. Zhang, Bone Biologics, Inc.: Ownership or Partnership. K. Ting, Bone Biologics, Inc.: Ownership or Partnership.

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