Biomedical subjects

J M Sautier

Publications and source records attributed to J M Sautier.

At least 19 recordsLinked to original sources

PubMed2001

Bioactive glass stimulates in vitro osteoblast differentiation and creates a favorable template for bone tissue formation.

In this study, we have investigated the behavior of fetal rat osteoblasts cultured on bioactive glasses with 55 wt% silica content (55S) and on a bioinert glass (60S) used either in the form of granules or in the form of disks. In the presence of Bioglass granules (55 wt% silica content), phase contrast microscopy permitted step-by-step visualization of the formation of bone nodules in contact with the particles. Ultrastructural observations of undecalcified sections revealed the presence of an electron-dense layer composed of needle-shaped crystals at the periphery of the material that seemed to act as a nucleating surface for biological crystals. Furthermore, energy dispersive X-ray (EDX) analysis and electron diffraction patterns showed that this interface contains calcium (Ca) and phosphorus (P) and was highly crystalline. When rat bone cells were cultured on 55S disks, scanning electron microscopic (SEM) observations revealed that cells attached, spread to all substrata, and formed multilayered nodular structures by day 10 in culture. Furthermore, cytoenzymatic localization of alkaline phosphatase (ALP) and immunolabeling with bone sialoprotein antibody revealed a positive staining for the bone nodules formed in cultures on 55S. In addition, the specific activity of ALP determined biochemically was significantly higher in 55S cultures than in the controls. SEM observations of the material surfaces after scraping off the cell layers showed that mineralized bone nodules remained attached on 55S surfaces but not on 60S. X-ray microanalysis indicated the presence of Ca and P in this bone tissue. The 55S/bone interfaces also were analyzed on transverse sections. The interfacial analysis showed a firm bone bonding to the 55S surface through an intervening apatite layer, confirmed by the X-ray mappings. All these results indicate the importance of the surface composition in supporting differentiation of osteogenic cells and the subsequent apposition of bone matrix allowing a strong bond of the bioactive materials to bone.

Animals↗

PubMed2000

In vitro bone formation on a bone-like apatite layer prepared by a biomimetic process on a bioactive glass-ceramic.

In this study we have investigated the behavior of fetal rat osteoblasts, cultured up to 23 days, on a bioactive apatite-wollastonite (AW) glass-ceramic and on the same material on which a carbonated apatite layer had been formed by a biomimetic process (AWa). At the last day of culture, the specific activity of alkaline phosphatase activity, as determined biochemically, was about 30% greater on AWa compared with AW disks. After the cell layers had been scraped off, scanning electron microscopic (SEM) observations of the materials' surfaces revealed that mineralized bone nodules remained attached to both surfaces but in larger amounts on AWa. X-ray microanalysis indicated the presence of calcium (Ca) and phosphorus (P) in the bone tissue throughout the AWa surface and Ca, P, and silicon (Si) on the AW surface. The AW/ and AWa/bone interfaces also were analyzed after fracturing of the disks. The interfacial analysis showed firm bone bonding to the AW and AWa surfaces, confirmed by the X-ray microanalytic mappings. These results indicate the importance of surface composition in supporting differentiation of osteogenic cells and the subsequent apposition of bone matrix, which allows a strong bond of the bioactive materials to the bone. Furthermore, prefabrication of a biologic apatite layer by a method that mimics biomineralization could find application to bone-repairing materials.

Alkaline Phosphatase↗

PubMed2000

Association of enhanced expression of gap junctions with in vitro chondrogenic differentiation of rat nasal septal cartilage-released cells following their dedifferentiation and redifferentiation.

The nasal septum is an important centre of endochondral ossification during the development of the facial region. Previous studies have shown that it is possible to recapitulate the differentiation programme of 21-day-old rat nasal chondrocytes in vitro. The purpose now was to investigate, in vitro, the cell condensation phase that represents the earliest morphological event associated with cartilage differentiation in skeletal development. The study focuses on the ability of the cells to form condensations before overt differentiation, with special emphasis on gap-junction expression. The gap-junction protein connexin 43 was localized by indirect immunofluorescence as primarily intracellular and, on day 5, at the condensation stage, as spot-like contacts between cells. Intracellular injection of the permeable dye Lucifer yellow led to the staining of up to 20 neighbouring cells, indicating functional gap junctions and coupling. In contrast, treatment of cultures with the gap-junction blocker glycyrrhetinic acid inhibited dye coupling and reduced cartilage differentiation. Northern blotting of connexin 43 mRNA showed a faint band during the first days of culture, with a striking increase after day 4. In addition, the mRNA of the homeodomain-containing gene Cart-1 began to be expressed in prechondrogenic condensations and corresponded to the expression of type II collagen mRNA. These data indicate that the early stage of in vitro chondrocyte differentiation is the formation of cell condensations and the ability to establish cell-to-cell communication. Connexin 43, together with other molecular mechanisms, mediates the condensation phase of chondrogenesis and sets up the optimal environment in which nasal septal cells may terminally differentiate into chondrocytes.

Animals↗

PubMed2000

Phenotypic modulation of nasal septal chondrocytes by cytoskeleton modification.

The reversion to the initial round shape of chondrocytes in monolayer cultures is one of the initial events required for the expression of cartilage-specific macromolecules. Thus, considerable research efforts have focused on developing reliable procedures to maintain a round morphology of cultured chondrocytes. Our study focuses on evaluating the response of dedifferentiated fetal rat chondrocytes to cytochalasin D, an actin-disrupting agent, with special emphasis on the morphological events. Immediately after exposure to the drug, cells round up but flatten again after removing the agent. However, immunocytochemical procedures revealed a disorganization of microfilaments and intermediate filaments. Phase-contrast and scanning electron microscopic observations revealed that on day 6 of culture, cells located at the top of the cell layer adopted a spherical morphology. Prominent differences were noted in control cultures where cells had to aggregate prior to overt chondrogenesis. These morphological changes occurred parallel to the expression of type II collagen, marker of the chondrocytes phenotype, strongly expressed in experimental cultures, but relatively weak in control cultures, and only restricted on areas of polygonal cellular aggregates. Furthermore, [35S]-sulphate incorporation into sulphated glycosaminoglycans increased rapidly with the period of culture to a maximum after 7 days and was then two-fold in treated cultures. Taken together, these findings indicated that cytochalasin-D stimulates chondrogenesis in response to modification of cytoskeleton architecture and the subsequent rounding up of the cells.

Animals↗

PubMed2000

[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. Dental tissues organisation is also based on specific cellular programs of morpho-differentiation (polarity) and on expression patterns of proteins implicated in mineralisation. The regulation of gene expression in tooth has been analysed regarding various hormones such as vitamin D in a first step and recently transcription factors (Osf-2/Cbfa1/Aml3). Other molecular families encoded by divergent homeobox genes (Msx and Dlx) are implicated in the determinism of this gene regulation and of early development. Genetic and hormonal abnormalities of dental mineralized tissues should now be interpreted thanks to the recent availability of cellular models and of odontogenic protein promoter structure.

Animals↗

PubMed1998

Cartilage formation by fetal rat chondrocytes cultured in alginate beads: a proposed model for investigating tissue-biomaterial interactions.

Chondrocytes from 21-day-old rat fetal nasal cartilage were cultured in alginate beads for up to 20 days. It was found that chondrocytes retained their spherical shape and typical chondrocytic appearance. During the culture time, chondrocytes underwent differentiation, as demonstrated by the alkaline phosphatase-specific activity and rate of proteoglycan synthesis. Morphological data confirmed chondrocyte differentiation with the appearance of hypertrophic chondrocytes scattered in the alginate gel and a dense extracellular matrix containing filamentous structures and matrix vesicles. In addition, Northern blot analysis performed on day 8 of culture showed that chondrocytes cultured in alginate beads expressed type II collagen mRNA. The alginate bead method also appeared to be suitable for testing biomaterials, and the ready dissolution of the alginate beads by chelating agents provided a simple means for the rapid recovery of encapsulated chondrocytes. Powdered glass-ceramic particles entrapped in the alginate gel were colonized by chondrocytes, which then proliferated and formed a tissue similar to a true calcified cartilaginous structure. These results indicate that the alginate system represents a relevant model for studies of chondrogenesis and endochondral ossification. Furthermore, the encapsulation method could prove useful for studies of tissue-biomaterial interactions in an in vitro environment which more closely mirrors the cartilage matrix than other culture methods.

Animals↗

PubMed1997

Behavior of fetal rat chondrocytes cultured on a bioactive glass-ceramic.

We examined the behavior of fetal rat chondrocytes cultured on a bioactive glass-ceramic containing apatite and wollastonite (A.W.G.C.). Biomaterial surface topography and profiles were evaluated by bidimensional profilometry and revealed a rough surface for the glass-ceramic compared to the plastic coverslips used as controls. Chondrocyte attachment was evaluated by measuring the number of attached cells after one day of culture and by morphological observations. Chondrocytes attached in great numbers to the material surface by means of focal contacts containing vinculin and beta1-integrin. Fluorescent labeling of actin and vimentin revealed a poor spreading of chondrocytes on the bioactive glass-ceramic compared to the plastic coverslips, where the cells appeared to adhere intimately to the surface and exhibited polygonal arrays of stress fibers. During the following days of culture, chondrocytes proliferated, colonized the surface of the material, and, finally, on day 10, formed nodular structures composed of round cells separated by a dense extracellular matrix. Furthermore, these clusters of round cells were positive for type II collagen and chondroitin sulfate, both hard markers of the chondrocyte phenotype. In addition, protein synthesis, alkaline phosphatase activity, and proteoglycan production were found to increase gradually during the culture period with a pattern similar to that observed on control cultures. These results demonstrate that the bioactive glass-ceramic tested in this study appears to be a suitable substrate for in vitro chondrocyte attachment, differentiation, and matrix production.

Actins↗

PubMed1995

Cytochalasin D induces changes in cell shape and promotes in vitro chondrogenesis: a morphological study.

One of the initial events required for the expression of cartilage-specific macromolecules in monolayer cultures is the reversion to the initial round shape of chondrocytes. Thus, considerable research efforts have focused on developing reliable procedures to maintain a round morphology of cultured chondrocytes. Our study focuses on evaluating the response of dedifferentiated fetal rat chondrocytes to cytochalasin D, an actin-disrupting agent, with special emphasis on the morphological events. Immediately after exposure to the drug, cells round up but flatten again after removing the agent. However, immunocytochemical procedures revealed a disorganization of microfilaments and intermediate filaments. Phase-contrast and scanning electron microscopic observations revealed that on day 6 of culture, cells located at the top of the cell layer adopted a spherical morphology. Prominent differences were noted in control cultures where cells had to aggregate prior to overt chondrogenesis. Transmission electron microscopy confirmed the round morphology of the cells situated at the top layer but also revealed the presence of cell contacts between the cells. In addition, cells located at the central part of the cell layer displayed a typical morphology of mature chondrocytes, separated by an extensive extracellular matrix. These morphological changes occurred parallel to the expression of type II collagen and chondroitin sulfate, both hallmarks of the chondrocyte phenotype strong in experimental cultures, relatively weak in control cultures, and only restricted on areas of polygonal cellular aggregates. Furthermore, [35S]-sulfate incorporation into sulfated glycosaminoglycans increased rapidly with the period of culture to a maximum after 7 days and was then two-fold in treated cultures. Taken together, these findings indicated that cytochalasin D stimulates chondrogenesis in response to modification of cytoskeleton architecture and the subsequent rounding up of the cells.

Actin Cytoskeleton↗

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

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

[Root growth].

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Cementogenesis↗

PubMed1989

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. Microcinematographic analysis showed the formation of bone nodules within 24 h. The initial event observed was the change in the top cells layer which became alkaline phosphatase positive. Matrix synthesis occurred a few hours after this. The autoradiographic results demonstrated the formation of an integrated system where osteoblasts and osteocytes were active and synthesized a collagen matrix and mineralized it in a similar time sequence than in vivo.

Alkaline Phosphatase↗