Nell-1-Induced Bone Regeneration in Calvarial Defects
2006; Elsevier BV; Volume: 169; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2006.051210
ISSN1525-2191
AutoresTara Aghaloo, Catherine M. Cowan, Yu‐Fen Chou, Xinli Zhang, Haofu Lee, Steve Miao, Nichole Hong, Shun’ichi Kuroda, Benjamin M. Wu, Kang Ting, Chia Soo,
Tópico(s)Bone and Dental Protein Studies
ResumoMany 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-β1 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. 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-β1 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. Clinical problems requiring bone regeneration are diverse and especially challenging within the craniofacial complex. The majority of craniofacial defects have skeletal components and require extensive surgery and bone grafting procedures.1Canady JW Zeitler DP Thompson SA Nicholas CD Suitability of the iliac crest as a site for harvest of autogenous bone grafts.Cleft Palate Craniofac J. 1993; 30: 579-581Crossref PubMed Scopus (100) Google Scholar Although autografts are the gold standard, they are clinically limited by availability and donor site morbidity.2Schlegel KA Donath K Rupprecht S Falk S Zimmermann R Felszeghy E Wiltfang J De novo bone formation using bovine collagen and platelet-rich plasma.Biomaterials. 2004; 25: 5387-5393Crossref PubMed Scopus (124) Google Scholar Therefore, clinicians and researchers are continuously looking for ways to treat bony defects without autogenous grafting. Growth factors such as bone morphogenetic proteins (BMPs) have demonstrated great in vivo osteogenic potential3Kang Q Sun MH Cheng H Peng Y Montag AG Deyrup AT Jiang W Luu HH Luo J Szatkowski JP Vanichakarn P Park JY Li Y Haydon RC He TC Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery.Gene Ther. 2004; 11: 1312-1320Crossref PubMed Scopus (490) Google Scholar and are already approved for spinal fusion and long bone fractures in humans. Unfortunately, these applications currently require superphysiological doses4Boden SD Kang J Sandhu H Heller JG Use of recombinant human bone morphogenetic protein-2 to achieve posterolateral lumbar spine fusion in humans: a prospective, randomized clinical pilot trial: 2002 Volvo Award in clinical studies.Spine. 2002; 27: 2662-2673Crossref PubMed Scopus (580) Google Scholar and have been reported to induce undesirable heterotopic bone formation away from sites of administration as well as pleiotropic nonbone-specific effects.5Valentin-Opran A Wozney J Csimma C Lilly L Riedel GE Clinical evaluation of recombinant human bone morphogenetic protein-2.Clin Orthop. 2002; 1: 110-120Crossref Scopus (262) Google Scholar These concerns have limited the surgical techniques, site selection, and reinforce the need for osteoblast-specific stimulants. Core-binding factor α 1/runt-related transcription factor 2 (Cbfa1/Runx2), an essential transcription factor in osteoblast differentiation and bone formation,6Franceschi RT Xiao G Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways.J Cell Biochem. 2003; 88: 446-454Crossref PubMed Scopus (454) Google Scholar has been actively investigated for potential translational applications because it signals downstream of BMPs and is osteoblast-specific. However, because Cbfa1/Runx2 is a transcription factor, its use is restricted to gene therapy approaches as opposed to recombinant protein delivery. Thus, osteogenic proteins that are downstream of Cbfa1/Runx2 and more specific to bone formation may be viable alternatives to BMPs. NELL-1 [NEL-like molecule-1; NEL (a protein strongly expressed in neural tissue encoding epidermal growth factor-like domain)], isolated and characterized in craniosynostosis patients as specifically up-regulated within prematurely fusing sutures,7Zhang X Kuroda S Carpenter D Nishimura I Soo C Moats R Iida K Wisner E Hu FY Miao S Beanes S Dang C Vastardis H Longaker M Tanizawa K Kanayama N Saito N Ting K Craniosynostosis in transgenic mice overexpressing Nell-1.J Clin Invest. 2002; 110: 861-870Crossref PubMed Scopus (129) Google Scholar, 8Ting K Vastardis H Mulliken JB Soo C Tieu A Do H Kwong E Bertolami CN Kawamoto H Kuroda S Longaker MT Human NELL-1 expressed in unilateral coronal synostosis.J Bone Miner Res. 1999; 14: 80-89Crossref PubMed Scopus (145) Google Scholar may represent such an exciting alternative. NELL-1 is highly conserved across species, with human and rat Nell-1 being 90% homologous at the genetic level and 93% homologous at the protein level.9Watanabe TK Katagiri T Suzuki M Shimizu F Fujiwara T Kanemoto N Nakamura Y Hirai Y Maekawa H Takahashi E Cloning and characterization of two novel human cDNAs (NELL1 and NELL2) encoding proteins with six EGF-like repeats.Genomics. 1996; 38: 273-276Crossref PubMed Scopus (109) Google Scholar, 10Kuroda S Oyasu M Kawakami M Kanayama N Tanizawa K Saito N Abe T Matsuhashi S Ting K Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2.Biochem Biophys Res Commun. 1999; 265: 79-86Crossref PubMed Scopus (112) Google Scholar The phenotype of the Nell-1 transgenic overexpression mouse revealed cranial suture overgrowth similar to human craniosynostosis,7Zhang X Kuroda S Carpenter D Nishimura I Soo C Moats R Iida K Wisner E Hu FY Miao S Beanes S Dang C Vastardis H Longaker M Tanizawa K Kanayama N Saito N Ting K Craniosynostosis in transgenic mice overexpressing Nell-1.J Clin Invest. 2002; 110: 861-870Crossref PubMed Scopus (129) Google Scholar suggesting a distinct role for Nell-1 in bone formation. Conversely, a mouse model with mutated N-ethyl-N-nitrosourea-induced alleles, including Nell-1, resulted in cranial and other skeletal defects.11Desai J, Hughes L, Millsaps J, Stanford B, Kerley M, Carpenter D, Rinchik E, Culiat C: NELL-1, a gene coding for a novel PKC-binding protein is a candidate for late-gestation recessive lethal mutations at the L7R6 locus. Presented at The 16th International Mouse Genome Conference, San Antonio, TX, 2002Google Scholar Furthermore, Nell-1 is preferentially expressed in neural crest-derived tissues, suggesting its specificity for the craniofacial region. In osteoblasts, Nell-1 up-regulation accelerates differentiation and bone formation, whereas Nell-1 down-regulation inhibits osteoblast differentiation.7Zhang X Kuroda S Carpenter D Nishimura I Soo C Moats R Iida K Wisner E Hu FY Miao S Beanes S Dang C Vastardis H Longaker M Tanizawa K Kanayama N Saito N Ting K Craniosynostosis in transgenic mice overexpressing Nell-1.J Clin Invest. 2002; 110: 861-870Crossref PubMed Scopus (129) Google Scholar, 8Ting K Vastardis H Mulliken JB Soo C Tieu A Do H Kwong E Bertolami CN Kawamoto H Kuroda S Longaker MT Human NELL-1 expressed in unilateral coronal synostosis.J Bone Miner Res. 1999; 14: 80-89Crossref PubMed Scopus (145) Google Scholar, 10Kuroda S Oyasu M Kawakami M Kanayama N Tanizawa K Saito N Abe T Matsuhashi S Ting K Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2.Biochem Biophys Res Commun. 1999; 265: 79-86Crossref PubMed Scopus (112) Google Scholar, 12Kuroda S Tanizawa K Involvement of epidermal growth factor-like domain of NELL proteins in the novel protein-protein interaction with protein kinase C.Biochem Biophys Res Commun. 1999; 265: 752-757Crossref PubMed Scopus (65) Google Scholar, 13Zhang X Carpenter D Bokui N Soo C Miao S Truong T Wu B Chen I Vastardis H Tanizawa K Kuroda S Ting K Overexpression of Nell-1, a craniosynostosis-associated gene, induces apoptosis in osteoblasts during craniofacial development.J Bone Miner Res. 2003; 18: 2126-2134Crossref PubMed Scopus (57) Google Scholar Interestingly, we have recently shown that human NELL-1 is directly regulated by Cbfa1/Runx2,14Truong S Zhang X Miao Y Chiu R Ting K Cbfa1 regulation of the human Nell-1 promoter.J Dent Res. 2004; 83: 1307Google Scholar confirming its osteochondral specificity. Finally, because Nell-1 is a secreted protein, controlled delivery of Nell-1 may be a possible modality to regenerate craniofacial bony defects.15Truong S, Zhang X, Pathmanathan D, Soo C, Ting K: Runt homology domain transcription factor Runx2 mediates induction of the Nell-1 promoter. J Biol Chem (in press)Google Scholar In the current research, wild-type and transgenic Nell-1 newborn (with mild sagittal synostosis) mouse calvarial explants demonstrated patent and dramatic bone overlap within sagittal sutures, respectively. Parietal bone overgrowth and overlap within the sagittal suture has been described by other transgenic mouse models as murine craniosynostosis and has been compared to the human disease of craniosynostosis.16Liu YH Kundu R Wu L Luo W Ignelzi Jr, MA Snead ML Maxson Jr, RE Premature suture closure and ectopic cranial bone in mice expressing Msx2 transgenes in the developing skull.Proc Natl Acad Sci USA. 1995; 92: 6137-6141Crossref PubMed Scopus (200) Google Scholar, 17Zhou YX Xu X Chen L Li C Brodie SG Deng CX A Pro250Arg substitution in mouse Fgfr1 causes increased expression of Cbfa1 and premature fusion of calvarial sutures.Hum Mol Genet. 2000; 9: 2001-2008Crossref PubMed Scopus (198) Google Scholar Investigations into Nell-1 signaling pathways revealed a transcriptional regulation by transforming growth factor (TGF)-β1 and fibroblast growth factor (FGF)-2, but not bone morphogenetic protein (BMP)-2. Although all three growth factors are known to regulate bone formation18Bonewald L Bilezikian J Riaisz L Rodan G Transforming growth factor-beta. Principles of Bone Biology. Academic Press, San Diego2002: 903Google Scholar, 19Vehof JW Fisher JP Dean D van der Waerden JP Spauwen PH Mikos AG Jansen JA Bone formation in transforming growth factor beta-1-coated porous poly(propylene fumarate) scaffolds.J Biomed Mater Res. 2002; 60: 241-251Crossref PubMed Scopus (106) Google Scholar, 20Gosain AK Song L Yu P Mehrara BJ Maeda CY Gold LI Longaker MT Osteogenesis in cranial defects: reassessment of the concept of critical size and the expression of TGF-beta isoforms.Plast Reconstr Surg. 2000; 106: 360-372Crossref PubMed Scopus (101) Google Scholar, 21Gong Z Zhou S Cao J Gu X Effects of recombinant human basic fibroblast growth factor on cell proliferation during mandibular fracture healing in rabbits.Chin J Traumatol. 2001; 4: 110-112PubMed Google Scholar and the protein kinase C (PKC) activity22Rosado E Schwartz Z Sylvia VL Dean DD Boyan BD Transforming growth factor-beta1 regulation of growth zone chondrocytes is mediated by multiple interacting pathways.Biochim Biophys Acta. 2002; 1590: 1-15Crossref PubMed Scopus (29) Google Scholar that is indicated for Nell-1 activation,12Kuroda S Tanizawa K Involvement of epidermal growth factor-like domain of NELL proteins in the novel protein-protein interaction with protein kinase C.Biochem Biophys Res Commun. 1999; 265: 752-757Crossref PubMed Scopus (65) Google Scholar only increased FGF-2 signaling, which has multiple defined pathways leading to PKC activation, has been clearly associated with craniosynostosis.23Greenwald JA Mehrara BJ Spector JA Warren SM Crisera FE Fagenholz PJ Bouletreau PJ Longaker MT Regional differentiation of cranial suture-associated dura mater in vivo and in vitro: implications for suture fusion and patency.J Bone Miner Res. 2000; 15: 2413-2430Crossref PubMed Scopus (80) Google Scholar Furthermore, Nell-1 reduced the transcription of early regulators and induced that of intermediate and late markers of osteogenic differentiation. Finally, bioactive recombinant trimeric Nell-1 protein, with a modified signaling peptide, was generated using the baculoviral expression system. Grafting Nell-1-coated PLGA scaffolds into 3-mm calvarial defects revealed the osteogenic potential of Nell-1 to induce in vivo bone regeneration equivalent to BMP-2. Insights into Nell-1 regulated osteogenesis coupled with its ability to induce specific localized bone regeneration in vivo, indicates a potential alternative therapeutic role to the currently accepted techniques for bone regeneration. B6C3 F1 wild-type mice were purchased from Charles River (Wilmington, MA), and Nell-1 transgenic mice were generated as previously described.7Zhang X Kuroda S Carpenter D Nishimura I Soo C Moats R Iida K Wisner E Hu FY Miao S Beanes S Dang C Vastardis H Longaker M Tanizawa K Kanayama N Saito N Ting K Craniosynostosis in transgenic mice overexpressing Nell-1.J Clin Invest. 2002; 110: 861-870Crossref PubMed Scopus (129) Google Scholar Animals were housed and experiments were performed in accordance with guidelines of the Chancellor's Animal Research Committee of the Office for Protection of Research Subjects at the University of California, Los Angeles. For the calvarial explant stimulation, calvaria of wild-type and Nell-1 transgenic newborn littermates (p1) were harvested and cultured in osteogenic differentiation media containing 50 μg/ml of ascorbic acid and 10 mmol/L of β-glycerol phosphate in BGJb media without serum.13Zhang X Carpenter D Bokui N Soo C Miao S Truong T Wu B Chen I Vastardis H Tanizawa K Kuroda S Ting K Overexpression of Nell-1, a craniosynostosis-associated gene, induces apoptosis in osteoblasts during craniofacial development.J Bone Miner Res. 2003; 18: 2126-2134Crossref PubMed Scopus (57) Google Scholar The calvarial explants from newborn Nell-1 transgenic mice (n = 3) and wild-type littermates (n = 4) were cultured for 9 days and harvested for histological analysis. Calvaria cultured for more than 9 days became nonviable. Calvaria were fixed and stained with Alizarin Red and Alcian Blue. Five-μm-thick sections of the sagittal sutures were analyzed histologically using a fluorescent microscope. Fetal rat calvarial cells were harvested as previously described.7Zhang X Kuroda S Carpenter D Nishimura I Soo C Moats R Iida K Wisner E Hu FY Miao S Beanes S Dang C Vastardis H Longaker M Tanizawa K Kanayama N Saito N Ting K Craniosynostosis in transgenic mice overexpressing Nell-1.J Clin Invest. 2002; 110: 861-870Crossref PubMed Scopus (129) Google Scholar Subconfluent fetal rat calvarial cells were stimulated with rhBMP-2 (100 ng/ml), FGF-2 (10 ng/ml), or TGF-β1 (5 ng/ml) (all from Sigma-Aldrich, St. Louis, MO) for 24 and 48 hours. Total RNA was purified in Trizol reagent (Invitrogen, Carlsbad, CA). MC3T3-E1 cells were purchased from the American Type Culture Collection (Rockville, MD) for plasmid transfection and adenoviral transduction. Cells were transfected with pcDNA-Cbfa1 or pcDNA3.1 (Invitrogen) for 3 hours with lipofectamine reagent and cultured in standard media for 24 hours before total RNA was harvested in Trizol reagent. Additional subconfluent MC3T3-E1 cells were infected with an adenovirus overexpressing rat Nell-1 (AdNell-1) driven by a CMV promoter7Zhang X Kuroda S Carpenter D Nishimura I Soo C Moats R Iida K Wisner E Hu FY Miao S Beanes S Dang C Vastardis H Longaker M Tanizawa K Kanayama N Saito N Ting K Craniosynostosis in transgenic mice overexpressing Nell-1.J Clin Invest. 2002; 110: 861-870Crossref PubMed Scopus (129) Google Scholar at 20 PFU/cell. Cells were then maintained for 48 hours before switched to osteogenic differentiation medium.13Zhang X Carpenter D Bokui N Soo C Miao S Truong T Wu B Chen I Vastardis H Tanizawa K Kuroda S Ting K Overexpression of Nell-1, a craniosynostosis-associated gene, induces apoptosis in osteoblasts during craniofacial development.J Bone Miner Res. 2003; 18: 2126-2134Crossref PubMed Scopus (57) Google Scholar AdLacZ was used as a control. RNA was harvested on days 0, 3, and 6. Total RNA was extracted by Trizol reagent, and DNase-treated RNA was tested for its integrity by agarose gel electrophoresis. One μg of DNase I-treated total RNA was used for reverse transcription as previously described.7Zhang X Kuroda S Carpenter D Nishimura I Soo C Moats R Iida K Wisner E Hu FY Miao S Beanes S Dang C Vastardis H Longaker M Tanizawa K Kanayama N Saito N Ting K Craniosynostosis in transgenic mice overexpressing Nell-1.J Clin Invest. 2002; 110: 861-870Crossref PubMed Scopus (129) Google Scholar The product of reverse transcription was used for both conventional PCR and real-time PCR. PCR products were separated on an agarose gel. The primers for mouse genes are as follows: Cbfa1/Runx2 (forward: 5′-CCGCACGACAACCGCACCAT-3′, reverse: 5′-CGCTCCGGCCCACAAATCTC-3′), Nell-1 (forward: 5′-TGCCACTGTGAGAAGACCTG-3′, reverse: 5′-TGCACAGGAAGTGAGTCTGG-3′), osteocalcin (OCN) (forward: 5′-CAAGTCCCACACAGCAGCTT-3′, reverse: 5′-AAAGCCGAGCTGCCAGAGTT-3′), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward: 5′-CCATCCACAGTCTTCTGA-3′, reverse: 5′-CACCACCATGGAGAAGGC-3′). For protein-stimulated fetal rat calvarial cells, PCR products were then blotted to nitrocellulose membrane for hybridization with product-specific oligonucleotide probe labeled with 32P. The specific primer and probe sequences for Nell-1 were designed according to accession number NM031096 (forward: 5′-CTGTGTGGCTCCTAACAAGTGTG-3′, reverse: 5′-GGATTCTGGCAATCACAAGCTGCT-3′, probe: 5′-CCTACTCACTGTCCGGGGAGTCCTGC-3′). Real-time PCR analysis of bone marker genes, including alkaline phosphatase (ALP), osteopontin (OPN), OCN, Nell-1, osterix (OSX), and GAPDH, performed for MC3T3-E1 transduction with AdNell-1 and AdLacZ with ABI Prism 7300 real-time PCR system, and the primers and probes were purchased as TaqMan primer-probe sets (Applied Biosystems, Foster City, CA). Analysis was based on calculating the relative expression level of the gene of interest compared to GAPDH,24Heid CA Stevens J Livak KJ Williams PM Real time quantitative PCR.Genome Res. 1996; 6: 986-994Crossref PubMed Scopus (4940) Google Scholar then normalized to the expression induced by AdLacZ control at corresponding time points (n = 4). Student's t-test was used to assess significant differences of gene expression on days 3 or 6 as compared to day 0 with P ≤ 0.05 considered significant. Five hundred-μm-thick 85/15 poly(lactic-co-glycolic acid) (PLGA) scaffolds were fabricated by solvent casting and a particulate leaching process (inherent viscosity = 0.62 dl/g; Absorbable Polymers, Pelham, AL) as previously described.25Cowan C Shi Y Aalami O Chou Y Mari C Thomas R Quarto N Contag C Wu B Longaker M Adipose-derived adult stromal cells heal critical-sized mouse calvarial defects.Nature Biotechnol. 2004; 22: 560-567Crossref Scopus (818) Google Scholar The porogen (NaCl, 200 to 300 μm) and PLGA/chloroform solution were packed into a 3-mm diameter Teflon mold to achieve 92% porosity (volume fraction). After porogen leaching and ethanol sterilization, scaffolds were verified by scanning electron microscope (FEI/Phillips XL-30; New York, NY). For growth factor coating, 200 ng/scaffold of Nell-110Kuroda S Oyasu M Kawakami M Kanayama N Tanizawa K Saito N Abe T Matsuhashi S Ting K Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2.Biochem Biophys Res Commun. 1999; 265: 79-86Crossref PubMed Scopus (112) Google Scholar or BMP-2 (Sigma-Aldrich) was diluted in 0.025% type I collagen solution (Cohesion, Palo Alto, CA) and adsorbed onto each scaffold. For production of the C-terminally FLAG-tagged Nell-1 protein, a pIZT-FLC-Nell-1 plasmid was constructed by inserting the rat Nell-1 cDNA linked to a FLAG epitope sequence derived from the pTB701-FLC-Nell-1 plasmid into baculoviral vector pIZT/V5-His (Invitrogen).12Kuroda S Tanizawa K Involvement of epidermal growth factor-like domain of NELL proteins in the novel protein-protein interaction with protein kinase C.Biochem Biophys Res Commun. 1999; 265: 752-757Crossref PubMed Scopus (65) Google Scholar The recombinant rat Nell-1 protein was purified from the culture medium of zeocin-resistant high five cells by anion-exchange chromatography using a UNO Q-6 column (Bio-Rad, Hercules, CA). Sprague-Dawley rats were purchased from Charles River Laboratories. Calvaria of male 3-month-old Sprague-Dawley rats were exposed to a trephine drill, under constant irrigation, to create 3-mm full-thickness craniotomy defects in each parietal bone with care to avoid injury to the underlying dura mater. Each defect was flushed with saline to remove bone debris and then implanted with scaffolds. A small animal microCT imaging system was used to examine bone formation throughout time in individual rats, as previously described.26Berger F Lee YP Loening AM Chatziioannou A Freedland SJ Leahy R Lieberman JR Belldegrun AS Sawyers CL Gambhir SS Whole-body skeletal imaging in mice utilizing microPET: optimization of reproducibility and applications in animal models of bone disease.Eur J Nucl Med Mol Imaging. 2002; 29: 1225-1236Crossref PubMed Scopus (53) Google Scholar Rats containing control (n = 9), Nell-1 (n = 3)-, or BMP-2 (n = 2)-coated scaffolds were anesthetized and placed in a microCT scanner (Imtek Inc., Knoxville, TN) to acquire three-dimensional morphometric data. Images were acquired with the X-ray source biased at 35 kVp and 400 μA. Data sets for rats were acquired and reconstructed with resolutions of 100 μm. Visual analyses of the CT data were performed using AVS/Express (version 5.1; Advanced Visual Systems Inc., Waltham, MA) Density measurements were taken at each 50 μm distance throughout the thickness of the calvaria. Analyses performed included mineralization density and calvarial thickness. A high-resolution microCT (μCT40; Scanco USA, Inc., Southeastern, PA) was used as previously published.7Zhang X Kuroda S Carpenter D Nishimura I Soo C Moats R Iida K Wisner E Hu FY Miao S Beanes S Dang C Vastardis H Longaker M Tanizawa K Kanayama N Saito N Ting K Craniosynostosis in transgenic mice overexpressing Nell-1.J Clin Invest. 2002; 110: 861-870Crossref PubMed Scopus (129) Google Scholar MicroCT data were collected at 50 kVp and 160 μA. Visualization, reconstruction, and volume analysis of the data were performed using the MetaMorph Imaging System (Universal Imaging Corp., Downingtown, PA) of defects containing control (n = 10 per time point), Nell-1 (n = 5 per time point)-, or BMP-2 (n = 5 per time point)-coated scaffolds. Bone-specific analyses included: new bone area using Image Pro Plus version 5.0 (Media Cybernetics, Carlsbad, CA) and new bone volume/tissue volume, number of bone voxels in the volume of interest divided by the total number of tissue voxels in the volume of interest.27Borah B Dufresne TE Cockman MD Gross GJ Sod EW Myers WR Combs KS Higgins RE Pierce SA Stevens ML Evaluation of changes in trabecular bone architecture and mechanical properties of minipig vertebrae by three-dimensional magnetic resonance microimaging and finite element modeling.J Bone Miner Res. 2000; 15: 1786-1797Crossref PubMed Scopus (64) Google Scholar Ten-μm-thick paraffin sections (microtome; McBain Instruments, Chatsworth, CA) of decalcified samples were stained with hematoxylin and eosin25Cowan C Shi Y Aalami O Chou Y Mari C Thomas R Quarto N Contag C Wu B Longaker M Adipose-derived adult stromal cells heal critical-sized mouse calvarial defects.Nature Biotechnol. 2004; 22: 560-567Crossref Scopus (818) Google Scholar and Masson-Goldner trichrome stain according to standard protocols. Additional sections were incubated with anti-bone sialoprotein (BSP), anti-OSX, anti-BMP-7, anti-OCN, and anti-calcitonin receptor (anti-CTR) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and biotinylated anti-rabbit or anti-goat IgG secondary antibody (Vector Laboratories, Burlingame, CA). Positive immunoreactivity was detected using Vectastain ABC and AEC kits (Vector Laboratories). Controls for each antibody consisted of incubation with secondary antibody in the absence of primary antibody. In addition, tartrate-resistant acid phosphatase staining was used to examine osteoclast activity within frozen sections using an acid phosphatase, leukocyte kit (Sigma Diagnostics, St. Louis, MO) according to the manufacturer's protocol. Photomicrographs were taken with a Leica DMLB microscope (McBain Instruments) and using BioQuant software (R&M Biometrics, Nashville, TN). To investigate Nell-1-induced sagittal suture overgrowth, wild-type and Nell-1 transgenic newborn mouse calvaria were harvested and cultured ex vivo for 9 days with the dura mater detached. Whole mount staining revealed that newborn wild-type mice had patent sagittal and posterior frontal sutures (Figure 1A), which remained patent after 9 days in culture (Figure 1B). Nell-1 transgenic mice displayed mild synostosis of the sagittal suture at the time of harvest (Figure 1D) with further sagittal suture overgrowth after 9 days of culture (Figure 1E). Compared to wild-type controls, the sagittal suture of Nell-1 transgenic mice demonstrated overlapping calvaria osteogenic fronts that extended into the suture mesenchyme, and increased mineralization after 9 days in culture (Figure 1, C and F). These observations demonstrate the osteogenic potential of Nell-1 in calvarial tissues. Because the Nell-1 osteoinductive pathway is primarily unknown, our first goal was to determine whether Nell-1 was regulated by common osteoinductive growth factors, such as BMPs and other members of the TGF-β and FGF superfamilies, which reportedly play major roles in the regulation of osteogenesis.18Bonewald L Bilezikian J Riaisz L Rodan G Transforming growth factor-beta. Principles of Bone Biology. Academic Press, San Diego2002: 903Google Scholar, 19Vehof JW Fisher JP Dean D van der Waerden JP Spauwen PH Mikos AG Jansen JA Bone formation in transforming growth factor beta-1-coated porous poly(propylene fumarate) scaffolds.J Biomed Mater Res. 2002; 60: 241-251Crossref PubMed Scopus (106) Google Scholar, 20Gosain AK Song L Yu P Mehrara BJ Maeda CY Gold LI Longaker MT Osteogenesis in cranial defects: reassessment of the concept of critical size and the expression of TGF-beta isoforms.Plast Reconstr Surg. 2000; 106: 360-372Crossref PubMed Scopus (101) Google Scholar, 21Gong Z Zhou S Cao J Gu X Effects of recombinant human basic fibroblast growth factor on cell proliferation during mandibular fracture healing in rabbits.Chin J Traumatol. 2001; 4: 110-112PubMed Google Scholar, 28Vehof JW Mahmood J Takita H van't Hof MA Kuboki Y Spauwen PH Jansen JA Ectopic bone formation in titanium mesh loaded with bone morphogenetic protein and coated with calcium phosphate.Plast Reconstr Surg. 2001; 108: 434-443Crossref PubMed Scopus (74) Google Scholar, 29Bouletreau PJ Steinbrech D Spector JA Warren SM Greenwald JA Mehrara BJ Detch RC Longaker MT Gene expression of transforming growth factor-beta 3 and tissue inhibitor of metalloproteinase type 1 during membranous bone healing in rats.J Craniofac Surg. 2000; 11: 521-526Crossref PubMed Scopus (8) Google Scholar Because BMP-2, FGF-2, and TGF-β1 are known to directly regulate the expression of human Cbfa1/Runx2,6Franceschi RT Xiao G Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways.J Cell Biochem. 2003; 88: 446-454Crossref PubMed Scopus (454) Google Scholar, 30Lee KS Hong SH Bae SC Both the Smad and
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