Biomedical subjects

J Amédée

Publications and source records attributed to J Amédée.

At least 19 recordsLinked to original sources

PubMed2006

Functionalization of biomaterials and cell to cell communication.

In the field of osseous substitution, the possibilities being offered to the surgeons prove sometimes difficult to apply in particular in the case of great losses of osseous substance. For these reasons, it is necessary to develop innovative techniques to satisfy the request increasing for substitutes and to see appearing on the market solutions combining availability, perenniality and biosecurity of the implants. The implantation of stem cells in a biomaterial opens a way of development of therapeutic substitute. Moreover, in order to optimize the rehabitation of the biomaterials by the cells and the host tissues, the second approach consists in modifying the surface of materials by the coating or the grafting of adhesive factors in order to stimulate their colonization. At least, one cannot consider a tissue mechanism of repair without a better knowledge of the respective role of the various cell populations implied in the rebuilding of this tissue and their cell to cell communication processes.

Alloys↗

PubMed2005

Effect of several sterilisation techniques on homogeneous self assembled monolayers.

Understanding how cells sense their environment and are able to regulate their metabolism is of great importance for the success of biomaterials implantation. Self assembled monolayers (SAMs) are in use nowadays to model the surface of such materials. They permit the control of different surface parameters (like chemistry, surface energy and topography) enabling to get a greater insight in cells behaviour when interacting with surfaces and thus, in the future, to enhance surface properties of biomaterials. As sterilisation is the compulsory step for in vitro and in vivo assays with living biological materials, it is important to know how SAMs react under sterilisation techniques in use on biomaterials. In this work, the effect of three types of sterilisation techniques: gamma-irradiation, mostly used on biomaterials, dry heat and steam autoclaving, have been investigated on NH2 and CH3 terminated SAMs. Gamma-irradiation destructs drastically the NH2 and partially the CH3 monolayers by producing oxidative compounds (COOH, C=O, C-OH). The main product induced by gamma-irradiation on NH2 monolayers is carboxylic acid, whereas CH3 shows an important increase in the amount of alcoholic groups. This difference in deterioration is assumed to be due to the higher stability of the CH3 monolayer. Steam autoclaving to a lesser extent gives the same results on NH2 monolayers. Dry heat seems to be the most reliable technique, which can be used on such surfaces as it removes physically adsorbed organic contaminants without affecting the integrity of the surface.

Biocompatible Materials↗

PubMed2004

Cyclo-(DfKRG) peptide grafting onto Ti-6Al-4V: physical characterization and interest towards human osteoprogenitor cells adhesion.

In the present paper, specific interest has been devoted to the design of new hybrid materials associating Ti-6Al-4V alloy and osteoprogenitor cells through the grafting of two RGD containing peptides displaying a different conformation (linear RGD and cyclo-DfKRG) onto titanium surface. Biomimetic modification was performed by means of a three-step reaction procedure: silanization with APTES, cross-linking with SMP and finally immobilization of peptides thanks to thiol bonding. The whole process was performed in anhydrous conditions to ensure homogeneous biomolecules layout as well as to guarantee a sufficient amount of biomolecules grafted onto surfaces. The efficiency of this new route for biomimetic modification of titanium surface was demonstrated by measuring the adhesion between 1 and 24 h of osteoprogenitor cells isolated from HBMSC. Benefits of the as-proposed method were related to the high concentration of peptides grafted onto the surface (around 20 pmol/mm(2)) as well as to the capacity of cyclo-DfKRG peptide to interact with integrin receptors. Moreover, High Resolution beta-imager (using [(35)S]-Cys) has exhibited the stability of peptides grafted onto the surface when treated in harsh conditions.

Alloys↗

PubMed2004

Grafting RGD containing peptides onto hydroxyapatite to promote osteoblastic cells adhesion.

Ceramics possess osteoconductive properties but exhibit no intrinsic osteoinductive capacity. Consequently, they are unable to induce new bone formation in extra osseous sites. In order to develop bone substitutes with osteogenic properties, one promising approach consists of creating hybrid materials by associating in vitro biomaterials with osteoprogenitor cells. With this aim, we have developed a novel strategy of biomimetic modification to enhance osseointegration of hydroxyapatite (HA) implants. RGD-containing peptides displaying different conformations (linear GRGDSPC and cyclo-DfKRG) were grafted onto HA surface by means of a three-step reaction procedure: silanisation with APTES, cross-linking with N-succinimidyl-3-maleimidopropionate and finally immobilisation of peptides thanks to thiol bonding. Whole process was performed in anhydrous conditions to ensure the reproducibility of the chemical functionalisation. The three-step reaction procedure was characterised by high resolution X-ray photoelectron spectroscopy. Efficiency of this biomimetic modification was finally demonstrated by measuring the adhesion of osteoprogenitor cells isolated from HBMSC onto HA surface.

Amino Acid Sequence↗

PubMed2004

Heterogeneous cell mechanical properties: an atomic force microscopy study.

Atomic force microscopy (AFM) is a non-invasive microscopy to explore living biological systems like cells in liquid environment. Thus AFM is an appropriate tool to investigate surface chemical modification and its influence on biological systems. In particular, control over biomaterial surface chemistry can result in a regulated cell response. This report investigates the influence of adhesive and non-adhesive surfaces on the cell morphology and the influence of the cytoskeleton structure on the local mechanical properties. In this study, the main work concerns a thorough investigation of the height images obtained with an AFM as therecorded images provide the evolution of the mechanical properties of the cell as function of its local structure. Information on the cell elasticity due to the cytoskeleton organization is deduced when comparing the AFM tip indentation depth versus the distance between the cytoskeleton bundles for the different samples.

Cell Adhesion↗

PubMed2003

Characterization of dynamic cellular adhesion of osteoblasts using atomic force microscopy.

BACKGROUND: Atomic force microscopy (AFM) can be used to visualize the cell morphology in an aqueous environment and in real time. It also allows the investigation of mechanical properties such as cell compliance as a function of cell attachment. This study characterized and evaluated osteoblast adhesion by AFM. METHODS: Human bone marrow stromal cells were cultured on two types of surface to induce weak and strong cellular adhesions. RESULTS: Cells were considered as spreading if they had a flattened and lengthened shape and a cytoskeletal organization in the submembrane cytosolic region. Cell detachment demonstrated different adhesion states between adherent cells to be distinguished. The stability of the cytoskeletal fibers indicated that cells were adherent. The elastic modulus was estimated by two complementary approaches. The values deduced were between 3 x 10(2) and 2 x 10(5) Nm(-2) according to the state of cell adhesion and the approaches used to measure this elastic modulus. CONCLUSIONS: Although the results were qualitative, a relation may be deduced between the elasticity of living cells as demonstrated by cytoskeletal organization and the state of cell adhesion. The technique could be used to determine the adhesion state of an adherent osteoblast observed under AFM.

Bone Marrow Cells↗

PubMed2002

RGD-functionalized spherulites as targeted vectors captured by adherent cultured cells.

Spherulites are multilamellar vesicles consisting of concentric shells that can encapsulate small organic molecules or macromolecules. We investigate the possibility of targeting neutral spherulites to adherent culture cells by functionalizing their surface with RGD-containing ligands. The strength and specificity of association of RGD spherulites with several cell lines (EAhy 926 endothelial cell line, human umbilical vein endothelial cell (HUVEC) and human osteoprogenitor (HOP) primary cells) was studied, and the molecular interaction of RGD spherulites with the EAhy 926 cell surface was investigated. We show that, after binding to cells, spherulites are internalized.

Biological Transport↗

PubMed2002

Function of linear and cyclic RGD-containing peptides in osteoprogenitor cells adhesion process.

Cell adhesion directly influences cell growth, differentiation and migration as well as morphogenesis, integrity and repair. The extracellular matrix (ECM) elaborated by osteoblast cells constitutes a regulator of the cell adhesion process and then of the related phenomenon. These regulatory effects of ECM are mediated through integrins and some of them are able to bind RGD sequences. The aim of this study was to determine the role of the sequence and the structure of RGD-containing peptides (linear and cyclic) as well as their role in the cell adhesion process. Cell adhesion assays onto ECM proteins coated surfaces were performed using a range of linear and cyclic RGD-containing peptides. We showed a different human osteoprogenitor cell adhesion according to the coating for ECM proteins and for RGD-peptides. Inhibition assays using peptides showed different responses depending on the coated protein. Depending on the amino-acid sequence and the structure of the peptides (cyclic linear), we observed 100% inhibition of cell adhesion onto vitronectin. These results suggest the importance of sequence, structure and conformation of the peptide, which may play a crucial function in the ligand/receptor interaction and/or in the stability of the interaction.

Adult↗

PubMed2002

Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication.

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Our aim was to investigate the interaction between human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC). Cell differentiation analysis performed with different cell culture models revealed that alkaline phosphatase activity and type I collagen synthesis were increased only by the direct contact of HUVEC with HBMSC. This "juxtacrine signaling" could involve a number of different heterotypic connexions that require adhesion molecules or gap junctions. A dye coupling assay with Lucifer yellow demonstrated a functional coupling between HUVEC and HBMSC. Immunocytochemistry revealed that connexin43 (Cx43), a specific gap junction protein, is expressed not only in HBMSC but also in the endothelial cell network and that these two cell types can communicate via a gap junctional channel constituted at least by Cx43. Moreover, functional inhibition of the gap junction by 18alpha-glycyrrhetinic acid treatment or inhibition of Cx43 synthesis with oligodeoxyribonucleotide antisense decreased the effect of HUVEC cocultures on HBMSC differentiation. This stimulation could be mediated by the intercellular diffusion of signaling molecules that permeate the junctional channel.

Bone Marrow Cells↗

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

[Engineering of osseous cells and bioartificial tissues].

The association of osteogenic stem cells to a synthetic carrier makes possible the elaboration of bioartificial tissue. Numerous phosphocalcic ceramics does not trigger a foreign body reaction when implanted in bone tissue and thus, a number of materials are available osteogenic stem cell carriers to replace the bone tissue. Several methods can be used to harvest these cells. Their multiplication in vitro can lead to the appearance of anomalies of their metabolism or their karyotype. The culture method also seems to have a major influence on their appearance. The presence of these anomalies could explain the variability of results in terms of bone extracellular matrix synthesis after cell reimplantation. The surgical technique used for the implantation is also of influence. A method suppressing the in vitro period has been developed to avoid any cell metabolism modification. This method allows for a very reproducible bone synthesis in ectopic site. The availability of human embryonic stem cells could help to develop cell graft techniques for bone reconstruction.

Biocompatible Materials↗

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↗

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↗

PubMed1997

Opposing actions of BMP3 and TGF beta 1 in human bone marrow stromal cell growth and differentiation.

BMP3 and TGF beta 1 were found to induce opposite effects on human bone marrow stromal cells both in cell proliferation and cell differentiation and on calcium deposition onto the extracellular matrix. Moreover, BMP3 may exert, in part, the inhibitory effect of TGF beta 1 through decreasing the affinity of TGF-beta for its receptors which are now identified in these human bone cells.

Activin Receptors, Type I↗

PubMed1996

Ability of various inserts to promote endothelium cell culture for the establishment of coculture models.

To select an insert suitable for human umbilical vein endothelial cell (HUVEC) culture, we compared several available inserts of 0.2 to 0.45 micron porosity: Cellagen (ICN), Transwell-COL (Costar), Millicell-HA and CM (Millipore), Anopore (Nunc), Cyclopore (Falcon) in comparison with a control surface (Thermanox). The requirements were: (i) to promote attachment, adhesion and proliferation of HUVEC (judged by [3H]thymidine incorporation into DNA at days 1, 3, 7); (ii) to allow HUVEC visualization by inverted, fluorescence microscopy for uptake of DiI-Ac-LDL and scanning electron microscopy, performed at day 9 after seeding. Because Transwell and Cellagen are collagen precoated and CM has to be coated for cell culture, we performed collagen coating (types I + III or IV) for non-pretreated inserts for the purpose of comparison. Our preferences comprise Transwell-COL, Cyclopore not coated or coated (whatever the collagen type), and Cellagen. However, on a quality/price ratio criterion, Cyclopore, even uncoated, is the insert of choice. The HA, CM and Anopore inserts, even coated, do not allow HUVEC growth but do not alter positive uptake of acetylated LDL.

Cells, Cultured↗

PubMed1995

The resorption of bone-implanted corals varies with porosity but also with the host reaction.

Three different exoskeletons of coral species Porites asteroides (P), Montastrea annularis (M), and Dichocoenia stokesi (D) were implanted for 2-20 weeks in rabbits. At 2, 4, 8, or 20 weeks, the exoskeletons presented variations in their resorptions depending on the species. To understand the variations in the decreasing speed of the implants despite their similar chemical composition, a study of the surface and architecture of the coral was carried out using scanning electronic microscopy, porosity was evaluated, and growth and differentiation of osteogenic cells cultured in vitro were observed for more than 1 month. At the cellular level, the surface of the implants was identical. Three-dimensional structures of the implants were variable, but the porosity values [P = 42.7%, M = 40.7%, and D = 17.4%] could not completely account for the differences in the resorbing process of the species. Standard histologic studies performed at 2, 4, 8, and 20 weeks after implantation produced the same pattern with P or M, showing aspects of rapid resorption; however, with D there were images resembling those of a foreign-body reaction. It seems that when resorption is not quick enough, a foreign body reaction develops which further slows down the process. This work focuses on the importance of porosity when using coral as bone substitute.

Animals↗

PubMed1994

Osteogenin (bone morphogenic protein 3) inhibits proliferation and stimulates differentiation of osteoprogenitors in human bone marrow.

Treatment of human bone marrow osteoprogenitors with osteogenin (BMP-3; at 1, 2.5 and 10 ng/ml) caused dose- and time-dependent inhibition of DNA synthesis and cell proliferation. Simultaneously, osteogenin stimulated type I collagen synthesis and cAMP production. Addition of osteogenin to the cell culture increased intracellular alkaline phosphatase activity and osteocalcin synthesis, with maximal stimulation at 2.5 ng/ml. Simultaneous addition of 2.5 ng/ml osteogenin and 1,25 dihydroxy vitamin D3 (10(-8) M) enhanced the stimulation observed in osteocalcin synthesis. The experiments reported here demonstrate the significant "in vitro" influence of osteogenin in the stimulation of osteogenic phenotype in osteoprogenitor cells which have been isolated from human bone marrow and cloned. These results support a reciprocal relationship between cell growth inhibition and expression of osteoblast differentiation.

Adult↗