FGF2 promotes Msx2 stimulated PC‐1 expression via Frs2/MAPK signaling

PC‐1 is an enzymatic generator of pyrophosphate and a critical regulator of tissue mineralization. We previously showed that fibroblast growth factor‐2 (FGF2) specifically induces PC‐1 expression in calvarial pre‐osteoblasts and that this occurs via a transcriptional mechanism involving Runx2. Because aberrant FGF signaling and Msx2 activity result in similar craniofacial skeletal defects and because Msx2 is an established regulator of osteoblastic gene expression, here we investigate Msx2 as an additional mediator of PC‐1 gene expression. mRNA analysis and experiments utilizing PC‐1 gene promoter/luciferase reporter constructs demonstrate that Msx2 promotes transcription of the PC‐1 gene downstream of FGF2. Results indicate that both Msx2 and Runx2 are recruited to a conserved core Msx2 binding site within the PC‐1 gene promoter upon FGF2 stimulation, and that Msx2 and Runx2 function together to induce PC‐1 gene expression in osteoblastic cells. Here we show that FGF signaling promotes Msx2 transcriptional activity on the PC‐1 gene promoter via the Frs2/MAPK signaling pathway. To our knowledge, this is the first report of Msx2 functioning as a transcriptional enhancer downstream of FGF2 in calvarial pre‐osteoblasts. As activating mutations in FGF receptors and Msx2 result in similar craniofacial skeletal disorders, our findings support the idea that FGF signaling and Msx2 activity influence cranial osteogenesis via the same molecular mechanism. J. Cell. Biochem. 111: 1346–1358, 2010. © 2010 Wiley‐Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92096/1/22861_ftp.pd

Impact of pharmacologic inhibition of tooth movement on periodontal and tooth root tissues during orthodontic force application

ObjectiveThe goal of this study was to investigate potential negative sequelae of orthodontic force application ±delivery of an osteoclast inhibitor, recombinant osteoprotegerin protein (OPG‐Fc), on periodontal tissues.Setting and Sample PopulationSprague Dawley rats from a commercial supplier were investigated in a laboratory setting.Materials and MethodsRats were randomly divided into four groups (n = 7 each): one group with no orthodontic appliances and injected once prior to the experimental period with empty polymer microspheres, one group with orthodontic appliances and injected once with empty microspheres, one group with orthodontic appliances and injected once with polymer microspheres containing 1 mg/kg of OPG‐Fc, and one group with orthodontic appliances and injected with non‐encapsulated 5 mg/kg of OPG‐Fc every 3 days during the experimental period. The animals were euthanized after 28 days of tooth movement for histomorphometric analyses.ResultsRoot resorption, PDL area and widths were similar in animals without appliances and animals with appliances plus high‐dose OPG‐Fc. PDL blood vessels were compressed and decreased in number in all animals that received orthodontic appliances, regardless of OPG‐Fc. Hyalinization was significantly increased only in animals with orthodontic appliances plus multiple injections of 5 mg/kg non‐encapsulated OPG‐Fc when compared to animals without appliances.ConclusionsResults of this study indicate that while pharmacological modulation of tooth movement through osteoclast inhibition is feasible when delivered in a locally controlled low‐dose manner, high‐dose levels that completely prevent tooth movement through bone may decrease local blood flow and increase the incidence of hyalinization.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153668/1/ocr12350_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153668/2/ocr12350-sup-0001-FigS1-S2.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153668/3/ocr12350.pd

Dental and craniofacial defects in the Crtap−/− mouse model of osteogenesis imperfecta type VII

BackgroundInactivating mutations in the gene for cartilage‐associated protein (CRTAP) cause osteogenesis imperfecta type VII in humans, with a phenotype that can include craniofacial defects. Dental and craniofacial manifestations have not been a focus of case reports to date. We analyzed the craniofacial and dental phenotype of Crtap−/− mice by skull measurements, micro‐computed tomography (micro‐CT), histology, and immunohistochemistry.ResultsCrtap−/− mice exhibited a brachycephalic skull shape with fusion of the nasofrontal suture and facial bones, resulting in mid‐face retrusion and a class III dental malocclusion. Loss of CRTAP also resulted in decreased dentin volume and decreased cellular cementum volume, though acellular cementum thickness was increased. Periodontal dysfunction was revealed by decreased alveolar bone volume and mineral density, increased periodontal ligament (PDL) space, ectopic calcification within the PDL, bone‐tooth ankylosis, altered immunostaining of extracellular matrix proteins in bone and PDL, increased pSMAD5, and more numerous osteoclasts on alveolar bone surfaces.ConclusionsCrtap−/− mice serve as a useful model of the dental and craniofacial abnormalities seen in individuals with osteogenesis imperfecta type VII.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155878/1/dvdy166.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155878/2/dvdy166_am.pd

FGF2 Stimulation of the Pyrophosphate-Generating Enzyme, PC-1, in Pre-Osteoblast Cells Is Mediated by RUNX2

Pyrophosphate is an established inhibitor of hydroxyapatite deposition and crystal growth, yet when hydrolyzed into phosphate, it becomes a substrate for hydroxyapatite deposition. Pyrophosphate-generating enzyme (PC-1), Ank, and tissue nonspecific alkaline phosphatase (Tnap) are three factors that regulate extracellular pyrophosphate levels through its generation, transport, and hydrolysis. We previously showed that fibroblast growth factor 2 (FGF2) induces PC-1 and Ank while inhibiting Tnap expression and mineralization in MC3T3E1(C4) calvarial pre-osteoblast cells. In this study, we showed similar FGF2 regulation of these genes in primary pre-osteoblast cultures. In contrast to Ank and Tnap that are regulated by FGF2 in multiple cell types, we found regulation of PC-1 to be selective to pre-osteoblastic cells and to require the osteoblast-related transcription factor, Runx2. Specifically, FGF2 was unable to induce PC-1 expression in Runx2-negative nonbone cells or in calvarial cells from Runx2-deficient mice. Transfection of these cells with a Runx2 expression vector restored FGF2 responsiveness. FGF2 was also shown to stimulate recruitment of Runx2 to the endogenous PC-1 promoter in MC3T3E1(C4) cells, as measured by chromatin immunoprecipitation. Taken together, our results establish that FGF2 is a specific inducer of PC-1 in pre-osteoblast cells and that FGF2 induces PC-1 expression through a mechanism involving Runx2

Control of Orthodontic Tooth Movement by Nitric Oxide Releasing Nanoparticles in Sprague-Dawley Rats

Orthodontic treatment commonly requires the need to prevent movement of some teeth while maximizing movement of other teeth. This study aimed to investigate the influence of locally injected nitric oxide (NO) releasing nanoparticles on orthodontic tooth movement in rats. Materials and Methods: Experimental tooth movement was achieved with nickel-titanium alloy springs ligated between the maxillary first molar and ipsilateral incisor. 2.2 mg/kg of silica nanoparticles containing S-nitrosothiol groups were injected into the mucosa just mesial to 1st molar teeth immediately prior to orthodontic appliance activation. NO release from nanoparticles was measured in vitro by chemiluminescence. Tooth movement was measured using polyvinyl siloxane impressions. Bones were analyzed by microcomputed tomography. Local tissue was assessed by histomorphometry. Results: Nanoparticles released a burst of NO within the first hours at approximately 10 ppb/mg particles that diminished by 10 × to approximately 1 ppb/mg particles over the next 1–4 days, and then diminished again by tenfold from day 4 to day 7, at which point it was no longer measurable. Molar but not incisor tooth movement was inhibited over 50% by injection of the NO releasing nanoparticles. Inhibition of molar tooth movement occurred only during active NO release from nanoparticles, which lasted for approximately 1 week. Molar tooth movement returned to control levels of tooth movement after end of NO release. Alveolar and long bones were not impacted by injection of the NO releasing nanoparticles, and serum cyclic guanosine monophosphate (cGMP) levels were not increased in animals that received the NO releasing nanoparticles. Root resorption was decreased and periodontal blood vessel numbers were increased in animals with appliances that were injected with the NO releasing nanoparticles as compared to animals with appliances that did not receive injections with the nanoparticles. Conclusion: Nitric oxide (NO) release from S-nitrosothiol containing nanoparticles inhibits movement of teeth adjacent to the site of nanoparticle injection for 1 week. Additional studies are needed to establish biologic mechanisms, optimize efficacy and increase longevity of this orthodontic anchorage effect

Viral delivery of tissue nonspecific alkaline phosphatase diminishes craniosynostosis in one of two FGFR2C342Y/+ mouse models of Crouzon syndrome.

Craniosynostosis is the premature fusion of cranial bones. The goal of this study was to determine if delivery of recombinant tissue nonspecific alkaline phosphatase (TNAP) could prevent or diminish the severity of craniosynostosis in a C57BL/6 FGFR2C342Y/+ model of neonatal onset craniosynostosis or a BALB/c FGFR2C342Y/+ model of postnatal onset craniosynostosis. Mice were injected with a lentivirus encoding a mineral targeted form of TNAP immediately after birth. Cranial bone fusion as well as cranial bone volume, mineral content and density were assessed by micro CT. Craniofacial shape was measured with calipers. Alkaline phosphatase, alanine amino transferase (ALT) and aspartate amino transferase (AST) activity levels were measured in serum. Neonatal delivery of TNAP diminished craniosynostosis severity from 94% suture obliteration in vehicle treated mice to 67% suture obliteration in treated mice, p<0.02) and the incidence of malocclusion from 82.4% to 34.7% (p<0.03), with no effect on cranial bone in C57BL/6 FGFR2C342Y/+ mice. In contrast, treatment with TNAP increased cranial bone volume (p< 0.01), density (p< 0.01) and mineral content (p< 0.01) as compared to vehicle treated controls, but had no effect on craniosynostosis or malocclusion in BALB/c FGFR2C342Y/+ mice. These results indicate that postnatal recombinant TNAP enzyme therapy diminishes craniosynostosis severity in the C57BL/6 FGFR2C342Y/+ neonatal onset mouse model of Crouzon syndrome, and that effects of exogenous TNAP are genetic background dependent

IGF-1 TMJ injections enhance mandibular growth and bone quality in juvenile rats

ObjectivesDentofacial orthopaedic treatment of mandibular hypoplasia has unpredictable skeletal outcomes. Although several biomodulators including insulin-like growth factor 1 (IGF-1) are known to contribute to chondrocyte proliferation, their efficacy in modulating mandibular growth has not been validated. The aim of this study was to determine the effect of locally delivered IGF-1 on mandibular growth and condylar bone quality/quantity in juvenile rats.Setting and sample populationInstitutional vivarium using twenty-four 35-day-old male Sprague-Dawley rats.MethodsPBS or 40��g/kg (low-dose) IGF-1 or 80��g/kg (high-dose) IGF-1 was injected bilaterally into the temporomandibular joints of the rats at weekly intervals for four weeks. Cephalometric and micro-computed tomography measurements were used to determine mandibular dimensions. Bone and tissue mineral density, volume fraction and mineral content were determined, and serum IGF-1 concentrations assayed.ResultsIntra-articular administration of high-dose IGF-1 contributed to a significant 6%-12% increase in mandibular body and condylar length compared to control and low-dose IGF-1-treated animals. Additionally, IGF-1 treatment resulted in a significant decrease in the angulation of the lower incisors to mandibular plane. Condylar bone volume, bone volume fraction, mineral content and mineral density were significantly increased with high-dose IGF-1 relative to control and low-dose IGF-1 groups. Serum IGF-1 levels were similar between all groups confirming limited systemic exposure to the locally administered IGF-1.ConclusionLocal administration of high-dose 80��g/kg IGF-1 enhances mandibular growth and condylar bone quality and quantity in growing rats. The findings have implications for modulating mandibular growth and potentially enhancing condylar bone health and integrity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/172354/1/ocr12524.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/172354/2/ocr12524_am.pd