Cephalometric assessment of craniofacial dysmorphologies in relation with Msx2 mutations in mouse.

Objectives: To determine the role of Msx2 in craniofacial morphology and growth, we used a mouse model and performed a quantitative morphological characterization of the Msx2 (-/-) and the Msx2 (+/-) phenotype using a 2D cephalometric analysis applied on micrographs.

Materials And Methods: Forty-four three-and-a-half-month-old female CD1 mice were divided into the following three groups: Msx2 (+/+) (n = 16), Msx2 (+/-) (n = 16), and Msx2 (-/-) (n = 12). Profile radiographs were scanned.

Osteoclasts in the dental microenvironment: a delicate balance controls dental histogenesis.

The impact of osteoclast activity on dental development has been previously analyzed but in the context of severe osteopetrosis. The present study sought to investigate the effects of osteoclast hypofunction,present in Msx2 gene knockin mutant mice (Msx2-/-), and hyperfunction, in transgenic mice driving RANK over-expression in osteoclast precursors (RANK(Tg)), on tooth development. In Msx2-/- mice, moderate osteopetrosis was observed, occurring exclusively in the periodontal region.

Differential impact of MSX1 and MSX2 homeogenes on mouse maxillofacial skeleton.

Craniofacial development involves a large number of genes involved in a complex time- and site-specific cascade of cellular crosstalk. Msx homeobox genes are expressed very early and have been implicated in multiple signaling processes. However, little is known about their role in postnatal growth and at adult stages.

Msx2 -/- transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodental osteopetrosis.

The physiological function of the transcription factor Msx2 in tooth and alveolar bone was analysed using a knock-in transgenic mouse line. In this mouse line, the beta-galactosidase gene was used to disrupt Msx2: thus, beta-galactosidase expression was driven by the Msx2 promoter, but Msx2 was not produced. This allowed to monitor Msx2 expression using a beta-galactosidase assay.

[Genetic and experimental approach to bony craniofacial growth: the role of the divergent homeobox gene Msx1].

Tooth agenesis and clef palate are associated to the mutation of the Msx1 homeobox genes, highlighting the pivotal role of homeobox genes during the initial development of the craniofacial skeleton. Msx1 also controls the terminal differentiation of mineralised tissues forming cells. Recently, a Msx1 antisense RNA has been identified which inhibits Msx1 protein expression in odontoblastic cells.

Msx1 is a regulator of bone formation during development and postnatal growth: in vivo investigations in a transgenic mouse model.

The present study is devoted to Msx1 distribution and function from birth to 15 months, events and periods still unexplored in vivo using Msx1 knock in transgenic mice. The study is focused on the mandible, as an exemplary model system for Msx1-dependent neural crest-derived skeletal unit. The transgenic line enabled study of morphological abnormalities in Msx1 null mutation mice and Msx1 protein expression in Msx1+/- heterozygous mice.

Expression of a 1,25-dihydroxyvitamin D3 membrane-associated rapid-response steroid binding protein during human tooth and bone development and biomineralization.

The calciotropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has been established to control skeletal tissue formation and biomineralization via the regulation of gene expression. This action involves the well-characterized nuclear 1,25(OH)2D3 receptor. However, it has been recognized that several cellular responses to 1,25(OH)2D3 may not to be related to the exclusive nuclear receptor.

Postnatal Msx1 expression pattern in craniofacial, axial, and appendicular skeleton of transgenic mice from the first week until the second year.

Phenotypes associated with Msx1 mutations have established the prominent role of this divergent homeogene in skeletal patterning. Previous studies have been achieved during antenatal development in relation with the early death of null mutant mice. Therefore, the present study is devoted to Msx1 homeogene in the postnatal craniofacial, axial, and appendicular skeleton.

[Mineralized dental tissues: a unique example of skeletal biodiversity derived from cephaic neural crest].

Molecular and structural biodiversity characterises dental mineral tissues. Groups of matrix proteins belong specifically to each tissue; amelogenins to enamel, DSPP to dentine and CAP to cementum. A wide group of proteins is also shares with other mineralized tissues such as calcium (calbindins) and phosphate (alkaline phosphatase) handling proteins.

Activated adult human alveolar bone cells: a new model of matrix mineralization.

Activated adult human alveolar bone cells were isolated from 2-wk-old osteogenic tissue recuperated from dental implant surgeries following a two-step procedure. Osteogenic tissues were cultured as explant for 2 months. Cells began to migrate in the first 3 d and were confluent in 3-4 wk.

Sequential expression of bone matrix proteins during rat calvaria osteoblast differentiation and bone nodule formation in vitro.

We investigated the expression of osteocalcin (OC), bone sialoprotein (BSP), osteonectin (ON), and alkaline phosphatase (ALP) during cell differentiation and bone nodule formation by fetal rat calvaria cells, using immunofluorescent and immunogold techniques at light and electron microscopic levels. Six hours after plating all proteins were expressed in calvaria cells. However, expression was not detected during the proliferation phase after plating.

Rapid nodule evaluation computer-aided image analysis procedure for bone nodule quantification.

Using bone cell cultures, the effects of drugs on cell activities such as proliferation, differentiation, matrix formation, and mineralization can be explored. To quantify these parameters accurately and quickly, a kinetic reproducible computed image analysis procedure of culture dishes is proposed which could be conjointly used with biochemical analysis of the medium. In the present article, different mathematical procedures coupled either with or without histochemical staining are investigated and analyzed.

Role of beta-GP-derived Pi in mineralization via ecto-alkaline phosphatase in cultured fetal calvaria cells.

The permissive effect of beta-GP on mineralization in cultured rat fetal calvaria cells was investigated in relationship with phosphohydrolase activity of ecto-ALP at physiological pH range. Beta-GP present in the culture medium for 8 days exerted a stimulatory effect on 45Ca incorporation into matrix cell layers while the ecto-ALP activity level measured on intact cells with a saturating concentration of pNPP was similar for cells grown either in the presence or absence of beta-GP. In both types of cultures, beta-GP addition inhibited pNPP hydrolysis in a competitive and reversible manner and increased Pi concentration in the medium.

Bioactive glass-ceramic containing crystalline apatite and wollastonite initiates biomineralization in bone cell cultures.

Rat bone cells were cultured in the presence of bioactive glass-ceramic containing crystalline apatite and wollastonite. Scanning electron microscopy observations of the surface of the seeded ceramic disks revealed that cells attached, spread, and proliferated on the material surface. Soaking in cell-free culture medium showed that no change occurred in the surface structure.

In vitro differentiation and mineralization of cartilaginous nodules from enzymatically released rat nasal cartilage cells.

Nasal cartilage cells from 21-day-old rat fetuses were cultured at high density in the presence of ascorbic acid and beta-glycerophosphate over a 12-day period. Immediately after plating, the cells exhibited a fibroblastic morphology, lost their chondrocyte phenotype and expressed type I collagen. On day 3, clusters of enlarged polygonal cells were found.

[Cell culture model and concept of bone surface].

Cellular differentiation areas leading to bone nodular formation from rat bone calvaria cells were studied under optic and electronic transmission microscope. 3H-thymidine labeling, BrdU proliferating cells and alkaline phosphatase cytoenzymatic reaction allowed us to dynamically describe the development of a cellular group called "Active Osteogenic Unit" (AOU) responsible for bone nodule formation. This AOU was formed by synchronized, localized and increased cell surface proliferation allowing a three dimensional cellular organization leading to an underneath osteoblastic cell proliferation.

Mineralization and bone formation on microcarrier beads with isolated rat calvaria cell population.

Using enzymatically isolated rat bone cells in the presence of cytodex microcarrier beads, osteoblastic cell differentiation and bone nodule formation were studied at the optical and electron microscopic level. Cytochemical method showed an intense alkaline phosphatase activity mainly around the microcarriers where the cells have formed multilayers on day 4 of cultures. On day 7 of experiment cultures, Von Kossa method stained positively only the cytodex microcarriers.

Localization of malachite green positive lipids in the matrix of bone nodule formed in vitro.

An electron histochemical study was carried out on bone nodules formed in vitro in collagenase-released calvarial cells in order to visualize the lipid components of the extracellular matrix (EM). The malachite green aldehyde fixative technique, which allows both preservation and staining of some phospholipids of the extracellular matrix, was used. Controls were performed on sections demineralized, and then submitted to lipid extraction with a chloroformmethanol mixture (2/1 v/v) and to glycosaminoglycans digestion with 0.

Expression of collagen, osteocalcin, and bone alkaline phosphatase in a mineralizing rat osteoblastic cell culture.

Rat calvaria bone cells isolated by collagenase digestion form a bone-like matrix which mineralizes in vitro in the presence of beta-glycerophosphate, in less than 2 weeks. The purpose of this work was to investigate, in this mineralizing rat osteoblastic cell culture, the synthesis of collagen, osteocalcin, and bone alkaline phosphatase (ALP). The results obtained indicate (1) After 15 days in culture, the extracellular-matrix contains collagen type I, V, and to some extent type III.

Surface-reactive biomaterials in osteoblast cultures: an ultrastructural study.

The tissue/biomaterial interface reactions of three biomaterials selected as candidates for hard tissue replacement were studied at the electron microscopical level after incubation with enzymatically isolated rat bone cells. An electron-dense layer was routinely observed between hydroxyapatite, coral, cytodex polymer and the neighbouring cells. This layer was visible before bone formation occurred, and was collagen free.

How osteoblasts become osteocytes: a decreasing matrix forming process.

Osteocyte matrix inclusion process was studied in an in vitro woven bone nodule formation model where a large number of osteocytes at different degrees of maturation were examined. This work focused on early stages of osteocyte inclusion. This matrix inclusion occurred without a matrix synthesis inversion by the future osteocyte and with maintenance of close cell contacts with the replacing cell.

In vitro bone formation on coral granules.

We investigated the ability of fetal rat bone cells isolated after collagenase digestion to differentiate in vitro and to produce a mineralized matrix on coral granules. Scanning electron microscopy examination of the surface of the seeded coral granules revealed that cells attached, spread, and proliferated on the material surface. Bone nodule formation was studied in this in vitro system by direct examination under an inverted phase contrast microscope.

Effects of acidic fibroblast growth factor and epidermal growth factor on fetal rat calvaria cell cultures.

An inhibitory effect of alkaline phosphatase (LP) activity on short and long term fetal rat calvaria cell cultures was recorded with both acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF) at a concentration of 30 ng ml-1. This inhibition was well correlated with the nodule number on long term culture, except for EGF treatment in subconfluent cell culture.

Effects of acidic fibroblast growth factor and epidermal growth factor on subconfluent fetal rat calvaria cell cultures: DNA synthesis and alkaline phosphatase activity.

The effects of acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF) were examined in subconfluent fetal rat calvaria cell cultures, in the presence of 2% serum. Maximal effect of aFGF and EGF on DNA synthesis measured by [3H]thymidine incorporation was observed after 18 h. aFGF stimulated DNA synthesis by 3.

Microcinematographic and autoradiographic kinetic studies of bone cell differentiation in vitro: matrix formation and mineralization.

Matrix formation and mineralization have been reported in vitro with cells isolated from rat calvaria bones by collagenase digestion (Nefussi et al., 1985). In the current study, kinetics of bone nodule formation and osteoblastic cell differentiation were studied in this in vitro system using an improved microcinematographic device and flash and follow-up labeling autoradiographic techniques.