Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo.

Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells.

We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKO(ob)) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKO(ob) mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKO(ob) osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA(+) MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells.

NFI-C2 temporal-spatial expression and cellular localization pattern during tooth formation.

Currently, little is known regarding critical signaling pathways during later stages of tooth development, especially those associated with root formation. Nfi-c null mice, lacking molar roots, have implicated the transcription factor NFI-C as having an essential role in root development. Previously, we identified three NFI-C isoforms expressed in dental tissues with NFI-C2 being the major transcript. However, the expression pattern of the NFI-C2 protein is not characterized. In this study we performed in situ hybridization and immunohistochemistry using isoform specific probes. We show the production of a NFI-C2 peptide antibody, its characterization, the temporal-spatial expression pattern of the NFI-C2 protein during odontogenesis and sub-cellular localization in dental cells. Moderate NFI-C2 staining, as early as bud stage, was detected mostly in the condensing dental ectomesenchyme. This staining intensified within the dental pulp at later stages culminating in high expression in the dentin producing odontoblasts. The dental epithelium showed slight staining until cytodifferentiation of enamel organ into ameloblasts and stratum intermedium. During root formation NFI-C2 expression was high in the Hertwig's epithelial root sheath and later was found in the fully developed root and its supporting tissues. NFI-C2 cellular staining was cytosolic, associated with the Golgi, and nuclear. These data suggest a broader role for NFI-C during tooth formation than limited to root and periodontal ligament development.

Abnormalities in the enamel in bmp2-deficient mice.

Tooth development is regulated by epithelial-mesenchymal interactions and their reciprocal molecular signaling. Bone morphogenetic protein 2 (Bmp2) is essential for tooth formation. However, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in the regulation of postnatal enamel formation was investigated via the conditional ablation of Bmp2 in enamel using the (Osx-Cre) mouse. Bmp2 gene ablation was confirmed by PCR analysis in Osx-Cre, Bmp2(flox/flox) mice. Bmp2-null mice displayed a severe and profound tooth phenotype with asymmetric and open forked incisors. Microradiographs revealed broken incisor tips and dental pulp chamber exposure. The enamel layer of incisors and molars was thin with hypomineralization. Scanning electron microscopy analysis showed that the enamel surface was rough with chipping and the enamel lacked a typical prismatic architecture. These results demonstrate that Bmp2 is essential for enamel formation.

Immortalized mouse floxed Bmp2 dental papilla mesenchymal cell lines preserve odontoblastic phenotype and respond to BMP2.

Bone morphogenetic protein 2 (Bmp2) is essential for odontogensis and dentin mineralization. Generation of floxed Bmp2 dental mesenchymal cell lines is a valuable application for studying the effects of Bmp2 on dental mesenchymal cell differentiation and its signaling pathways during dentinogenesis. Limitation of the primary culture of dental mesenchymal cells has led to the development of cell lines that serve as good surrogate models for the study of dental mesenchymal cell differentiation into odontoblasts and mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 dental papilla mesenchymal cell lines, which were isolated from 1st mouse mandibular molars at postnatal day 1 and immortalized with pSV40 and clonally selected. These transfected cell lines were characterized by RT-PCR, immunohistochemistry, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iBmp2-dp, displayed a higher proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers as well as demonstrated the ability to differentiate and form mineralized nodules. In addition, iBmp2-dp cells were inducible and responded to BMP2 stimulation. Thus, we for the first time described the establishment of an immortalized mouse floxed Bmp2 dental papilla mesenchyma cell line that might be used for studying the mechanisms of dental cell differentiation and dentin mineralization mediated by Bmp2 and other growth factor signaling pathways.

Tissue-specific expression of dentin sialophosphoprotein (DSPP) and its polymorphisms in mouse tissues.

Dentin sialophosphoprotein (DSPP) consists of dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). DSPP is highly expressed in mineralized tissues. However, recent studies have shown that DSPP is also expressed in several active metabolic ductal epithelial tissues and exists in a variety of sequences. We have investigated DSPP expression in various mouse tissues using RT-PCR, in situ hybridization and immunohistochemical analyses. To identify DSPP gene polymorphisms, we screened a mouse tooth cDNA library as well as isolated and characterized DSPP variations. Our results show that DSPP is predominantly expressed in teeth and moderately in bone tissues. We also have characterized a full-length DSPP cDNA clone with an open-reading frame of 940 codons and this polyadenylation signal. Compared to previously reported mouse DSPP cDNAs, 13 sequence variations were identified, including 8 non-synonymous single nucleotide polymorphisms and an in-frame indel (8 amino acids) at DPP domain of the mouse DSPP. These 8 amino acids are rich in aspartic acid and serine residues. Northern blot assay showed a prominent band at 4.4kb. RT-PCR demonstrated that this mouse DSPP gene was dominantly expressed in teeth. The predicted secondary structure of DPP domain of this DSPP showed differences from the previously published mouse DPPs, implying that they play different roles during tooth development and formation.

Mechanical loading stimulates expression of connexin 43 in alveolar bone cells in the tooth movement model.

Bone osteoblasts and osteocytes express large amounts of connexin (Cx) 43, the component of gap junctions and hemichannels. Previous studies have shown that these channels play important roles in regulating biological functions in response to mechanical loading. Here, we characterized the distribution of mRNA and protein of Cx43 in mechanical loading model of tooth movement. The locations of bone formation and resorption have been well defined in this model, which provides unique experimental systems for better understanding of potential roles of Cx43 in bone formation and remodeling under mechanical stimulation. We found that mechanical loading increased Cx43 mRNA expression in osteoblasts and bone lining cells, but not in osteocytes, at both formation and resorption sites. Cx43 protein, however, increased in both osteoblasts and osteocytes in response to loading. Interestingly, the upregulation of Cx43 protein by loading was even more pronounced in osteocytes compared to other bone cells, with an appearance of punctate staining on the cell body and dendritic process. Cx45 was reported to be expressed in several bone cell lines, but here we did not detect the Cx45 protein in the alveolar bone cells. These results further suggest the potential involvement of Cx43-forming gap junctions and hemichannels in the process of mechanically induced bone formation and resorption.

MEPE Localization in the Craniofacial Complex and Function in Tooth Dentin Formation.

Matrix extracellular phosphoglycoprotein (MEPE) is an extracellular matrix protein found in dental and skeletal tissues. Although information regarding the role of MEPE in bone and disorders of phosphate metabolism is emerging, the role of MEPE in dental tissues remains unclear. We performed RNA in situ hybridization and immunohistochemistry analyses to delineate the expression pattern of MEPE during embryonic and postnatal development in craniofacial mineralizing tissues. Mepe RNA expression was seen within teeth from cap through root formation in association with odontoblasts and cellular cementoblasts. More intense expression was seen in the alveolar bone within the osteoblasts and osteocytes. MEPE immunohistochemistry showed biphasic dentin staining in incisors and more intense staining in alveolar bone matrix and in forming cartilage. Analysis of Mepe null mouse molars showed overall mineralized tooth volume and density of enamel and dentin comparable with that of wild-type samples. However, Mepe(-/-) molars exhibited increased thickness of predentin, dentin, and enamel over controls and decreased gene expression of Enam, Bsp, Dmp1, Dspp, and Opnby RT-PCR. In vitro Mepe overexpression in odontoblasts led to significant reductions in Dspp reporter activity. These data suggest MEPE may be instrumental in craniofacial and dental matrix maturation, potentially functioning in the maintenance of non-mineralized matrix.

Mechanical loading stimulates dentin matrix protein 1 (DMP1) expression in osteocytes in vivo.

Dentin matrix protein 1 (DMP1) was originally postulated to be dentin specific. Further analysis showed that DMP1 is also expressed in mature cartilage and bone. In bone tissue, DMP1 is expressed predominantly in late osteoblasts and osteocytes. DMP1 belongs to the SIBLING (Small Integrin Binding Ligand N-linked Glycoprotein) family of cellular matrix proteins that also includes osteopontin, bone sialoprotein, dentin sialophosphoprotein, and others. In this study, we examined the effect of mechanical loading on expression of DMP1 mRNA and DMP1 protein in alveolar bone in the mouse tooth movement model by in situ hybridization and immunocytochemistry. The expression of DMP1 mRNA was determined quantitatively in mechanically loaded and control sites of dento-alveolar tissue at several time points from 6 h to 7 days after loading. The tooth movement model allows simultaneous evaluation of bone resorption and bone formation sites. Expression of DMP1 mRNA in osteocytes increased 2-fold as early as 6 h after treatment in both the bone formation and bone resorption sites. After 4 days, DMP1 expression in osteocytes increased to a maximum of 3.7-fold in the bone formation sites and 3.5-fold in the resorption sites. Osteoblasts responded in the opposite manner and showed a transient 45% decrease of DMP1 mRNA in bone formation sites and a constant decrease of DMP1 mRNA during the entire course of treatment in the bone resorption sites, with a peak inhibition of 67% at day 2. By immunocytochemistry using a C-terminal region peptide antibody to DMP1, we found that there was a transient decrease in immunoreactivity at 3 days after treatment on both the formation side and the resorption side compared with the matched contralateral control tissue. However by 7 days of loading, there was a dramatic increase in DMP1 protein immunoreactivity on both the formation side and the resorption side. These results represent changes in epitope availability using this antibody or true changes in protein levels. The observations imply that the DMP1 protein is undergoing dynamic changes in either synthesis or other protein/matrix interaction after mechanical loading of alveolar bone. The findings indicate that DMP1 is involved in the responses of osteocytes and osteoblasts to mechanical loading of bone. These results support the hypothesis that osteocytes alter their matrix microenvironment in response to mechanical loading.

Enamel matrix derivative: protein components and osteoinductive properties.

BACKGROUND: Although enamel matrix derivative (EMD) has demonstrated the ability to promote angiogenesis and osteogenesis both in vitro and in vivo, the specific elements within the EMD compound responsible for these effects remain unknown. METHODS: Nine different protein pools from a commercially produced EMD were collected based on molecular weight. Six of these pools, along with the complete EMD unfractionated compound and positive and negative controls, were tested for their ability to induce bone formation in a calvarial induction assay. Immunocytochemistry of phosphorylated SMAD1/5/8 (phospho-SMAD), osterix, and vascular endothelial growth factor A (VEGF-A) was carried out at selected time points. Finally, proteomic analysis was completed to determine the specific protein-peptide content of the various osteoinductive pools. RESULTS: One of the lower-molecular-weight pools tested, pool 7, showed bone induction responses significantly greater than those of the other pools and the complete EMD compound and was concentration dependent. Dynamic bone formation rate analysis demonstrated that pool 7 was optimally active at the 5- to 10-µg concentration. It was demonstrated that EMD and pool 7 induced phospho-SMAD, osterix, and VEGF-A, which is indicative of increased bone morphogenetic protein (BMP) signaling. Proteomic composition analysis demonstrated that pool 7 had the highest concentration of the biologically active amelogenin-leucine-rich amelogenin peptide and ameloblastin 17-kDa peptides. CONCLUSIONS: These studies demonstrate that the low-molecular-weight protein pools (7 to 17 kDa) within EMD have greater osteoinductive potential than the commercially available complete EMD compound and that the mechanism of action, in part, is through increased BMP signaling and increased osterix and VEGF-A. With this information, selected components of EMD can now be formulated for optimal osteo- and angio-genesis.

Bone morphogenetic protein-2 gene controls tooth root development in coordination with formation of the periodontium.

Formation of the periodontium begins following onset of tooth-root formation in a coordinated manner after birth. Dental follicle progenitor cells are thought to form the cementum, alveolar bone and Sharpey's fibers of the periodontal ligament (PDL). However, little is known about the regulatory morphogens that control differentiation and function of these progenitor cells, as well as the progenitor cells involved in crown and root formation. We investigated the role of bone morphogenetic protein-2 (Bmp2) in these processes by the conditional removal of the Bmp2 gene using the Sp7-Cre-EGFP mouse model. Sp7-Cre-EGFP first becomes active at E18 in the first molar, with robust Cre activity at postnatal day 0 (P0), followed by Cre activity in the second molar, which occurs after P0. There is robust Cre activity in the periodontium and third molars by 2 weeks of age. When the Bmp2 gene is removed from Sp7(+) (Osterix(+)) cells, major defects are noted in root, cellular cementum and periodontium formation. First, there are major cell autonomous defects in root-odontoblast terminal differentiation. Second, there are major alterations in formation of the PDLs and cellular cementum, correlated with decreased nuclear factor IC (Nfic), periostin and α-SMA(+) cells. Third, there is a failure to produce vascular endothelial growth factor A (VEGF-A) in the periodontium and the pulp leading to decreased formation of the microvascular and associated candidate stem cells in the Bmp2-cKO(Sp7-Cre-EGFP). Fourth, ameloblast function and enamel formation are indirectly altered in the Bmp2-cKO(Sp7-Cre-EGFP). These data demonstrate that the Bmp2 gene has complex roles in postnatal tooth development and periodontium formation.

Meox2Cre-mediated disruption of CSF-1 leads to osteopetrosis and osteocyte defects.

CSF-1, a key regulator of mononuclear phagocyte production, is highly expressed in the skeleton by osteoblasts/osteocytes and in a number of nonskeletal tissues such as uterus, kidney and brain. The spontaneous mutant op/op mouse has been the conventional model of CSF-1 deficiency and exhibits a pleiotropic phenotype characterized by osteopetrosis, and defects in hematopoiesis, fertility and neural function. Studies to further delineate the biologic effect of CSF-1 within various tissues have been hampered by the lack of suitable models. To address this issue, we generated CSF-1 floxed/floxed mice and demonstrate that Cre-mediated recombination using Meox2Cre, a Cre line expressed in epiblast during early embryogenesis, results in mice with ubiquitous CSF-1 deficiency (CSF-1KO). Homozygous CSF-1KO mice lacked CSF-1 in all tissues and displayed, in part, a similar phenotype to op/op mice that included: failure of tooth eruption, osteopetrosis, reduced macrophage densities in reproductive and other organs and altered hematopoiesis with decreased marrow cellularity, circulating monocytes and B cell lymphopoiesis. In contrast to op/op mice, CSF-1KO mice showed elevated circulating and splenic T cells. A striking feature in CSF-1KO mice was defective osteocyte maturation, bone mineralization and osteocyte-lacunar system that was associated with reduced dentin matrix protein 1 (DMP1) expression in osteocytes. CSF-1KO mice also showed a dramatic reduction in osteomacs along the endosteal surface that may have contributed to the hematopoietic and cortical bone defects. Thus, our findings show that ubiquitous CSF-1 gene deletion using a Cre-based system recapitulates the expected osteopetrotic phenotype. Moreover, results point to a novel link between CSF-1 and osteocyte survival/function that is essential for maintaining bone mass and strength during skeletal development.

Differential expression of intestinal genes in opossums with high and low responses to dietary cholesterol.

High and low responding opossums (Monodelphis domestica) differ in their plasma very low density lipoprotein and low density lipoprotein (VLDL+LDL) cholesterol concentrations when they consume a high cholesterol diet, which is due in part to absorption of a higher percentage of dietary cholesterol in high responders. We compared the expression of a set of genes that influence cholesterol absorption in high and low responders fed a basal or a high cholesterol and low fat (HCLF) diet. Up-regulation of the ABCG5, ABCG8, and IBABP genes by the HCLF diet in high and low responders may reduce cholesterol absorption to maintain cholesterol homeostasis. Differences in expression of the phospholipase genes (PLA2 and PLB) and phospholipase activity were associated with differences in cholesterol absorption when opossums were fed cholesterol-enriched diets. Higher PLA2 and PLB mRNA levels and higher phospholipase activity may increase cholesterol absorption in high responders by enhancing the release of cholesterol from bile salt micelles for uptake by intestinal cells.

Glucocorticoid-induced autophagy in osteocytes.

Glucocorticoid (GC) therapy is the most frequent cause of secondary osteoporosis. In this study we have demonstrated that GC treatment induced the development of autophagy, preserving osteocyte viability. GC treatment resulted in an increase in autophagy markers and the accumulation of autophagosome vacuoles in vitro and in vivo promoted the onset of the osteocyte autophagy, as determined by expression of autophagy markers in an animal model of GC-induced osteoporosis. An autophagy inhibitor reversed the protective effects of GCs. The effects of GCs on osteocytes were in contrast to tumor necrosis factor α (TNF-α), which induced apoptosis but not autophagy. Together this study reveals a novel mechanism for the effect of GC on osteocytes, shedding new insight into mechanisms responsible for bone loss in patients receiving GC therapy.

Bone morphogenetic protein 2 mediates dentin sialophosphoprotein expression and odontoblast differentiation via NF-Y signaling.

Dentin sialophosphoprotein (DSPP), an important odontoblast differentiation marker, is necessary for tooth development and mineralization. Bone morphogenetic protein 2 (BMP2) plays a vital role in odontoblast function via diverse signal transduction systems. We hypothesize that BMP2 regulates DSPP gene transcription and thus odontoblast differentiation. Here we report that expression of BMP2 and DSPP is detected during mouse odontogenesis by in situ hybridization assay, and BMP2 up-regulates DSPP mRNA and protein expression as well as DSPP-luciferase promoter activity in mouse preodontoblasts. By sequentially deleting fragments of the mouse DSPP promoter, we show that a BMP2-response element is located between nucleotides -97 and -72. By using antibody and oligonucleotide competition assays in electrophoretic mobility shift analysis and chromatin immunoprecipitation experiments, we show that the heterotrimeric transcription factor Y (NF-Y) complex physically interacts with the inverted CCAAT box within the BMP2-response element. BMP2 induces NF-Y accumulation into the nucleus increasing its recruitment to the mouse DSPP promoter in vivo. Furthermore, forced overexpression of NF-Y enhances promoter activity and increases endogenous DSPP protein levels. In contrast, mutations in the NF-Y-binding motif reduce BMP2-induced DSPP transcription. Moreover, inhibiting BMP2 signaling by Noggin, a BMP2 antagonist, results in significant inhibition of DSPP gene expression in preodontoblasts. Taken together, these results indicate that BMP2 mediates DSPP gene expression and odontoblast differentiation via NF-Y signaling during tooth development.

Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin.

Sclerostin, the protein product of the Sost gene, is a potent inhibitor of bone formation. Among bone cells, sclerostin is found nearly exclusively in the osteocytes, the cell type that historically has been implicated in sensing and initiating mechanical signaling. The recent discovery of the antagonistic effects of sclerostin on Lrp5 receptor signaling, a crucial mediator of skeletal mechanotransduction, provides a potential mechanism for the osteocytes to control mechanotransduction, by adjusting their sclerostin (Wnt inhibitory) signal output to modulate Wnt signaling in the effector cell population. We investigated the mechanoregulation of Sost and sclerostin under enhanced (ulnar loading) and reduced (hindlimb unloading) loading conditions. Sost transcripts and sclerostin protein levels were dramatically reduced by ulnar loading. Portions of the ulnar cortex receiving a greater strain stimulus were associated with a greater reduction in Sost staining intensity and sclerostin-positive osteocytes (revealed via in situ hybridization and immunohistochemistry, respectively) than were lower strain portions of the tissue. Hindlimb unloading yielded a significant increase in Sost expression in the tibia. Modulation of sclerostin levels appears to be a finely tuned mechanism by which osteocytes coordinate regional and local osteogenesis in response to increased mechanical stimulation, perhaps via releasing the local inhibition of Wnt/Lrp5 signaling.

Differential regulation of dentin sialophosphoprotein expression by Runx2 during odontoblast cytodifferentiation.

Dentin sialophosphoprotein (DSPP) consists of dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). The spatial-temporal expression of DSPP is largely restricted during differentiational stages of dental cells. DSPP plays a vital role in tooth development. It is known that an osteoblast-specific transcription factor, Runx2, is essential for osteoblast differentiation. However, effects of Runx2 on DSPP transcription remain unknown. Here, we studied different roles of Runx2 in controlling DSPP expression in mouse preodontoblast (MD10-F2) and odontoblast (MO6-G3) cells. Two Runx2 isoforms were expressed in preodontoblast and odontoblast cells, and in situ hybridization assay showed that DSPP expression increased, whereas Runx2 was down-regulated during odontoblast differentiation and maturation. Three potential Runx2 sites are present in promoters of mouse and rat DSPP genes. Runx2 binds to these sites as demonstrated by electrophoretic mobility shift assay and supershift experiments. Mutations of Runx2 sites in mouse DSPP promoter resulted in a decline of promoter activity in MD10-F2 cells compared with an increase of its activity in MO6-G3 cells. Multiple Runx2 sites were more active than a single site in regulating the DSPP promoter. Furthermore, forced overexpression of Runx2 isoforms induced increases of endogenous DSPP protein levels in MD10-F2 cells but reduced its expression in MO6-G3 cells consistent with the DSPP promoter analysis. Thus, our results suggest that differential positive and negative regulation of DSPP by Runx2 is dependent on use of cytodifferentiation of dental ectomesenchymal-derived cells that may contribute to the spatial-temporal expression of DSPP during tooth development.

Dentin matrix protein 1 gene cis-regulation: use in osteocytes to characterize local responses to mechanical loading in vitro and in vivo.

Dentin matrix protein 1 (DMP1) is highly expressed in osteocytes and is mechanically responsive. To study osteocyte-specific and mechanically regulated DMP1 gene expression, the transcriptional activity of three cis-regulatory regions was first examined in an osteoblast differentiation model in vitro using a green fluorescent protein (GFP) reporter. Expression of the -9624 to +1996 bp (10 kb) and -7892 to +4439 bp (8 kb) DMP1 cis-regulatory regions dramatically increased in areas of mineralized matrix, in dendritic, osteocyte-like cells. Mineralizing cultures expressing the 8-kb construct show dramatic GFP increases in response to loading in cells with a dendritic morphology. Transgenic mice expressing the 8-kb DMP1-GFP and -2433 to +4439 bp (2.5 kb) DMP1-LacZ were generated. Osteocyte-specific expression was found with the 8 kb but not with the 2.5 kb in postnatal animals. However, the 2.5 kb could support expression in rapidly forming osteoblasts and pre-osteocytes in the embryo. Primary calvarial osteoblast cultures demonstrated that the 2.5 kb supports weak expression in a subset of osteoblasts and pre-osteocytes, but not in mature osteocytes. However, the 8 kb supports robust expression in primary bone marrow cultures. Therefore the region -7892 to -2433 bp, termed a 5.5-kb "Osteocyte Enhancer Module," appears to be required for osteocyte specificity. Ulnae of mice with the 8-kb DMP1-GFP were subjected to mechanical loading where GFP expression increased selectively and locally in osteocytes, distal to the mid-shaft and near the surface of the bone. Thus, the 8-kb region of the DMP1 gene is a target for mechanotransduction in osteocytes, and its cis-regulatory activity may be correlated to local strain in bone.

Orthodontically stressed periodontium of transgenic mouse as a model for studying mechanical response in bone: The effect on the number of osteoblasts.

A better understanding of cellular and molecular mechanisms involved in response to mechanical stress is a prerequisite for future improvements in orthodontic treatment. To expand the application of molecular biology techniques in this area of research, we developed and characterized a mouse tooth movement model. The aim of this study was to biomechanically characterize this model and to evaluate the effect of orthodontic stress on the proliferation of periodontal osteoblasts. We used an orthodontic coil spring appliance with a low force/deflection rate, which produced an average force of 10-12 g. This design provided a predictable tipping movement of the molar with the center of rotation at the level of root apices. Histological observations of paradental tissues revealed a response favoring a fast onset of tooth movement and deposition of new osteoid starting after 3 days of treatment. The effect of treatment on the histomorpometric parameter of the number of osteoclasts per unit bone perimeter was determined after 1, 2, 3, 4, 6, and 12 days of treatment. Starting with day 2, the osteoblast number showed a modest but consistent increase in treated periodontal sites at all time-points, ranging from 14 to 39% and becoming significant only at day 6. Only a moderate increase in the number of osteoblasts in the areas of otherwise intense bone matrix synthesis suggests that, during bone formation, proliferation of cells has a smaller role compared to a marked increase in differentiation of individual cells. The mouse model, which allows for a controlled, reproducible, orthodontic mechanical loading, can be applied to both wild-type and transgenic animals and should enhance the research of the transduction of mechanical orthodontic signal into a biological response.

Orthodontically stressed periodontium of transgenic mouse as a model for studying mechanical response in bone: The effect on the number of osteoblasts.

A better understanding of cellular and molecular mechanisms involved in response to mechanical stress is a prerequisite for future improvements in orthodontic treatment. To expand the application of molecular biology techniques in this area of research, we developed and characterized a mouse tooth movement model. The aim of this study was to biomechanically characterize this model and to evaluate the effect of orthodontic stress on the proliferation of periodontal osteoblasts. We used an orthodontic coil spring appliance with a low force/deflection rate, which produced an average force of 10-12 g. This design provided a predictable tipping movement of the molar with the center of rotation at the level of root apices. Histological observations of paradental tissues revealed a response favoring a fast onset of tooth movement and deposition of new osteoid starting after 3 days of treatment. The effect of treatment on the histomorpometric parameter of the number of osteoclasts per unit bone perimeter was determined after 1, 2, 3, 4, 6, and 12 days of treatment. Starting with day 2, the osteoblast number showed a modest but consistent increase in treated periodontal sites at all time-points, ranging from 14 to 39% and becoming significant only at day 6. Only a moderate increase in the number of osteoblasts in the areas of otherwise intense bone matrix synthesis suggests that, during bone formation, proliferation of cells has a smaller role compared to a marked increase in differentiation of individual cells. The mouse model, which allows for a controlled, reproducible, orthodontic mechanical loading, can be applied to both wild-type and transgenic animals and should enhance the research of the transduction of mechanical orthodontic signal into a biological response.