Biomedical subjects

J R Nefussi

Publications and source records attributed to J R Nefussi.

At least 19 recordsLinked to original sources

PubMed2002

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. In Msx1 null mutation, the most striking feature was an inhibition of the mandibular basal convexity, the absence of teeth and alveolar bone processes, and absence of endochondral ossification in the mandibular condyle. At birth, in Msx1+/- heterozygous animals, we identified for the first time a double Msx1 aboral-oral and disto-proximal gradient field developmental pattern located in the low border of the mandibular bone in relation with this bone segment modeling. Msx1 expression involved both osteoblast and osteoclast cells. A distinct pattern characterized bone surfaces: Periosteum osteoblast differentiation was related to Msx1 down-regulation, while in the endosteum both differentiated osteoblasts and osteoclasts expressed the homeoprotein. In postnatal stages, Msx1 expression was maintained in the alveolar bone processes and dento-alveolar cells in relation with tooth function. Our data suggest that Msx1 play a role in a site-specific manner not only in early patterning but also in skeletal growth and modeling by acting on heterogenous bone cell populations.

Animals↗

PubMed2001

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. A knock-in transgenic mouse line was studied from the first postnatal week until 15 months. Whole-mount beta-galactosidase enzymology identified Msx1 protein expression pattern. Maintained expression of Msx1 was observed in growing and adult mice, specifically in the sites where Msx1 plays an early morphogenetic role during initial skeletal patterning. These included the craniofacial sutures, autopodium, mandible, and alveolar bone. Furthermore, active membranous and endochondral bone formation involved Msx1 in the entire skeleton. Histologic sections showed that progenitor as well as differentiating and differentiated cells of all the bone cell lineages could express the Msx1 protein (chondrocytes, osteoblasts, tartrate-resistant acid phosphatase positive osteoclasts and chondroclasts). Recent developments in the genetic and developmental biology of skeletal morphogenesis demonstrate that genes critical for development are jointly expressed in discrete embryonic signalling and growth centers, the enamel knot in teeth, the cranial suture in skull morphogenesis, and the progress zone in the limb buds. The present study suggests that these signalling pathways are jointly important throughout the entire lifetime with an exquisite site-specificity spatially related to early patterning.

Animals↗

PubMed1998

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. However, adjacent to the explants, multicellular cell layers began to form in 10 d, and matrix mineralization was observed by 4 wk in these areas. These formations enlarged and by the end of the culture period, large diffuse matrix mineralization areas were observed. Light and electron microscopic observations confirmed the presence of a collagen matrix undergoing a mineralization process but showing important differences with the mineralized matrix tissue formed with a rat embryo calvaria bone cell system. This new model, using activated human alveolar bone cells, may provide a tool to investigate alveolar bone development and physiology and to set up new therapeutic approaches.

Adult↗

PubMed1997

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. Using serial cross sections and microradiographies of bone nodules, we demonstrated that the gray-level parameter is well correlated with bone mass and/or the mineralization status of the nodules. The procedure selected is a multistep procedure called rapid nodule evaluation (RNE), which uses a binary reconstruction program with different thresholds. To challenge this RNE procedure with the classical Von Kossa staining and quantification procedure, we cultured the cells in the presence of 10 nmol/L dexamethasone and compared the results using the two procedures. The RNE procedure appeared to be accurate and reproducible, and also has the advantage of speed and dynamic analysis over the classical Von Kossa quantification procedure.

Animals↗

PubMed1997

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. Cell morphological modifications were observed in osteoblastic cells expressing ALP, OC, and BSP, but not ON. During the matrix formation phase, all proteins were expressed with various intensities and OC was limited to differentiated osteoblastic cells. EM observations demonstrated that BSP was selectively associated with clusters of needle-like crystals, but not with collagen fibers, in mineralization foci and in the mineralized matrix. OC was localized intracellularly and in all the extracellular compartments, and was concentrated at the mineralization front. ON was distributed uniformly throughout the osteoid and mineralized matrix, which was intensely labeled. The results show that the expression of bone matrix proteins during differentiation of calvaria cells and nodule formation in vitro duplicate what is observed during osteogenesis in vivo.

Alkaline Phosphatase↗

PubMed1996

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. The dose dependency of the effect of beta-GP on 45Ca incorporation and generation of Pi was similar (k phi = 3 mM). Levamisole, but not dexamisole, inhibited both pNPP and beta-GP hydrolyses, which were likely catalyzed by the same ecto-enzyme. The rate of 45Ca incorporation into matrix cell layers, which was high (0.90 mumol/4h/mg cell protein) in cells grown in the absence of beta-GP, was inhibited by 50% by levamisole. In cells grown in the absence of beta-GP, the 45Ca incorporation rate increased progressively after beta-GP addition, reaching after 12 h the value of cultures grown in the presence of beta-GP, the increase being totally inhibited by levamisole. In both types of cells, addition of exogenous Pi at concentrations corresponding to medium levels of beta-GP-derived Pi rapidly led to high 45Ca incorporation rate which was unaffected by levamisole. beta-GP removal from cultures grown in its presence reduced by 50% the 45Ca incorporation rate which recovered the initial value after exogenous Pi addition independently of levamisole presence. Thus, mineral deposition did not affect the level and catalytic efficiency of ecto-ALP to hydrolyze beta-GP in cultured fetal calvaria cells, yet it influenced the beta-GP-stimulatory effect on mineralization so as to render this process not sensitive to high medium Pi levels.

Alkaline Phosphatase↗

PubMed1994

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. However, when cultured with bone cells and observed under a transmission electron microscope, an electron-dense layer was noted initially at the surface of the material, before bone formation occurred. In addition, energy-dispersive X-ray microanalysis demonstrated the presence of calcium and phosphorus in this layer. Progressively, during the following days of culture, active osteoblasts synthetized and laid down an osteoid matrix composed of numerous collagen fibrils arranged either parallel or perpendicularly to the first-formed electron-dense layer. Mineralization initiated on the ceramic surface dispersed then along the collagenous fibrils, leading to a mineralized matrix which surrounded the ceramic particles. These results demonstrate the capacity of apatite-wollastonite glass ceramic to initiate biomineralization in osteoblast cultures and to achieve a direct bond between the surface apatite layer of the bioactive glass-ceramic and the mineralized bone matrix.

Animals↗

PubMed1993

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. These cell clusters synthetized type II collagen and formed an alcian-blue-positive matrix. The following days, a progressive increase in the number of cells positive for type II collagen was noted and, on day 8, typical cartilaginous nodules were formed. These nodules increased in size and number, spreading outward, laying down a dense matrix which mineralized. Light and electron microscopy observations of cross-sections of nodules confirmed the cartilaginous nature of this tissue formed in vitro with typical chondrocytes embedded in a hyaline matrix. Furthermore, at the electron microscopic level, matrix vesicles were seen in extracellular matrix associated with the initiation of mineralization. Typical rod-like crystals were present in the intercellular spaces along the collagen fibers. These results indicated that in a specific environment, dedifferentiated chondrocytes were able to redifferentiate and to form nodular structures with morphological ultrastructure of calcified cartilage observed in vivo.

Alcian Blue↗

PubMed1993

[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. The osteocyte embedding process observed secondly are in relation with the cell heterogeneity forming the AOU. AOU's final cell activity might be a triggering factor in bone remodeling.

Alkaline Phosphatase↗

PubMed1992

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 0.5% bovine testicular hyaluronidase to verify specificity for lipid staining. This allowed us to visualize the lipids (1) in the osteoid as granules associated to ribbon-like structures connected to the collagen fibers, (2) as electrondense deposits seen as dots on the outer surface membrane of the matrix vesicles, and (3) in the mineralized matrix as roundish patches formed of needle-shaped materials and at the mineralization front as individual ones. This study demonstrated that at the EM level, the lipids are present in the osteoid at locations very similar to what have been observed for the glycosaminoglycans, and in the mineralized matrix as components of the crystal ghosts.

Animals↗

PubMed1992

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. Metabolic labeling at day 14, during the phase of nodules mineralization as well as new nodules formation, shows that collagen types I and type V are synthesized; (2) During the phase of cell growth, no osteocalcin could be detected in the medium, however, at the point of nodule formation, the osteocalcin level reached values of 3.55 +/- 1.39 ng/ml, followed by a 30-fold increase after nodules became mineralized. At day 14, after metabolic labeling, de novo synthesized osteocalcin was chromatographed on an immunoadsorbing column. With urea-SDS PAGE the apparent molecular weight was determined to be 9,000 daltons. (3) Specific activity of ALP was found to be 10 nmol/min/mg of proteins at cell confluence. At day 15, when nodules are mineralized, this activity was increased by 40-fold. The Michaelis constant was 1.58 10(-3) M/L. ALP was inhibited by L-homoarginine and levamisole but not by L-phenylalanine. ALP was shown to be heat sensitive at 56 degrees C with two slopes of inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1992

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. During the following days, bone nodule formation was closely associated with cytodex microcarriers. In contrast, in control cultures with negatively charged glass beads, cells failed to pile up around the glass beads, and bone nodule formation occurred randomly in the culture dishes with 24 hour delay. Light microscopy observations of experiment cultures revealed the formation of nodular structures, with active osteoblastic cells forming a mineralized matrix in which osteocytes were present. Transmission electron microscopy revealed first, a mineralization process of the surface of the cytodex microcarriers which appeared like a granular electron-dense, collagen-free layer followed by the deposit of a collagenous matrix. These results indicated that cytodex microcarriers provided an excellent matrix for bone cell differentiation and mineralization.

Animals↗

PubMed1992

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. The ultrastructural features revealed a needle-shaped filamentous layer continuous with coral material, whereas hydroxyapatite or cytodex/tissue interface was granular in appearance. These different structures may indicate reactive surfaces, depending on the composition of the substrate.

Animals↗

PubMed1991

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. A passive matrix embedding process related to a decreased activity of the osteoblast-osteocyte cell is proposed as a comprehensive pathway from osteoblast to osteocyte. The formation of the osteocyte is therefore presented as a very coordinated space and time related cell-cell interaction between cells of the three cell pools of the bone.

Animals↗

PubMed1990

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. The initial event observed was the appearance of cells with phosphatase alkaline activity arranged in several layers and forming a three-dimensional organization around the coral particles. By Day 7, nodule formation began and a refringent material appeared and extended to the background cells during the following days. By Day 15, some coral granules were embedded in a mineralized matrix. Histologic results demonstrated the formation of a mineralized tissue with the appearance of woven bone.

Animals↗

PubMed1990

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 3.5-fold with an ED50 of 0.75 ng/ml while a 2.3-fold EGF stimulation was recorded with an ED50 of 0.067 ng/ml. 5-Bromo-2-deoxyuridine staining showed a higher stimulation of proliferation in the scattered cells than in the cell clusters. An 18 h aFGF or EGF treatment decreased alkaline phosphatase (ALP) activity by 40 and 23%, respectively, as compared with control cultures. This inhibition was more pronounced after 48 h in the presence of the effectors but no modification of the ALP electrophoretic mobility was observed. These data suggest that aFGF is a less potent mitogen than EGF and a higher inhibitor of ALP activity in fetal rat calvaria cell culture.

Alkaline Phosphatase↗

PubMed1990

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.

Alkaline Phosphatase↗

PubMed1989

A comparative ultrahistochemical study of glycosaminoglycans with cuprolinic blue in bone formed in vivo and in vitro.

Histochemical and morphological studies have shown that proteoglygans (PG) are involved in mineralization process in vivo but such studies have not yet been conducted in vitro. A comparative histochemical study in electronic microscopy of the localization, organization, and morphology of the PG was performed with bones of calvaria rat formed in vivo and bone nodules formed in vitro from osteoblastic cells in culture. For this investigation, we used a cationic phthalocyanin dye, cuprolinic blue, in a critical electrolyte concentration which simultaneously stained the glycosaminoglycans and demineralized the bone. This histochemical technique demonstrated (1) osteoblast cells in vitro synthesized PG which were included in the matrix formed. (2) These PG were found in the calcified and uncalcified matrix both in vivo and in vitro. In the uncalcified matrix, PG were either free with a granular or rodlike structure or tightly connected to the periphery of the collagen fiber. Contrarily, in the calcified matrix, PG formed dense filamentous reticular patches between the collagen fibers. (3) Similarities in localization, organization, and morphology were noted in PG of bone formed de novo in vitro and in vivo with the exception of the mineralization front, where the staining in vivo compared with in vitro was faint or absent.

Animals↗