F. Rouais

Human bone marrow stromal cells express an osteoblastic phenotype in culture

SummaryThis study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two “clonings” and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [3H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D3 by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of β-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by “budding” structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with45Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.

Cellulose phosphates as biomaterials. Mineralization of chemically modified regenerated cellulose hydrogels

Femoral implantation of regenerated cellulose hydrogels revealed their biocompatible and osteoconductive properties, but a complete osseointegration could not be observed. Phosphorylation was therefore envisaged as the means to enhance cellulose bioactivity. Once implanted, phosphorylated cellulose could promote the formation of calcium phosphates, having therefore closer resemblance to bone functionality and assuring a satisfactory bonding at the interface between hard tissue and biomaterial. In the present work, regenerated cellulose hydrogels were surface modified via phosphorylation. Phosphorylated materials, having varying degrees of substitution, were soaked in a Simulated Body Fluid (SBF) solution in order to investigate their ability to induce the formation of a calcium phosphate layer. Mineralization was assessed by Scanning Electron Microscopy (SEM), Energy Dispersive Spectroscopy (EDS) and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. It was demonstrated that the calcium salt of cellulose phosphates mineralized at a higher extent than materials only phosphorylated. The degree of phosphorylation influenced the extent of surface mineralization. Moderate degrees of surface phosphorylation promoted the highest extent of mineralization. This was attributed to inadequate functionality of the surface in terms of density of PO4 groups and overall surface charge, in the case of low and high phosphate contents.

TiN coating : surface characterization and haemocompatibility

The left ventricular assist device under consideration is based on the principle of the Maillard-Wankel rotary pump. The construction materials must meet stringent requirements. Titanium nitride was chosen for its surface properties and graphite for its bulk characteristics. The purpose of this study was to characterize the chemical vapour deposition titanium nitride coating via morphology, roughness, crystallinity, chemical composition, to report and discuss the results of in vitro haemocompatibility tests (protein adsorption, platelet retention, haemolysis) and to discuss physico-chemical and biological results. This chemical vapour deposition titanium nitride coating is well tolerated by the blood despite its surface irregularities, and appears as a good candidate material after improvements.

Preliminary report on a new composite material made of calcium phosphate, elastin peptides and collagens

With a view to elaborating a bioactive bone substitute, the association of an artificial extracellular matrix, basically constituted of elastin-solubilized peptides (ESP) and type I+III collagens, with different types of calcium phosphates, was investigated. This paper describes the selective adsorption of ESP on some calcium phosphate samples, and the further association of the adsorbed peptides with type I+III collagens. A preliminary study of the material cytotoxicity was carried out, investigating the behaviour of human osteoblast cells in contact with the yielded composite material.

Biocompatibility testing of a bovine hydroxy—apatite ceramic material with the use of osteo-progenitor cells isolated from human bone marrow

Bioactive materials such as hydroxyapatite ceramics are known to show a stable interfacial bond with tissues. The objective of this study was to investigate the behaviour of human bone differentiated cells isolated from bone marrow on the surface of a hydroxyapatite ceramic obtained from bovine spongiosa. Scanning electron microscopy and DNA synthesis analysis assessed by [3H]-thymidine incorporation showed cell colonization of the whole material. Immunological studies using monoclonal antibodies to osteocalcin and osteonectin and cytochemical analysis of alkaline phosphatase activity indicated that these cells did not lose their osteoblastic phenotype after 28 days of culture. Furthermore, this study demonstrates the in vitro interface between the material and human cells, which is reporduced in vivo.

In vitro evaluation of an epoxy resin's cytocompatibility using cell lines and human differentiated cells

The cytocompatibility of a polyepoxy resin (Elf Aquitaine) has been studied using both cell lines and human differentiated cell cultures. The human models were gingival fibroblasts and bone osteoblasts, while the cell lines were Hela cells and 3T3 Balb/c cells. Basal cytocompatibility was assessed by estimation of the cell proliferation, total cell protein content, cell membrane sub-lysis, and cell attachment and spreading. Specific cytocompatibility concerning human osteoblasts, from both alveolar and trabecular bone, was determined by measuring the intracellular alkaline phosphatase activity. Resin colonization by the cells was studied by both TEM and SEM. The behaviour of the two cell lines reveals a significant level of discrepancy, whereas the behaviour of human cells, whatever the model, is comparable; however, osteoblasts look more sensitive. Moreover, the results show that this epoxy resin exhibits a moderate cytocompatibility which could be the result of the cytotoxicity of early released products, associated with the considerable surface roughness.

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 μm 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.

Physico-chemistry and cytotoxicity of ceramics: Part I – Characterization of ceramic powders

The morphology of Al2O3, ZrO2/Y2O3, AIN, B4C, BN, SiC, Si3N4, TiB2, TiC, TiN ceramic, graphite and diamond powders has been studied by scanning electron microscopy (SEM) and the specific area of each powder was determined with the BET method. X-ray diffraction (XRD) investigations have been carried out in order to evaluate the crystallinity and determine the constitutive phases. The chemical composition was assessed by classical chemical analyses and by X-ray microprobe; some powders were studied by the laser micro-Raman technique. Correlations have been established between all these results.

Endothelial cell compatibility testing of various prosthetic surfaces

Numerous methods are proposed to reduce the surface thrombogenicity of vascular prostheses, among them endothelialization of the lumen which has had clinical application since 1985. One of the problems is to collect enough cells to rapidly obtain a complete monolayer at the time of implantation. Thus, material improvements are necessary to enhance cell adhesion and spreading. A collaboration with the Bakoulev Institute (Moscow) gave us the opportunity to study the cytocompatibility of carbon coated materials (PAN and Vitlan®), polyester coated with albumin and/or synthetic polysaccharide. Studies were carried out with cultured human umbilical vein endothelial cells (HUVEC). Two steps are distinguished: indirect tests (medium added with materials extracts), then direct tests (cells directly seeded onto materials). Neither PAN, Vitlan® nor polyesters extracts have provoked a toxic effect on HUVEC. Concerning attachment on materials, a maximum of 60% of seeded cells is reached. Cells could proliferate and confluency is obtained between days 5 and 10 for the best materials. SEM corroborated these results. On polysaccharide-coated polyester (M. 11), HUVEC produced significant levels of vWF after thrombin stimulation (ELISA assay): vWF was functional (ristocetin cofactor activity). In conclusion, PAN and M. 11 gave encouraging results and further studies remain to be investigated.

A canine ex vivo shunt for isotopic hemocompatibility evaluation of a NHLBI DTB primary reference material and of a IUPAC reference material.

Factors determining the thrombogenic response to particular artificial surfaces were investigated ex vivo in a canine shunt model. Methods using radioisotopic tracers made it possible to dynamically monitor the deposition of labelled blood cells and proteins on a NHLBI.DTB primary reference material polydimethylsiloxane (PRM.PDMS) and on a IUPAC reference material polyvinyl chloride (IUPAC.PVC). On the one hand, leukocyte affinity tau s(leu) (number of deposited leukocytes mm-2s-1) was not significantly different between IUPAC.PVC (tau s(leu) = 1.2-2.5) and PRM.PDMS (tau s(leu) = 1.5-3.4) and the fibrinogen adsorption rate varied from 33 to 48.10(-5) micrograms mm-2s-1 for both these materials. On the other hand, platelet affinity tau s(plat) (number of deposited platelets mm-2s-1) was significantly different (p < 0.05) for IUPAC.PVC and PRM.PDMS (tau s(plat)PVC = 683 +/- 200 > tau s(plat)PDMS = 327 +/- 80). Scanning electron micrographs of adherent platelets, red cells and leukocytes after blood contact ex vivo were performed after each experiment. This preliminary work contributes not only to quantify the adsorption of different radiotracers, but also to evaluate the superficial distribution of the labelled biological species on the inner surface of the tested biomaterials.