Biomedical subjects

D Laurent-Maquin

Publications and source records attributed to D Laurent-Maquin.

At least 19 recordsLinked to original sources

PubMed2006

Interfacial reactions of glasses for biomedical application by scanning transmission electron microscopy and microanalysis.

Short-term physico-chemical reactions at the interface between bioactive glass particles and biological fluids are studied for three glasses with different bioactive properties; these glasses are in the SiO(2)-Na(2)O-CaO-P(2)O(5)-K(2)O-Al(2)O(3)-MgO system. Our aim is to show the difference between the mechanisms of their surface reactions. The relation between the composition and the bioactive properties of these glasses is also discussed. The elemental analysis is performed at the submicrometer scale by scanning transmission electron microscopy associated with energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy. After different immersion times (ranging from 0 to 96 h) of bioactive glass particles in a simulated biological solution, results show the formation of different surface layers at the glass periphery in the case of two bioactive glasses (A9 and BVA). For the third glass (BVH) we do not observe any surface layer formation or any modification of the glass composition. For the two other glasses (A9 and BVA), we observe the presence of different layers: an already observed (Si, O, Al) rich layer at the periphery, a previously demonstrated thin (Si, O) layer formed on top of the (Si, O, Al) layer and a (Ca, P) layer. We determine the different steps of the mechanisms of the surface reactions, which appear to be similar in these glasses, and compare the physico-chemical reactions and kinetics using the different immersion times. The A9 glass permits the observation of all important steps of the surface reactions which lead to bioactivity. This study shows the important relationship between composition and bioactivity which can determine the medical applicability of the glass.

Aluminum Oxide↗

PubMed2006

Sensitivity of bacterial biofilms and planktonic cells to a new antimicrobial agent, Oxsil 320N.

The effective concentrations of disinfectants were determined for planktonic bacteria using the norms EN 1040 and NF T 72-150. This concentration corresponds to biocide efficacy after 5 min of contact, followed by neutralization. However, micro-organisms often colonize a substratum and form microcolonies or biofilms where they are enclosed in exopolymer matrices. Biofilms are commonly resistant to a broad range of antimicrobial agents, and resistance mechanisms involve exopolymer matrices, changes in gene expression and metabolic alterations. Due to these different resistance mechanisms, it is difficult to select and titrate antimicrobial agents to be effective against biofilms. In this context, SODIFRA developed a new disinfectant, Oxsil 320N (French patent 94 15 193). Oxsil 320N is an association of three active principles: hydrogen peroxide, acetic acid/peracetic acid and silver. This biocide was tested on planktonic bacteria and on 24-h biofilms formed on AISI 304 stainless steel surfaces. The effective concentration of Oxsil 320N was also determined on biofilms using SODIFRA recommendations (without neutralization of the biocide). Data showed that the antimicrobial efficacy measured on planktonic bacteria is not a reliable indicator of performance when biofilm is present. When biofilms were exposed to Oxsil 320N, the concentration needed to achieve a 10(5)-fold decrease in concentration was 10 times higher than that for bacterial suspensions (0.313% Oxsil 320N). An effective concentration of Oxsil 320N of 3.13% was required.

Acetic Acid↗

PubMed2006

Behavior of human osteoblast-like cells in contact with electrodeposited calcium phosphate coatings.

Calcium-deficient hydroxyapatite (Ca-def-HAP) thin films were elaborated on Ti6Al4V substrates by electrodeposition. The coatings exhibit two different morphologies and crystallinities. Human osteoblast-like cells (MG-63) were cultured on the surfaces of these materials; the cell content and viability were evaluated up to 28 days. The scanning electron microscopy and biological investigations showed cells with a normal morphology, good proliferation, and viability from 7 to 21 days. But after 28 days, the number of live cells decreases in both cases; however, this decrease is less important in the case of calcium phosphate (CaP) coating surface when compared with the control (cell culture plastic). The cells cultured on Ca-def-HAP coating exhibit more cellular extensions and extracellular matrix. RT-PCR for type I collagen, alkaline phosphatase, and osteocalcin studies were also carried out, and was found that the CaP enhances gene expression of ALP and OC and thus the differentiation of osteoblast-like cells. Moreover, this study shows that the difference in the morphology of CaP coatings has no effect on the biocompatibility.

Calcium Phosphates↗

PubMed2005

STEM and EDXS characterisation of physico-chemical reactions at the periphery of sol-gel derived Zn-substituted hydroxyapatites during interactions with biological fluids.

With its good properties of biocompatibility and bioactivity hydroxyapatite (HA) is highly used as bone substitutes and as coatings on metallic prostheses. In order to improve the bioactive properties of HA, we have elaborated Zn2+ doped hydroxyapatite. Zn2+ ions substitute for Ca2+ cations in the HA structure and four Zn concentrations (Zn/Zn+Ca) were prepared at 0.5, 1, 2 and 5 at.%. To study physico-chemical reactions at the materials periphery, we immersed the bioceramics into biological fluids for intervals from 1 day to 20 days. The surface changes were studied at the nanometer scale by scanning transmission electron microscopy associated with energy dispersive X-ray spectroscopy. After 20 days of immersion, we observed the formation of a calcium-phosphate layer at the periphery of the HA doped with 5% zinc. This layer contains magnesium and its thickness was around 200 nm. Formation of this Ca-P-Mg layer represents the bioactive properties of 5% Zn-substituted hydroxyapatite. This biologically active layer improves the properties of HA and will permit a chemical bond between the ceramic and bone.

Body Fluids↗

PubMed2005

Preparation and characterization of an electrodeposited calcium phosphate coating associated with a calcium alginate matrix.

A new way of optimizing osteoconduction of biomaterials is to bring to them biological properties. In this work, we associated a novel release system with an electrodeposited calcium phosphate (CaP) coated titanium alloy Ti6Al4V. The characterization of this material was performed by means of light microscopy, scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM) and X-ray energy dispersive spectroscopy (EDXS). The electrodeposited CaP coating was a tricalcium phosphate, and the release system was composed of microcapsules entrapped in an alginate film. We observed that the alginate matrix had a close contact with the coating. An intermediate layer containing calcium and phosphorus appeared at the interface between the alginate matrix and the CaP coating. These results allowed us to conclude that the association of two techniques, i.e. electrodeposition followed by deposition of a calcium alginate matrix, led to the elaboration of a new biomaterial.

Alginates↗

PubMed2004

Matrix metalloproteinases MMP-2, -9 and tissue inhibitors TIMP-1, -2 expression and secretion by primary human osteoblast cells in response to titanium, zirconia, and alumina ceramics.

Osteogenic properties of bone cells are a key parameter governing osseointegration of implant devices. In this context, osteoblasts have a central role via extracellular matrix synthesis and remodeling that they regulate through different protease activity. In this study, we have analyzed the expression of two matrix metalloproteinases (MMPs): MMP-2 (72 kDa) and MMP-9 (92 kDa) and their specific tissue inhibitors TIMP-1 and TIMP-2 in primary human osteoblastic cells. The effect of titanium, zirconia, and alumina ceramics on the synthesis of these proteases was assessed using reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and zymographic analysis. Our results showed that osteoblasts express MMP-2 and -9 mRNA. Furthermore, MMP-2 mRNA expression was decreased by titanium and increased by alumina whereas zirconia did not have any significant effect. Conversely, MMP-9 mRNA expression was stimulated by titanium but decreased with zirconia, whereas alumina induced no significant changes. Zymographic analysis has evidenced pro-MMP-2 gelatinolytic activity in all cell populations with time-dependent increase profile; pro-MMP-9, however, was not detected. Enzyme-linked immunosorbent assay data confirmed the production of MMP-2 and very low levels of MMP-9. In addition, TIMP-1 was secreted in 24-h-cultured cells and increased to maximal level at 48-72 h whereas TIMP-2 levels were very low. The interactions between human osteoblasts and the studied biomaterials altered both MMP-2, -9 and TIMP-1expression indicating that biomaterials may influence osseointegration and bone remodeling.

Aluminum Oxide↗

PubMed2003

Influence of physicochemical reactions of bioactive glass on the behavior and activity of human osteoblasts in vitro.

Bioactive glasses are characterized by a bond to bone with a hydroxyl carbonate apatite layer. They enhance bone tissue formation and for this purpose are used in orthopedic surgery and in dental implantology. In the current work, we studied the biological response of human osteoblasts with a bioactive glass. This bioactive glass is based on 50% Si0(2), 20% Na(2)O, 16% CaO, 6% P(2)O(5), 5% K(2)0, 2% Al(2)O(3) and 1% MgO and designated A9. Cracks and irregularities were observed on the material surface when it was immersed in the culture medium. In addition, energy dispersive X-ray analyses highlighted a selective release of the elements at the surface of the bioactive glass, such as Na(+) and K(+) ions, released from the first day, contrary to the Si, Al, Ca, P, and Mg elements, which were released more slowly. Cell proliferation kinetics, total protein synthesis, and DNA content of the osteoblasts in contact with bioactive glass were similar to control cells. The morphological studies by light and scanning electron microscopy revealed an increasing cellular density in culture with bioactive glass without contact inhibition. The immunohistochemical studies highlighted the expression of types I, III, and V collagens by osteoblasts cultured in the presence of bioactive glass. The pH measurement of the culture medium in the presence of bioactive glass demonstrated a slight alkalinization. We thus conclude that human osteoblasts preserve their properties in the presence of bioactive glass (A9).

Biocompatible Materials↗

PubMed2003

Implementation of subcellular water mapping by electron energy loss spectroscopy in a medium-voltage scanning transmission electron microscope.

The water concentration in biological cells plays a predominant role in cellular life. Using electron energy loss spectroscopy, the feasibility to measure the water content in cells has already been demonstrated. In this paper, we present an upgrade of water measurement in hydrated cryosections by spectrum imaging mode in a medium-voltage scanning transmission electron microscope. The electron energy loss spectra are recorded in spectrum imaging mode in a 2(n)x2(n) pixels array. Each spectrum is processed in order to determine the water mass content in the corresponding pixel. Then a parametric image is obtained in which grey levels are related to water concentration. In this image, it is possible to recognize the different subcellular compartments. By averaging the water concentration over the relevant pixels, we can determine the water mass content in the concerned subcellular compartment. As an example, we present water mass content measurement at subcellular level in rat hepatocytes.

Animals↗

PubMed2001

Human monocyte-derived macrophages and dendritic cells as targets for biomaterial cytocompatibility studies using an improved in vitro culture system.

Since macrophage plays a key role in the biocompatibility process, neoplastic macrophage cell lines and human blood monocytes are commonly used as target cells for in vitro biomaterial tolerance evaluation. However, tumor cells profoundly differ from normal tissue cells and monocytes are only precursors of macrophages. It has become possible to generate recently, under adherent-free conditions, fully mature macrophages and dendritic cells from human blood monocytes in the presence of GM-CSF and GM-CSF + IL4 respectively. In the present work, we examined the effects of titanium-alloy on morphology, adhesion, cell phenotype and TNF-alpha release activity of such differentiated cells grown in hydrophobic teflon bags. Scanning electron microscopy showed that macrophages substantially adhered and spread on titanium-alloy surface throughout the culture period, whereas only a few dendritic cells were adherent. The phenotype of both cell types remained unchanged in the presence of the tested material. However, titanium-alloy stimulated the secretion of TNF-alpha by the macrophages of some donors. This model of culture may offer new insights into the biomaterial evaluation and may be useful for studying individual responses induced by biomaterials.

Journal Article↗

PubMed2000

Contribution of the plasmin/matrix metalloproteinase cascade to the retraction of human fibroblast populated collagen lattices.

To assess the contribution of the plasmin/matrix metalloproteinase cascade in lattices retraction, human gingival fibroblast-populated collagen lattices were supplemented with plasminogen. The rate of lattice retraction was enhanced by addition of plasminogen. This effect was concomitant to plasmin generation, prostromelysin-1 and procollagenase activation. Plasminogen-mediated initiation of that proteolytic cascade was accompanied by conspicuous changes in cell morphology and collagen fibers organization. At day 1 of culture fibroblasts shifted from a rounded (control) to an elongated (in presence of plgn) shape. At the latest stage of retraction, intense vacuolization around fibroblasts was noticed in plgn-supplemented lattices which paralleled the increased collagen degradation. Plgn-enhancing influence on the initial phase of lattice retraction could be totally annihilated by either aprotinin or Batimastat. Those data emphasize the crucial importance of the plasmin-MMP proteolytic cascade in granulation tissue retraction in a healing wound.

Cells, Cultured↗

PubMed2000

Nestin expression in embryonic and adult human teeth under normal and pathological conditions.

Nestin is an intermediate filament most related to neurofilaments and expressed predominantly in the developing nervous system and muscles. In the present study we examined the in vivo distribution of nestin in human teeth during embryonic development and in permanent teeth under normal and pathological conditions. The results show that nestin is first expressed at the bell stage and that its distribution is restricted in pulpal cells located at the cusp area of the fetal teeth. In young permanent teeth, nestin is found only in functional odontoblasts, which produce the hard tissue matrix of dentin. Expression is progressively down-regulated and nestin is absent from older permanent teeth. In carious and injured teeth, nestin expression is up-regulated in a selective manner in odontoblasts surrounding the injury site, showing a link between tissue repair competence and nestin up-regulation under pathological conditions. In an in vitro assay system of human dental pulp explants, nestin is up-regulated after local application of bone morphogenic protein-4. A similar effect is seen in cultures of primary pulp cells during their differentiation into odontoblasts. Taken together, these results suggest that nestin plays a potential role in odontoblast differentiation during normal and pathological conditions and that bone morphogenic protein-4 is involved in nestin up-regulation.

Adolescent↗

PubMed1999

In vitro reactions of human osteoblasts in culture with zirconia and alumina ceramics.

The biocompatibility of two implantable materials, zirconia and alumina ceramics, was investigated in vitro using human osteoblast cell cultures. The viability of osteoblast cells with the materials was evaluated by the methylthiazole sulfate test that revealed an absence of any cytostatic or cytotoxic effect. Cell proliferation kinetic and total protein synthesis in osteoblasts with zirconia or alumina were similar to that observed in control cells cultured on glass coverslips. Light and scanning electron microscopic examinations showed an intimate contact between osteoblasts and the substrates; well-spread cells were observed on the surfaces of both materials. Adhesion ability and morphological characteristics were preserved in osteoblast cultures with these substrates. Moreover, immunohistochemical staining in osteoblasts with zirconia and alumina showed the capacity of these cells to elaborate the extracellular matrix composed of types I and V collagen, osteocalcin, osteonectin, bone sialoprotein, and cellular fibronectin. Finally, DNA image cytometry and interphase silver-nucleolar organizer regions quantification were applied as complementary biocompatibility tests to detect any changes in DNA synthesis and cell proliferation, respectively. The results showed that neither material altered cell ploidy or cell growth rate in accordance with the absence of any inducing effect on DNA synthesis or proliferation.

Aluminum Oxide↗

PubMed1998

DNA image cytometry and Ag-NORs-staining application in biocompatibility studies on human osteoblast cells in vitro.

We report here the study of the biocompatibility of a bone graft material, the Pyrost, using a previously established in vitro model of human osteoblasts. The effect of this material on cell proliferation was evaluated by the MTS assay. Results indicated the absolute absence of cytotoxic or cytostatic effect of Pyrost on cultured osteoblasts. Viability rate was more than 90% in cells cultured with the material compared to the control. Morphological analysis, undertaken by scanning electron microscopy showed a good adhesion and a spreading of osteoblasts in contact with the material that was colonized by cultured cells. In the second part of this work, we have introduced two methods as complementary biocompatibility tests: DNA image cytometry and interphase Ag-NORs quantification. DNA content was measured in cells cultured with or without Pyrost for 3, 9, 15 and 30 days. The determination of DNA indicated that the majority of osteoblasts population was diploid without aneuploidy. The DNA index and cell distribution profile in DNA histograms were similar in all cell populations. The Ag-NORs amount was used as a parameter for cell kinetic evaluation. We have measured the Ag-NORs index like DNA quantification. The proliferation rate, evaluated by Ag-NORs counts in osteoblasts cultured with or without the material, was identical. However, a decrease in Ag-NORs index was observed from day 3 to day 15 of incubation. These results showed a satisfactory biocompatibility of the Pyrost in human osteoblasts culture. The material did not alter cell viability and had no inducing effect either on proliferation rate or on cell ploidy as demonstrated by DNA image cytometry and Ag-NORs proteins staining.

Aged↗

PubMed1998

Morphometric studies of collagen and fibrin lattices contracted by human gingival fibroblasts; comparison with dermal fibroblasts.

Cell shape variations and substratum re-organization during contraction of floating collagen and fibrin lattices seeded with human gingival fibroblasts were determined by computerized image analysis of light and scanning electron microscopic images. Data were compared with those obtained with lattices populated with human dermal fibroblasts. The extent of collagen lattice contraction was similar with both cell types, resulting in a two-fold decrease in the area fractions occupied by collagen fibers. Fibroblasts exhibited a rounded shape (form factors equal to 0.8 and 0.7 for gingival and dermal cells, respectively) at day 1 of culture; they possessed a more elongated appearance (with form factors equal to 0.3 and 0.15 for gingival and dermal cells, respectively) at day 7. Continuous (gingival) and discontinuous (dermal) layers of cells were evidenced at the cortex of lattices. Contractions were associated with a significant reduction of the diameters of collagen fibers. Re-organization of substratum, as analyzed by the "Rose of Directions" technique, was evidenced only at the vicinity of filopodia where fibers ran parallel to these protrusions. Several lysed matrix cavities were observed when fibrin lattices were populated with gingival but not dermal fibroblasts at day 5 of culture. Although cells in fibrin lattices exhibited morphometric parameters comparable with those in collagen lattices, no fibroblast layers could be demonstrated at gel peripheries. Fibrin matrices consisted of an isotropic network of entangled fibrin filaments from the start of culture, and only a slight reduction of the diameters of fibrin fibers could be evidenced in dermal fibroblast-populated lattices. Fibrinolysis at the vicinity of gingival fibroblasts led to an entire re-organization of substratum toward the formation of larger fibers. The differential behavior of gingival vs. dermal fibroblasts inside fibrin but not collagen matrices could therefore partly explain the increased rate of remodeling of gingiva as compared with dermis.

Adult↗

PubMed1998

[Biomaterials in an osteo-articular environment. Report of 129 anatomoclinical cases].

Actually, there is a range of biomaterials which are synthetic or metallic (or the both). They are employed as prosthesis (biostability property) or as bone graft (bioresorbability property). To understand the interactions between cells and such materials, we studied with human bone cellular cultures the cytologycal, immunohistochemical, cytogenetical and ultrastructural aspects of biomaterials in cell cultures. This paper concerns bioceramics like Pyrost, coral, biosorb, oxbone and polymers like polyethylene and silicones. The aim of this work is to evaluate the efficiency of some biomaterials. We found that porosity is primordial to promote biodegradation of bone substitutes. In fact, the biomaterials is integrated and lead to an osteoconduction, an osteoformation and finally an osteoinduction. Our observations show the implant resorption and ossification occurring in the matrix which penetrate it.

Biocompatible Materials↗

PubMed1996

Tissue origin and extracellular matrix control neutral proteinase activity in human fibroblast three-dimensional cultures.

Remodeling of the extracellular matrix by fibroblasts is an important step in the process of wound healing and tissue repair. We compared the behavior of fibroblasts from two different tissues, dermis and gingiva, in three-dimensional lattices made of two different extracellular matrix macromolecules, collagen and fibrin. Cells were grown in monolayer cultures from normal skin or gingiva and seeded in three-dimensional lattices made of either collagen of fibrin. Photonic and scanning electron microscopy did not reveal any morphological differences between the two types of fibroblasts in both sets of lattices. Both types of fibroblasts retracted collagen lattices similarly and caused only a slight degradation of the collagen substratum. By contrast, when seeded in fibrin lattices, gingival fibroblasts completely digested their substratum in less than 8 days, whereas only a slight fibrin degradation was observed with dermal fibroblasts. The ability of gingival but not dermal fibroblasts to express high levels of tissue plasminogen activators (tPA) when cultured in fibrin lattices was assessed on an immunological basis. Also, deprivation of plasminogen-contaminating fibrinogen preparations or use of tPA inhibitors markedly inhibited both fibrinolysis and retraction rates of fibrin lattices by gingival fibroblasts. Casein-zymography confirmed the intense proteolytic activity induced by fibrin in gingival fibroblasts. It was inhibited by aprotinin and phenyl methylsulfonyl fluoride (PMSF), two non-specific inhibitors of serine proteinases, and by epsilon-amino-caproic acid (epsilon ACA), an inhibitor of plasminogen activators. Monolayer cultures exhibited only trace amounts of caseinolytic activity. Our results demonstrate that the expression of proteinases by fibroblasts is dependent not only on their tissue origin but also on the surrounding extracellular matrix. The intense fibrinolytic activity of gingival fibroblasts in fibrin lattices may explain partially the high rate of healing clinically observed in gingiva.

Adult↗

PubMed1993

The influence of tissue pretreatment on the immunohistochemical demonstration of type I and III collagens and tenascin in fetal human tooth germs.

The influence of tissue pretreatment on the PAP immunostaining for type I and III collagens and tenascin was studied in formalin-fixed and paraffin-embedded human tooth germs at the 24th and 25th weeks of fetal life. Three variables were considered: the type of buffer used (PBS or Tris), pepsin digestion and the use of normal serum as a blocking agent prior to immunostaining. All three proteins needed an enzymatic digestion to be intensely revealed. Pepsin promoted, even at low concentrations, an intracellular staining of type I collagen in the secretory odontoblasts and in the pulpal fibroblasts. Normal serum partially blocked unspecific immunoreaction when polyclonal rabbit antibodies were used. The Tris buffer increased the staining intensity of the three macromolecules and revealed an unusual tenascin-like immunoreactivity in the ameloblasts. This study demonstrated that pepsin digestion and the use of normal serum and different buffers may influence the immunoreactivity of ECM proteins.

Buffers↗

PubMed1990

[After pulpectomy--dento-periodontal adaptation].

Recovery after an endodontic treatment can only be assessed clinically, on the basis of symptomatological and radiographic data, for lack of histo-clinical correlations. An anatomicopathological examination of damaged tissues pinpoints the anatomical obstacles and tissular modifications apt to undermine the success of root canal work. The aim of endodontic treatment is to maintain or reinstate an environment conducive to healing through the use of a non-traumatic endodontic technique, followed by functional rehabilitation of the dental organ in its periodontia structures.

Adaptation, Physiological↗