Biomedical subjects

R Bareille

Publications and source records attributed to R Bareille.

At least 37 records · Page 2Linked to original sources

PubMed2001

Study of a (trimethylenecarbonate-co-epsilon-caprolactone) polymer--part 2: in vitro cytocompatibility analysis and in vivo ED1 cell response of a new nerve guide.

Future surgical strategies to restore neurological function in peripheral nerve loss may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. Random copolymers of trimethylene carbonate and epsilon caprolactone (P(epsilonCL-TMC), 50: 50) have been synthesized by ring opening polymerization using rare earth alkoxides as initiator. Their potential use as nerve guide repairs has been assessed through indirect and direct in vitro biocompatibility tests and in vivo soft tissue response to EDI subclass macrophages. In vitro, we exposed monolayers of human skin fibroblasts and an established continuous cell line (Hela) to liquid extracts (either pure or diluted in the culture medium) of epsilonCL-TMC copolymer including positive (phenol) and negative controls. Then, colorimetric assays (Neutral red and MTT) were performed. The extracts of epsilonCL-TMC induced no significant cytotoxic effect. We also exposed in vitro Schwann cells to pieces of P(epsilonCL-TMC) and P(LA-GA) copolymers. We evaluated cell attachment at 1 and 3 h by measuring the activity of the lysosomal enzyme (N-acetyl-beta-hexosaminidase) and cell proliferation at 1, 3, 6 and 9 days by measuring the cell metabolic activity (MTT assay). Values for attachment slightly decreased between 1 and 3 h but were significantly higher than on agars (negative control). Cells plated on epsilonCL-TMC showed a rate of proliferation comparable with that of normalized controls and higher than on PGA-PLA at day 9. Finally, we evaluated in vivo the soft tissue response after implantation of cylindrical tubes of P(epsilonCL-TMC) and P(LA-GA) copolymers with an immunohistochemistry staining procedure for the newly recruited ED1 macrophages. An image analysis system automatically measured the optical density of labelled positive ED1 cells at 9, 21 and 60 days after implantation. epsilonCL-TMC copolymer showed a mild soft tissue reaction with no adverse chronic inflammatory reaction. These data allowed us to consider this conduit as a potential effective substitute in nerve repair. El sevier Science Ltd. All rights reserved.

Animals↗

PubMed2000

Effect of human endothelial cells on human bone marrow stromal cell phenotype: role of VEGF?

Angiogenesis is a tightly regulated process involved in growth, repair, and bone remodeling. Several studies have shown that there is a reciprocal regulation and functional relationship between endothelial cells and osteoblast-like cells during osteogenesis, where systemic hormones and paracrine growth factors play an active role. Angiogenesis is induced by a variety of growth factors; among them vascular endothelial growth factor (VEGF) may be an important mediator for the angiogenic process involved in bone physiology. We studied the VEGF effect on osteoblast progenitor cells (Human Bone Marrow Stromal Cells: HBMSE) cultured alone or associated with endothelial cells (Human Umbilical Vein Endothelial Cells: HUVEC) in different co-culture models (co-culture with or without direct contact, conditioned medium), to determine the influence of VEGF on these cells and on their relationship. In agreement with other studies, we show that HBMSC express and synthesize VEGF, HUVEC conditioned medium has a proliferative effect on them, and early osteoblastic marker (Alkaline phosphatase activity) levels increase when these cells are co-cultured with HUVEC only in direct contact. However, unlike previous studies, we did not find that VEGF increased these processes. These results suggest that the intercommunication between endothelial cells and osteoblastic-like cells requires not only diffusible factors, but also involving cell membrane proteins.

Alkaline Phosphatase↗

PubMed2000

Various evaluation techniques of newly formed bone in porous hydroxyapatite loaded with human bone marrow cells implanted in an extra-osseous site.

The purpose of this work was to develop qualitative methods for in situ analysis of bone formation in an osteoconductive hydroxyapatite matrix (ENDOBON), loaded with human bone marrow cells (HBMSC) implanted subcutaneously in athymic mice. Samples were taken before implantation (T0), 1, 2, 4 and 6 weeks after implantation. Bone-biomaterial interaction were investigated on undecalcified sections by histological, cytochemical, immunological and molecular biology methodologies. Histological observations were performed in order to observe inflammatory cells, vessels, newly formed bone, woven and lamellar bone. Enzymohistochemistry was carried out to detect positive tartrate resistant acid phosphatase activity (TRAP+). Immunohistochemistry using antibodies against type I collagen and osteocalcin permitted us to characterize the content of the matrix elaborated within the implant. Moreover, in situ hybridization was carried out to discriminate, the implanted human cells from the murine cells, and to evaluate the function of these human cells in osteogenesis. Results demonstrated an early formation of lamellar bone only in the pores of the studied HAP loaded with HBMSC. This bone contained a matrix showing positive reaction for type I collagen and osteocalcin. In situ hybridization identified some of these cells as human cells. At 6 weeks, examination of histological results showed persistance of lamellar bone in the implants. We only found TRAP+ activity in the materials loaded with human bone marrow cells. Molecular hybridization no longer revealed positive cells for the human DNA probe. All these results indicate that the various evaluation techniques performed on undecalcified sections, permit us to evaluate the response of human bone marrow cells in HAP implanted into mice.

Animals↗

PubMed2000

Cultured differentiated human urothelial cells in the biomaterials field.

To review the use of normal cultured differentiated human urothelial cells in the biomaterials field, we checked the literature for human urothelial cells in culture (HUC) both for their use in biocompatibility assessment and as bioartificial devices. The in vitro culture of differentiated human urothelium is now a simple and reliable procedure. These techniques provide new tools for biocompatibility assessment of urinary biomaterials, because for the rational design of a testing procedure, it is preferable that the particular cell culture models selected should be closely related to the end-use application. The emerging use of HUC culture should lead to the development of bioartificial tissue for urinary tract reconstruction. Tissue engineering techniques require urothelial cells and cell delivery matrices. The cytocompatibility of novel artificial delivery matrices should be assessed in vitro before implantation using cultured HUC to find the best material available.

Biocompatible Materials↗

PubMed2000

Cytotoxicity assessment of latex urinary catheters on cultured human urothelial cells.

PURPOSE: To study the toxicity of latex urinary catheters on cultured human urothelial cells (HUC). MATERIALS AND METHODS: We exposed monolayers of HUC (well characterized for their proliferation, qualitative evaluation and quantitative measurement of cytokeratins) to either pure or diluted liquid latex extracts, obtained under standard conditions or by direct contact with materials. RESULTS: The latex urinary catheter appears to be highly toxic since cell viability and metabolic activity were about 10% of those of negative controls for original extracts. Concerning direct contact, latex reduced cell viability, metabolic activity and cell proliferation of HUC on days 1, 3 and 8. CONCLUSION: The high toxicity of latex on HUC is confirmed for extracts and direct contact. Therefore, it should no longer be used for urinary catheters.

Cells, Cultured↗

PubMed1999

Effect of 1,25(OH)2D3 on bone morphogenetic protein-3 mRNA expression.

Bone morphogenetic proteins (BMPs) are members to the transforming growth factor-beta superfamily. They induce ectopic bone formation in rat and are pleiotropic initiators of inducible osteogenic precursor cells. A lot of reports have studied the presence of BMPs and their effects on bone marker expression in many different cell lines, however none describe the regulation of BMP3 by different factors and expression conditions. When a human bone marrow stromal cell (HBMSC) culture was treated simultaneously with 1,25(OH)2D3 (10(-8) M) and BMP3 (2.5 ng/ml), the total osteocalcin content in the cell layer and in the culture medium was higher than when the culture was treated with either factor alone (162%). To elucidate this synergistic activity, Northern blot analysis was done to study the effect of 1,25(OH)2D3 on BMP3 mRNA expression. Several human cell lines (MNNG, U-2OS, MG-63, KHOS, TE85, HOS) and HBMSC were treated by 1,25(OH)2D3 (10(-8) M for 24 h). Purified mRNA from treated and untreated cells were denatured using glyoxal and dimethylsulfoxide, and were fractionated on a 1% agarose gel. After electrophoresis, RNA were blotted onto a nylon membrane and incubated with 32P-labeled BMP3 and GAPDH riboprobes. Northern blot analysis revealed that, the BMP3 mRNA level was increased in a few cell lines (MG-63, HBMSC, HOS) after the addition of 1,25(OH)2D3 when compared to the untreated cells (127%+/-1; 130.5%+/-19.5; 207%+/-14). An higher stimulation was observed in HBMSC primary culture when compared to differentiated HBMSC. In view of these results, we now investigate the following hypothesis: does the BMP3 promoter exhibit the vitamin D receptor response like the osteocalcin gene?

Bone Morphogenetic Protein 3↗

PubMed1999

In vitro cytocompatibility of radio-opacifiers used in ureteral endoprosthesis.

Ureteral endoprostheses are urinary catheters made of polymeric biomaterials made radio-opaque through the addition of X-ray absorbing additives such as barium, bismuth, tantale or tungsten. The aim of this work was to study the in vitro toxicity of solutions of these radio-opacifiers using two cell culture models. Primary-cultures of human urothelial cells (HUC) arising from normal adult urinary tract and permanent urothelial cell line were used. Solutions at different dilutions were placed into the wells containing monolayers of confluent cells. After 24 h incubation period, the solutions were removed and cell viability and cell metabolic activity tests were performed (Neutral Red assay and MTT assay). At a concentration lower than 1 mg l(-1) the different radio-opacifiers used showed no toxicity. From 1 to 3 mg l(-1) one can note a significant dose-dependent decrease of cell metabolic activity of solely HUC for barium chloride. At 3 mg l(-1) one can note a significant deleterious effect on HUC metabolic activity, with bismuth and tantale. For tungsten, there is no deleterious effect, but on the contrary a significant increase in HUC metabolic activity at a 0.5 mg l(-1) concentration. None of the solutions did provoke alterations in HUC viability for concentrations less than 3 mg l(-1). Interestingly, for permanent cell line one can note a solely significant decrease of cell viability at 3 mg l(-1) for tantale. All the other tested salts on permanent cell line were not significantly different from controls for cell viability as well as cell metabolic activity. HUC culture model may be of relevance for the screening of radio-opacifiers intended for ureteral endoprostheses.

Adult↗

PubMed1999

In vitro assessment of endothelial cell adhesion mechanism on vascular patches.

Endothelial cell (EC) seeding of small caliber vascular grafts prior to their implantation has proved to significantly improve long-term patency in humans. We have previously demonstrated that a monolayer of EC could be obtained on type I collagen-coated knitted ultrathin polyster grafts (InterVascular, La Ciotat, France). Thus, the aim of the present work was to understand the nature of cell adhesion mechanisms involved in the cell /biomaterial interface, using HemaCarotid (InterVascular) patches made of type I collagen-coated knitted ultrathin polyster (type I collagen is used to coat patches to attain low permeability). By means of quantitative attachment tests, adhesion blocking assays, RT-PCR for the expression of beta1 integrin mRNA, indirect immunofluorescence with antivinculin antibody, we were able to show that EC are able to adhere to such surfaces by the means (non-unique) of cell surface receptors of the beta1 integrin group. However, the latter are probably downregulated at the cell/biomaterial interface.

Journal Article↗

PubMed1999

Clinical performance of vascular grafts lined with endothelial cells.

The replacement of arteries with purely synthetic vascular prostheses often leads to the failure of such reconstructions when small-diameter or low-flow locations are concerned, due in part to the thrombogenicity of the internal graft surface. In order to improve long-term patency of these grafts, the concept of endothelial cell seeding has been suggested because this metabolically active endothelial surface plays major roles in preventing in vivo blood thrombosis and because vascular grafts placed in humans do not spontaneously form an endothelial monolayer whereas they do in animal models. The composite structure resulting from the combination of biologically active cells to prosthetic materials thus creates more biocompatible vascular substitutes. To achieve endothelialization of synthetic vascular grafts, previous efforts aimed at "one-stage" procedure (adding autologous endothelial cells to the graft at the time of implantation) in the 1980's seemed clinically feasible but results of reported clinical trials were controversial and mostly disappointing. An alternative method is an in vitro complete and preformed endothelial lining at the time of implantation: the "two-stage" procedure which implies harvest and culture of autologous endothelial cells. Up to date, the latter approach demonstrated its superiority in terms of significantly increased patency of the grafts that underwent endothelialization eight years earlier.

Blood Vessel Prosthesis↗

PubMed1999

A novel potential application for 99mTc-HMPAO: endothelial cell labeling for in vitro investigation of cell-biomaterial interactions.

UNLABELLED: Good adherence of endothelial cells (ECs) seeded on vascular prostheses and cell retention under flow conditions are important factors to consider in the use of functionalized prostheses in vascular surgery. Because 111In-oxine radiolabeling presents disadvantages, we wondered whether, because of its well-known physical properties, 99mTc-hexamethyl propyleneamine oxime (HMPAO or exametazime) could be used. METHODS: The cytotoxicity of unlabeled HMPAO and 99mTc-HMPAO at increasing concentrations and activities was tested on monolayers of the EC line EA-hy-926. The influence of temperature and time on tracer incorporation into cells was also tested. The optimal labeling conditions were applied to evaluate the retention of ECs seeded on polyester grafts under flow conditions by gamma camera detection. RESULTS: The activity of 10 MBq/10(6) cells corresponding to 4.5 microg/10(6) cells of unlabeled HMPAO, applied for 3 h at 37 degrees C (cellular uptake = 18%), was the best compromise between the maintenance of cell viability and metabolic activity and efficient detection by the gamma camera. Spontaneous leakage was observed and analyzed by high-performance liquid chromatography. A cell loss of 13% after 180-min exposure to shear stress was obtained. CONCLUSION: Our data thus indicate the feasibility of using such a radiolabeling technique to investigate EC-biomaterial interactions.

Blood Vessel Prosthesis↗

PubMed1998

Synthesis of calbindin-D28K during mineralization in human bone marrow stromal cells.

1alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is known to modulate Ca2+ metabolism in several cell types. Vitamin-D-dependent calcium binding proteins such as calbindin-D28K (28 kDa calcium binding proteins) have been shown to be regulated by 1,25(OH)2D3 but the mechanisms controlling calbindin synthesis are still poorly understood in human osteoblast cell culture models. The human bone marrow stromal cells (HBMSC) described in this paper developed a calcified matrix, expressed osteocalcin (OC), osteopontin (OP) and responded to 1,25(OH)2D3. The expression of vitamin D receptor mRNA was demonstrated by reverse transcription-PCR. Calbindin-D28K protein was identified only in cells arising from the sixth subculture, which exhibited a calcified matrix and all of the osteoblastic markers, e.g. OC and OP. It was demonstrated by dot-immunodetection using immunological probes, and by in situ hybridization using labelled cDNA probes. Moreover, vitamin D3 enhanced calbindin-D28K synthesis as well as OC synthesis and alkaline phosphatase activity. Uptake of 45Ca induced into the matrix by 1,25(OH)2D3 supports the hypothesis that the calcium-enriched matrix could trap calbindin-D proteins. In conclusion, the studies in vitro described in the present paper indicate, for the first time, a possible role of calbindin-D28K in mineralized matrix formation in HBMSC.

Alkaline Phosphatase↗

PubMed1998

First use of cultured human urothelial cells for biocompatibility assessment: application to urinary catheters.

For several years, studies performed to estimate in vitro biocompatibility of urinary catheters have been carried out using permanent cell lines. But for a rational design of the testing procedure, the cell culture model should relate to the material application. This work presents the results of a probe study designed to obtain an in vitro model of normal human urothelial cells (HUC) and to test the relevance of this system in cytocompatibility experiments of urinary catheters currently used.A comparison is made with continuous cell lines, the use of which is recommended by normalization bodies. We exposed monolayers of HUC (well characterized for their proliferation, qualitative evaluation, and quantitative measurement of cytokeratins) and two continuous human cell lines to liquid extracts (either pure or diluted in the culture medium) of nine available catheters, including positive (latex) and negative controls, for a 24 h incubation. Then colorimetric assays (Neutral Red and MTT) were performed. The extracts of two polyurethanes provoked a significant toxic effect on HUC only, suggesting differences in sensitivity between the models used. This effect could be due to the presence of a great amount of barium (used as a radioopacifier) in extracts, as highlighted by results of absorption emission spectroscopy. A culture model of HUC may be of relevance for the screening of materials intended for urological practice.

Biocompatible Materials↗

PubMed1998

Evaluation of proliferation and protein expression of human bone marrow cells cultured on coral crystallized in the aragonite of calcite form.

The two crystalline forms of CaCO3, aragonite (from natural coral) and calcite (from natural limestone), have been used with success as bone graft substitutes. However, natural coral transformed into calcite by heating has never been tested. The objective of this work was to study the proliferation and alkaline phosphatase, osteonectin, and osteocalcin expression of human bone marrow cells cultured on CaCO3 crystallized both in the aragonite form (natural coral) and in the calcite form (natural coral modified by heating). The methods used to characterize calcite obtained from the coral were volumic porosimetry, scanning electron microscopy (SEM) and X-ray diffraction. Cell colonization of the material was assessed by SEM performed on days 1, 7, 20, and 30 and [3H]thymidine incorporation was performed on days 3, 7, 12, 18, 25, and 32. Phenotypic expression was assessed by using in situ cytochemistry (alkaline phosphatase), immunocytochemistry (osteonectin and osteocalcin), and hybridization (osteocalcin, beta-actin, and alkaline phosphatase mRNA). Results showed the transformation of aragonite into calcite after heating, the conservation of macroporosity, and a modification of the surface. Calcite appeared to have a smoother and more uniform surface than aragonite crystals. As for [3H]thymidine there was an increase incorporation from days 3 to 18, a stabilization from days 18 to 25, and a decrease from days 25 to 32. After 20 days of culture, immunological studies using monoclonal antibodies to osteocalcin, osteonectin, cytochemical analysis of alkaline phosphatase activity, and in situ hybridization using osteocalcin, beta-actin, and alkaline phosphatase cDNA indicated that the cells had not lost their osteoblastic phenotype. These experiments demonstrate that coral crystallized in the aragonite or calcite form present a similar degree of specific cytocompatibility.

Animals↗

PubMed1998

Endothelial cells lining polyester fabric express pro-coagulant phenotype in vitro.

The paper deals with the in vitro assessment of endothelial cell (EC) phenotype covering an albumin- and chitosan-coated polyester fabric and shows that resting ECs express a pro-coagulant phenotype by releasing a high von Willebrand factor level and expressing low thrombomodulin surface activity, despite maintaining an adequate response to stimulating agents.

Biocompatible Materials↗

PubMed1998

An in vitro biocompatibility evaluation of double-J stents.

OBJECTIVES: For several years, studies performed to estimate in vitro biocompatibility of urinary catheters have been carried out using permanent cell lines. However, for a rational design of the testing procedure, the cell culture model should depend on the material application. We assess the biocompatibility of 13 double-J stents using an in vitro model of normal human urothelial cells (HUC). This article aims to mimic in vitro, on HUC monolayers, the close contact existing in vivo between the urothelium and double-J stents and to evaluate the subsequent effect on these cells. METHODS: Fragments of each stent were deposited into the wells containing confluent HUC, with close contact maintained between the material and the cells. The same procedure with either no material or fragments of latex catheter was undertaken to provide the negative and positive controls, respectively. The contact was maintained for 1, 3, and 8 days. At the end of the incubation period, fragments of stent were removed and cell activity tests were performed (neutral red assay, MTT assay, and cell proliferation). RESULTS: One of the silicone stents is significantly deleterious on HUC as determined by three tests after 8 days of contact. For two copolymers, a tendency to increase cell proliferation was noted. Concerning polyurethanes, we observed significant decreases in HUC viability and cell metabolic activity for five stents after 8 days of contact. All seven polyurethane stents significantly inhibited cell proliferation. CONCLUSIONS: The HUC culture model may be of relevance for the screening of materials intended for use as double-J stents.

Biocompatible Materials↗

PubMed1998

Cellular biocompatibility and resistance to compression of macroporous beta-tricalcium phosphate ceramics.

The main problem for macroporous structures used as bone substitutes is their lower resistances when compared to that of cancellous bone. The present investigation aimed to improve the strength of ceramics with 65% porosities based on beta-TCP. The initial mixtures were rendered plastic by addition of non-ionic carbohydrate binders. Macropores were created using substances which were eliminated by heat. Mechanical tests indicated that the resistance of the ceramics depended more on the quantity than the nature of the binders. Porosity measurements were done with a mercury porosimeter, and cellular biocompatibility was evaluated by performing cellular attachment tests and observing the proliferation of differentiated cells.

Biocompatible Materials↗

PubMed1998

"In vitro" dissolution of coral in peritoneal or fibroblast cell cultures.

Previous studies have shown that in vivo coral resorption involves a biphasic process: First, the edges of the coral block become powdery, then extracellular fluid and phagocytosis contribute to the dissolution of the crystals. The authors examined some types of cells that could be involved in phagocytosis, particularly the ability of both dermal fibroblasts and mouse-resident peritoneal cells to phagocytose and dissolve coral powder "in vitro". Radioactive coral was incubated for 24, 48, or 72 hrs with cells in the presence or absence of cytochalasin B (a phagocytic inhibitor) or chloroquine (a lysosomotropic agent). Furthermore, to specify the role of crystal cell contacts in the solubilization process, they incubated radioactive coral in conditioned media (obtained from two-day human fibroblastic or macrophagic cell culture in the presence or absence of non-radioactive coral) or at a distance from the cells using culture inserts. Measurements of the radioactivity in the different supernatants were performed. Transmission electron microscopy was carried out on the cells cultivated in the presence or absence of radioactive coral. The data suggest that both fibroblasts and macrophages dissolve the coral, and that the intracellular degradation in phagolysosomes is one of the mechanisms explaining coral powder dissolution.

Animals↗

PubMed1998

[The biocompatibility of catheters and stents used in urology].

Biocompatibility can be interpreted as the optimal combination of a series of interactions occurring at the material-tissue interface as soon as these two systems are in contact. It is a multifactorial interface property which integrates all of the phenomena involved in a biological environment i.e. absence of toxicity of the material for the body and absence of degradation of the material by the body. Biocompatibility can be evaluated in a normative context by using in vivo techniques in animals or in vitro techniques using cell cultures allowing the study of cytotoxicity (related to a concept of safety) and cytocompatibility (related to biological acceptability) of a material. Because of their intimate contact with the urothelium throughout implantation, the biocompatibility of catheters and stents constitutes a major requirement. This review presents the current data reported in the literature concerning the evaluation of the biocompatibility of materials used in urology. The main problems encountered are alterations of the urothelium, such as erosions or, on the contrary, mucosal hyperplasia, and the existence of incrustations developing on these materials.

Animals↗