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. 2006 Sep;169(3):903-15.
doi: 10.2353/ajpath.2006.051210.

Nell-1-induced bone regeneration in calvarial defects

Tara Aghaloo  1 , Catherine M Cowan, Yu-Fen Chou, Xinli Zhang, Haofu Lee, Steve Miao, Nichole Hong, Shun'ichi Kuroda, Benjamin Wu, Kang Ting, Chia Soo
Affiliations

Nell-1-induced bone regeneration in calvarial defects

Tara Aghaloo et al. Am J Pathol. 2006 Sep.

Abstract

Many craniofacial birth defects contain skeletal components requiring bone grafting. We previously identified the novel secreted osteogenic molecule NELL-1, first noted to be overexpressed during premature bone formation in calvarial sutures of craniosynostosis patients. Nell-1 overexpression significantly increases differentiation and mineralization selectively in osteoblasts, while newborn Nell-1 transgenic mice significantly increase premature bone formation in calvarial sutures. In the current study, cultured calvarial explants isolated from Nell-1 transgenic newborn mice (with mild sagittal synostosis) demonstrated continuous bone growth and overlapping sagittal sutures. Further investigation into gene expression cascades revealed that fibroblast growth factor-2 and transforming growth factor-beta1 stimulated Nell-1 expression, whereas bone morphogenetic protein (BMP)-2 had no direct effect. Additionally, Nell-1-induced osteogenesis in MC3T3-E1 osteoblasts through reduction in the expression of early up-regulated osteogenic regulators (OSX and ALP) but induction of later markers (OPN and OCN). Grafting Nell-1 protein-coated PLGA scaffolds into rat calvarial defects revealed the osteogenic potential of Nell-1 to induce bone regeneration equivalent to BMP-2, whereas immunohistochemistry indicated that Nell-1 reduced osterix-producing cells and increased bone sialoprotein, osteocalcin, and BMP-7 expression. Insights into Nell-1-regulated osteogenesis coupled with its ability to stimulate bone regeneration revealed a potential therapeutic role and an alternative to the currently accepted techniques for bone regeneration.

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Figures

Figure 1
Figure 1
Sagittal suture overgrowth in Nell-1 transgenic calvarial explants. A: Newborn wild-type mouse calvarial explant demonstrating patent sagittal suture in low magnification (top) and high magnification (bottom). B: Wild-type mouse calvarial explant after 9 days in culture showing patent sagittal suture in low magnification (top) and high magnification (bottom). Arrow marks area of histological sections. C: Coronal sections of the wild-type sagittal suture after 9 days in culture under white light (top), Texas red filter (middle), or DAPI filter (bottom). Arrowheads indicate the edge of the calvaria within the suture. D: Newborn Nell-1 transgenic mouse calvarial explant demonstrating mild synostosis of the sagittal suture (top) and high magnification (bottom). E: Nell-1 transgenic mouse calvarial explant after 9 days in culture showing an overlapping sagittal suture in low magnification (top) and high magnification (bottom). Arrow marks area of histological sections. F: Coronal sections of the Nell-1 transgenic mouse sagittal suture after 9 days in culture under white light (top), Texas red filter (middle), or DAPI filter (bottom). Arrowheads indicate the osteogenic fronts of parietal bones. Note the overgrowth of the sagittal suture in Nell-1 transgenic calvarial explants. All specimens are stained with Alizarin Red and Alcian Blue. Original magnifications, ×100 (C, F).
Figure 2
Figure 2
In vitro analysis of Nell-1 signaling. A: Radioisotope hybridization analysis of Nell-1 expression in fetal rat calvarial cells stimulated with rhBMP-2, rhFGF-2, or rhTGF-β1 in vitro after 24 and 48 hours. B: Ethidium bromide staining of an agarose gel of PCR products for Cbfa1, Nell-1, OCN, and GAPDH on transfection in MC3T3-E1 cells with pcDNA3.1 or pcDNA-Cbfa1 plasmids after 24 hours. Real-time PCR analysis of Nell-1 (C), ALP (D), OPN (E), OCN (F), and OSX (G) transcription in MC3T3-E1 cells on infection with AdNell-1 for 0, 3, and 6 days. *Significant differences from control samples; P ≤ 0.05.
Figure 3
Figure 3
Live microCT analysis of bone formation and density throughout time. A: Live microCT analysis of control (C), Nell-1-implanted (N), or BMP-2-implanted (B) defects at weeks 0, 1, 2, 4, 8, and 12. Note the increasing bone formation throughout time in defects implanted with either Nell-1 or BMP-2. B: Density measurements through the center of calvarial defects from the endocranial to exocranial sides. Sixteen points were measured including the thickness of the calvaria and background on either side in 50-μm increments. Background density measured less than 20% of the maximal density of uninjured bone. Note the increasing bone density throughout time in defects implanted with either Nell-1 or BMP-2.
Figure 4
Figure 4
High-resolution microCT and quantification of bone regeneration. A: Quantification of percent area and volume bone regeneration in calvarial defects at weeks 1, 2, and 4. *Significant differences from control are noted; P < 0.05. B: Three-dimensional reconstruction of representative calvaria with Nell-1/control or BMP-2/control implants at each time point.
Figure 5
Figure 5
Histological analysis of bone regeneration in calvarial defects. A: Trichrome staining of calvarial defects demonstrated the presence of osteoid (blue) and mature bone (red) within defects. Increased mature bone formation was noted throughout time in Nell-1- and BMP-2-implanted defects, as compared to control defects. Arrows point to blue osteoid and arrowheads point to red mature bone. Immunohistochemistry on calvarial defect sections for CTR at week 4 (B), OSX at week 1 (C), BMP-7 at week 4 (D), BSP at week 4 (E), and OCN at week 4 (F). Areas within the back boxes are magnified below. Background staining (light brown) can be contrasted to positive staining (red or dark brown). Note the positive OSX staining located at the osteogenic fronts on control and BMP-2-implanted defects, but not Nell-1-implanted defects. In addition, positive CTR, BMP-7, BSP, and OCN staining is located within Nell-1- and BMP-2-implanted samples, as compared to control. BSP-producing cells surround the remaining scaffold and lay down bone matrix. As previously reported, mature bone did not stain for BSP. Original magnifications: ×40 (A); ×100 (B–F, top); ×400 (B–F, bottom).
Figure 6
Figure 6
Hypothetical model diagramming Nell-1’s regulation of osteogenic differentiation. Dashed line represents hypothetical modulation. Note that Nell-1 stimulates down-regulation of ALP and Col I but up-regulation of OPN and OCN during bone formation.
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