Marie-Françoise Harmand

Cytocompatibility of two coating materials, amorphous alumina and silicon carbide, using human differentiated cell cultures

The cytocompatibility of two coating materials, amorphous alumina and silicon carbide deposited by radio-frequency sputtering, was studied using alveolar bone osteoblasts and gingival fibroblasts from human healthy tissues. Cytocompatibility was assessed at the level of both the basic (attachment, proliferation and cell protein content) and the specific features (intracellular alkaline phosphatase activity and the cytoskeleton) of the cells in direct contact with the coating. Titanium was used as the reference material. The results showed that both silicon carbide and amorphous alumina are cytocompatible for human fibroblasts and osteoblasts, whereas titanium appears the least cytocompatible of all the three substrates. Moreover, the amorphous alumina coating seems slightly bioactive. It seems that these coatings, particularly amorphous alumina, could be used to protect alloys against corrosion, and consequently combine the good mechanical properties of the alloys with the good biocompatibility of the coatings. These coatings seem to perform more suitably than titanium if the strength of the bond between the coating and the underlying alloys is strong enough to give a stable composite material.

Effects of S-adenosylmethionine on human articular chondrocyte differentiation: An in vitro study

The effect of S-adenosyl-L-methionine (SAMe) on human articular osteoarthritic chondrocytes was studied using a thick-layer culture model. Three SAMe concentrations were tested (1, 10, and 100 micrograms/ml). For 10 micrograms/ml, the most efficient dose, a significant rise in the incorporation levels of 35S-sulfate and 3H-serine was observed, as was as an increase in the hexuronic acid content. All the parameters seem to express a more active protein synthesis, particularly for proteoglycans. Under the same conditions, the proliferation rate of chondrocytes does not undergo important variations. These results point to a direct action on the cell metabolism but little is known concerning the mechanism involved.

Fibrin seal in wound healing: Effect of thrombin and [Ca2+] on human skin fibroblast growth and collagen production

To assess the interaction of a fibrin sealant with wound healing, an in vitro study using human skin fibroblasts was carried out. The effect of thrombin and calcium concentrations in the sealant on the growth parameters and collagen synthesis by normal human skin fibroblasts was examined. The fibroblast proliferation was increased 3 times for 50 and 25 IU of thrombin/ml. However for 20 mM [Ca2+], this stimulating effect of thrombin was observed after an 8 day incubation period, whereas it was observed as soon as the 2nd day in the presence of 2 mM [Ca2+]. The high rate of [Ca2+] (20 mM) partly inhibited DNA synthesis: for 2 mM [Ca2+], the incorporation of [3H]-Thymidine was 4 times greater than for 20 mM [Ca2+]. Further experiments demonstrated that human skin fibroblasts in the presence of 50 IU of thrombin/ml and 2 mM [Ca2+] in fibrin seal could increase the type I and III collagen synthesis while increasing the ratio of type III to type I. Thus, our results suggest that in vivo wound healing which required fibroblast growth and collagen synthesis can be stimulated in the presence of fibrin glue which is in good accordance with previous clinical observations.

Production of epidermal sheets in a serum free culture system: a further appraisal of the role of extracellular calcium

Rhenwald and Greens technique is currently the standard method for growing stratifying epidermal cell cultures. The serum free system developed in Hams laboratory (MCDB 153) was designed to grow keratinocyte monolayers in clonogenic conditions. Our aim was to optimize conditions in serum-free MCDB 153 for culturing epidermal sheets from adult normal skin, and to assess the effect of extracellular calcium and temperature on proliferation and differentiation of cultured keratinocytes. Sixteen strains derived from plastic surgery specimens (mean age of donors 37 years; range 5-89) were used. Primary cultures were seeded at an optimal density of 8 x 10(4) cells/cm2 in primary cultures and 10(4) cells/cm2 in secondary cultures in complete medium including EGF, insulin, hydrocortisone and bovine pituitary extract, supplemented with isoleucine, tyrosine, methionine, phenylalanine, tryptophane and histidine. Amino acid (AA) supplementation allows a 5.8-fold increase in cell counts at confluency and monolayers with densely packed cells are obtained. In AA supplemented cultures, confluency is obtained in 16 +/- 3 days in primary cultures and in 13 +/- 0.5 days at first passages. Switches to 1.1 mM calcium at first or second passages resulted in a significant increase in cell counts (P less than 0.001), when compared with AA supplemented low calcium cultures. Low temperature/low calcium cultures resulted in a 50% decrease in cell counts. Low temperature/high calcium cultures gave similar cell counts as the 37 degrees C controls. AA and calcium supplemented cultures were evaluated for differentiation markers: involucrin expression was increased, keratins 5, 6, 14, 17 were expressed, and the sheets were 6-10 layers thick by electron microscopy, with keratohyalin granules and cornified envelopes appearing at layers 3-6 (from basal layer). Dispase treatment allowed an easy detachment of these sheets. These results show that the culture medium MCDB 153 can be adapted without serum supplementation to batch culture of human adult keratinocytes to produce epidermal sheets suitable for grafting. They also indicate that extracellular calcium in physiological range of concentration is not a sufficient signal for growth arrest when other growth conditions are optimized.

In vitro comparative evaluation under static conditions of the hemocompatibility of four types of tubing for cardiopulmonary bypass

A comparative in vitro assessment of 4 types of tubing representative of the materials currently used in cardiopulmonary bypass (CPB) procedures was conducted under static conditions using liquid extracts of the materials or direct contact with fresh human blood or serum. The parameters monitored were biomarkers of coagulation and fibrinolytic cascades, the complement system and cell activation. Silicone and PVC tubing were shown to be non-cytotoxic and non-hemolytic. Heparin-coated PVC tubing did present a certain degree of cytotoxicity especially when in direct contact. Thrombosis was found to be significantly lower with the same heparin-coated material. To a lesser extent, platinum-cured silicone also showed a reduced thrombotic tendency. None of the materials activated platelets or the complement system. With platinum-cured silicone tubing, constant and lower leukocyte adhesion was evidenced at the different experimental time points. This could reflect reduced cell activation.

The effect of recombinant human basic fibroblast growth factor rhfgf-2 on human osteoblast in growth and phenotype expression

This paper describes the studies of human recombinant basic fibroblast growth factor (rhFGF-2) for its effects on human osteoblast growth and phenotype expression. During a 24-h period of treatment, rhFGF-2 highly stimulated DNA synthesis in a dose-related fashion with a maximum stimulation of 150% for 1 ng/ml. On the other hand, rhFGF-2 decreases alkaline phosphatase activity, synthesis of type I collagen, and cumulative amount of osteocalcin. Moreover, rhFGF-2 provoked a threefold increase in the amount of intracellular cAMP. Scatchard plots show the presence of two classes of [125I] rhFGF-2 receptors. This data suggests that rhFGF-2 which stimulate cell replication may act indirectly as an anabolic agent and stimulate some of the phenotypic expression markers.

Proteoglycan synthesis in chondrocyte cultures from osteoarthrotic and normal human articular cartilage.

Chondrocytes derived by outgrowth from normal and osteoarthrotic human femoral head cartilage were grown in high density cultures for five passages. Cultures were analysed for their sulfated macromolecular components in medium, layer-matrix and intracellular compartments. Two fractions were obtained: typical proteoglycans and glycosaminoglycan-peptides (Mr approx. 60 000) which might result from an enzymatic degradation of proteoglycans. Proteoglycans from normal and osteoarthrotic cultures exhibited similar biochemical properties (size, protein:uronic acid ratio, glycosaminoglycan composition). Slightly less proteoglycans were aggregated with hyaluronic acid in osteoarthrotic than in normal cultures. Three populations of proteoglycan subunit were obtained under dissociative conditions (Sepharose CL-2B) in both normal and osteoarthrotic cultures: proteoglycan 1 (Kav=0.04), proteoglycan 2 (Kav=0.26) which were aggregated with hyaluronic acid in associative conditions, and proteoglycan 4 (Kav=0.48). A fourth population, proteoglycan 3 (Kav=0.33, Sepharose 2B and CL-2B) was intracellular in osteoarthrotic cultures. After a 4 day incubation period, about 60% more proteoglycans were found in osteoarthrotic than in normal cultures (medium+52%, layer-matrix + 44% and 17 times the normal value inside the cells). Proteoglycan distribution kinetics in the three compartments showed a higher net accumulation of proteoglycans in osteoarthrotic-derived cultures.

Polarization Phenomena in Collagens from Various Tissues

The various levels of organization of collagens extracted from different tissues (tendon, skin, cartilage) give rise to several polarization phenomena induced by molecular movements of dipolar entities. The corresponding dielectric relaxation time spectrum has been investigated by the Thermally Stimulated Current technique. The contribution of cooperative movements ofpolar and apolar sequences along the main chain has been identified: the corresponding relaxation times are given by Arrhenius equations following a compensation law. The relaxation times obey a Fulcher-Vogel equation and have been assigned to motions delocalized along the whole tropocollagen molecule. Special attention has been paid to dipolar reorientations associated with intra and intermolecular mobility which have been found to be specific of the type of collagen and of its interaction with the other constituents of the organic matrix.

Effect of a derivatized dextran on human osteoblast growth and phenotype expression.

Water soluble derivatized dextran named E9 with a molecular weight of 45,000 g l-1 containing 58% methyl carboxylic acid unit, 19% benzylamide unit, and 26% sulfonate with a specific anticoagulant activity of 0.29 IU mg-1 was studied for its effects on human osteoblast growth and phenotype expression for short-term treatment. At concentrations between 1 ng ml-1 and 1 microgram ml-1 E9 has no effect on DNA synthesis whereas at higher concentrations DNA synthesis is inhibited in a dose related fashion (87% for 400 micrograms ml-1). For concentrations which do not modify osteoblast growth, E9 promotes alkaline phosphatase activity, type I collagen and osteocalcin synthesis with a maximum effect for 0.1-1 microgram ml-1. It has a synergistic effect with hPTH increasing AMPc. Moreover, osteonectin synthesis was enhanced in a dose-dependent manner between 0.1 and 5 micrograms ml-1. These results seem to indicate that E9 is able to stimulate human osteoblast phenotype expression and could be useful in clinical applications.

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.