Hyaluronate-alginate combination for the preparation of new biomaterials: investigation of the behaviour in aqueous solutions.

With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated. Crossed techniques were used to assess the existence of polymer interactions in aqueous solutions up to 20 mg/ml. Alginate was obtained from algae and hyaluronate was purified from rooster comb. Viscometry measurements using the capillary technique or the Couette flow, together with circular dichroism investigations, evidenced the moderate significance of interactions between the two polysaccharides in dilute solutions. In addition, the case of more concentrated solutions and containing 20 mg/ml alginate was approached by rheological measurements in the flow mode; the behaviour of the polymer associations appeared as a compromise between those of individual polysaccharides.

Protein binding and stereoselectivity of nonsteroidal anti-inflammatory drugs.

Stereoselective binding of nonsteroidal anti-inflammatory drugs (NSAIDs) can be studied using various techniques. Thus the results obtained by different investigators may be poorly consistent and even contradictory. NSAIDs are bound stereoselectively to serum albumin to different degrees depending on the drug investigated (ibuprofen, indoprofen, carprofen, etodolac, ketoprofen and flurbiprofen). For other drugs, both enantiomers are bound to a similar extent (pirprofen, fenoprofen). This stereoselectivity could vary with experimental conditions, in particular with protein concentration (ketoprofen, etodolac), leading to individual differences. Finally, the stereoselectivity of protein binding and of pharmacokinetics can be compared: differences in binding between enantiomers can explain their differences in pharmacokinetics, once metabolic properties such as inversion have been taken into account.

Protein binding of indomethacin in human cerebrospinal fluid.

The binding of the non-steroidal anti-inflammatory drug indomethacin to proteins in human cerebrospinal fluid (CSF), drawn during lumbar puncture from 10 patients affected by lumbosciatica, was measured by equilibrium dialysis and spectrofluorimetry. Similar binding studies on human serum albumin solutions (0.5 and 1 g/L) were performed using the same techniques. The mean binding percentage of indomethacin determined by equilibrium dialysis was 40%. The results obtained by both techniques allowed us to conclude that the binding of indomethacin in CSF was essentially due to albumin.

Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification.

Endothelial cell seeding constitutes an appreciated method to improve blood compatibility of small-diameter vascular grafts. In this study, we report the development of a simple innovative technique based on multilayered polyelectrolyte films as cell adhesive substrates. Polyelectrolyte multilayered films ending by poly(sodium-4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) or poly(L-glutamic acid)/poly(D-lysine) (PGA/PDL) could enhance cell adhesion by modification of the physico-chemical properties of the surface. The biological responses of human umbilical vein endothelial cells seeded on the polyelectrolyte multilayer films, on PDL or PAH monolayers, and on control surfaces, were evaluated in terms of initial attachment, growth, cellular metabolic activity, endothelial phenotype, and adhesion. The results showed that polyelectrolyte multilayers neither induce cytotoxic effects nor alter the phenotype of the endothelial cells. The polyelectrolyte multilayered films enhanced initial cell attachment as compared to the polyelectrolyte monolayer. Cell growth observed on the films was similar to that on TCPS. Among the different coating tested, the film ending by PSS/PAH exhibited an excellent cellular biocompatibility and appeared to be the most interesting surface in terms of cellular adhesion and growth. Such films could be used to cover hydrophobic (cell resistant) substrates in order to promote cell colonization, thereby constituting an excellent material for endothelial cell seeding.

Evaluation of the effect of three surface treatments on the biocompatibility of 316L stainless steel using human differentiated cells.

AISI 316L stainless steel (SS) is widely used in orthopaedic implantology, although biological complications may result from its insufficient mechanical and tribological properties. In order to improve the wear and corrosion resistance as well as the hardness of 316L SS, three surface treatments, derived from those applied in mechanical engineering industries, were investigated: (1) glow discharge nitrogen implantation, (2) carbon-doped stainless steel coating sputtering and (3) low temperature plasma nitriding. Surface characterization according to the different heat treatments showed that corrosion and wear resistance were strongly improved, especially by ion implantation or carbon-doped SS coating sputtering. In the same way, microhardness was significantly increased after the three treatments. The effect of such treatments on the biocompatibility of 316L SS was studied with human osteoblast and fibroblast cultures. Basic and specific features of the cells showed that ion-implanted and carbon-doped stainless steels were biocompatible, whereas dramatic cellular reactions were noted when contacted with nitrided stainless steel. A hypothesis is given to explain this observation but further experiments are needed to optimize the nitriding process. Nitrogen implantation and carbon-doped layer deposition could be efficient means for improving the physical properties of stainless steel without affecting its biocompatibility. Such surface treatments may have relevance for increasing the life time of 316L biomedical devices.

Cytocompatibility of Ti-6Al-4V and Ti-5Al-2.5Fe alloys according to three surface treatments, using human fibroblasts and osteoblasts.

Titanium alloys are well known for their superior mechanical properties as well as for their good biocompatibility, making them desirable as surgical implant materials. However, these alloys have been proven to behave poorly in friction since wear particles were often detected in tissues and organs associated with titanium implants. In this paper, three surface treatments were investigated in order to improve the wear resistance and the hardness of Ti-6Al-4V and Ti-5Al-2.5Fe: (a) glow discharge nitrogen implantation (10(17) atoms cm-2), (b) plasma nitriding by plasma diffusion treatment (PDT) and (c) deposition of TiN layer by plasma-assisted chemical vapour deposition (PACVD) additionally to PDT. Surface characterization after the different treatments showed considerable improvement in surface hardness, especially after the two nitriding processes. Moreover, the good corrosion resistance of untreated alloys was maintained. A cell culture model using human cells was chosen to study the effect of such treatments on the cytocompatibility of these materials. The results showed that Ti-5Al-2.5Fe alloy was as cytocompatible as the Ti-6Al-4V alloy and the same surface treatment led to identical biological consequences on both alloys. Nitrogen implantation did not modify at all the cellular behaviour observed on untreated samples. After the two nitriding treatments, cell proliferation and viability appeared to be significantly reduced and the scanning electron microscopy study revealed somewhat irregular surface states. However, osteoblast phenotype expression and protein synthesis capacity were not affected. PDT and PACVD may be interesting alternatives to the physical vapour deposition technique.

Assay of circulating hyaluronic acid in the rat: study of diurnal variation and effect of anesthesia.

Serum hyaluronic acid (HA) may provide a good marker for the severity of joint disease in the rat since a positive correlation was observed in experimental models of arthritis. However, little is known about its physiological variation in rats. In the present work, we do not find any circadian rhythm of HA in healthy Sprague-Dawley rats in contrast to that observed in humans, whose serum levels vary during daytime. Furthermore, the influence of blood sampling conditions on HA concentrations was evaluated in conscious animals and by using different anesthetics. The greater reproducibility for the assay of HA is observed with the intracardiac puncture under ether inhalation. Blood sample collection in the absence of anesthesia leads to a significant increase in serum levels of HA, which could be attributed partly to enhanced joint movements generated by psychological stress.

Pharmacological aspects of chiral nonsteroidal anti-inflammatory drugs.

Most NSAIDs are chiral molecules: they exist under 2 configurations of non-superimposable mirror images which are termed enantiomers or optical isomers or optical antipodes. Direct or indirect (resolution) methods are used to separate this equal mixture of compounds. Some of the enantiomers of the NSAIDs are able to undergo chiral inversion from the inactive R(-) to the active S(+) form. The pharmacokinetics in terms of absorption, distribution, metabolism, protein binding and elimination may be different for the 2 enantiomers, leading to interindividual variability in clinical response and drug toxicity.

Representation of affinity in the case of co-operativity in protein-ligand binding.

We have already proposed a "Global Association Function" to represent the global affinity of proteins to a drug; it was first applied in the case of independent binding sites. In this paper, we show that this same function can also be used to assess interactions between sites by varying the number of interacting sites and their co-operativity level. The resulting curves in two application cases are given together with the corresponding Scatchard plot: i) in a system with one single class of identical and interacting sites, ii) in a system with two classes of sites in which either primary or secondary are interacting; unexpectedly, in this latter case we also observed that sometimes positive co-operativity occasionally resulted in a concave-up Scatchard plot which is unusually admitted. In addition, as described in one example, our function is assumption free; this might be an advantage over usual methods, such as discrete parameter methods, because they require additional and empirical hypotheses on their related binding model.

In vitro study of intracellular IL-1beta production and beta1 integrins expression in stimulated chondrocytes--effect of rhein.

The purpose of the present study was to investigate the intracellular IL-1beta production and beta1 integrins (alpha4/beta1 and alpha5/beta1) expression on chondrocytes. Chondroytes monolayer (human chondrosarcoma cell line HEM-C55) were incubated for 12, 24 and 48 hours in the presence of Tumor Necrosis Factor-alpha (TNF-alpha, Sigma, France) or recombinant human IL-1alpha (rh-IL1alpha, Becton Dickinson, France). After direct immunolabelling, cells were either analyzed on FACScan flow cytometer (Becton Dickinson, France), or observed under an epi-fluorescence inverted microscope equipped with the CellScan EPR optical scanning acquisition system (IPLab-Scanalytics, USA). We found that the IL-1beta mean fluorescence intensity in flow cytometry and in 3D microscopy was increased in the presence of TNF-alpha or rh-IL-1alpha, and alpha4/beta1 or alpha5/beta1 expression was higher on stimulated cells than on control cells. On the other hand, we have evaluated the in vitro effects of rhein (10(-5) M, Negma, France), an active metabolite of diacerein, on the intracellular IL-1beta and beta1 integrins expressed by stimulated or no-stimulated chondrocytes. The results indicated that rhein leads to a reduction of IL-1beta synthesis whereas a weak decrease of beta1 integrins receptors expression is observed. From this study, it seems that rhein partially reduce cytokine-induced intracellular IL-1beta production, and it has a weak action on alpha4/beta1 or alpha5/beta1 receptors.

Expression of adhesion molecules and collagen on rat chondrocyte seeded into alginate and hyaluronate based 3D biosystems. Influence of mechanical stresses.

Chondrocytes use mechanical signals, via interactions with their environment, to synthesize an extracellular matrix capable to withstanding high loads. Most chondrocyte-matrix interactions are mediated via transmembrane receptors such as integrins or non-integrins receptors (i.e. annexin V and CD44). The aim of this study was to analyze, by flow cytometry, the adhesion molecules (alpha5/beta1 integrins and CD44) on rat chondrocytes seeded into 3D biosystem made of alginate and hyaluronate. These biosystems were submitted to mechanical stress by knocking the biosystems between them for 48 hours. The expression of type I and type II collagen was also evaluated. The results of the current study showed that mechanical stress induced an increase of type II collagen production and weak variations of alpha5/beta1 receptors expression no matter what biosystems. Moreover, our results indicated that hyaluronan receptor CD44 expression depends on extracellular matrix modifications. Thus, these receptors were activated by signals resulted from cell environment variations (HA addition and modifications owing to mechanical stress). It suggested that this kind of receptor play a crucial role in extracellular matrix homeostasis. Finally, on day 24, no dedifferentiation of chondrocytes was noted either in biosystems or under mechanical stress. For all biosystems, the neosynthesized matrix contained an important level of collagen, which was type II, whatever biosystems. In conclusion, it appeared that the cells, under mechanical stress, maintained their phenotype. In addition, it seems that, on rat chondrocytes, alpha5/beta1 integrins did not act as the main mechanoreceptor (as described for human chondrocytes). In return, hyaluronan receptor CD44 seems to be in relation with matrix composition.

The role of 3D-microscopy in the study of chondrocyte-matrix interaction (alginate bead or sponge, rat femoral head cap, human osteoarthritic cartilage) and pharmacological application.

The potentialities of a new non-invasive optical scanning microscopy technique were evaluated through 3D analysis of chondrocyte-matrix interactions. Five different 2D or 3D culture systems were used: (1) MonoLayer (ML) of human chondrosarcoma cell line; (2) rat or human chondrocytes encapsulated in Alginate Bead (AB); (3) human chondrocytes encapsulated in Alginate Sponge (AS); (4) Rat Femoral Head Cap (RFHC); (5) slices of knee human Osteoarthritic Cartilage (HOAC). Chondrocytes ML, AB, RFHC were incubated for 24 h in vitro in the presence of recombinant human interleukin1-beta (rhIL1-beta) and the effects on cytoskeleton organisation (F-actin filament), Focal Adhesion Kinase (FAK) expression (tyrosine kinase), collagenase B expression (metalloprotease) were studied. Furthermore, the production of intracellular IL1-beta by LPS- or rhIL1-beta-stimulated chondrocytes was shown to be partly suppressed by rhein (active metabolite of diacerhein) in all culture systems. This high resolution light microscopy gave complementary information that could be important for a better understanding of the interaction of chondrocytes with the extracellular matrix in a variety of culture devices.

Cartilage repair: surgical techniques and tissue engineering using polysaccharide- and collagen-based biomaterials.

Lesions of articular cartilage have a large variety of causes among which traumatic damage, osteoarthritis and osteochondritis dissecans are the most frequent. Replacement of articular defects in joints has assumed greater importance in recent years. This interest results in large part because cartilage defects cannot adequately heal themselves. Many techniques have been suggested over the last 30 years, but none allows the regeneration of the damaged cartilage, i.e. its replacement by a strictly identical tissue. In the first generation of techniques, relief of pain was the main concern, which could be provided by techniques in which cartilage was replaced by fibrocartilage. Disappointing results led investigators to focus on more appropriate bioregenerative approaches using transplantation of autologous cells into the lesion. Unfortunately, none of these approaches has provided a perfect final solution to the problem. The latest generation of techniques, currently in the developmental or preclinical stages, involve biomaterials for the repair of chondral or osteochondral lesions. Many of these scaffolds are designed to be seeded with chondrocytes or progenitor cells. Among natural and synthetic polymers, collagen- and polysaccharide-based biomaterials have been extensively used. For both these supports, studies have shown that chondrocytes maintain their phenotype when cultured in three dimensions. In both types of culture, a glycosaminoglycan-rich deposit is formed on the surface and in the inner region of the cultured cartilage, and type II collagen synthesis is also observed. Dynamic conditions can also improve the composition of such three-dimensional constructs. Many improvements are still required, however, in a number of key aspects that so far have received only scant attention. These aspects include: adhesion/integration of the graft with the adjacent native cartilage, cell-seeding with genetically-modified cell populations, biomaterials that can be implanted without open joint surgery and combined therapies, aimed at disease modification, pain relief and reduction of inflammation.

The effect of alginate, hyaluronate and hyaluronate derivatives biomaterials on synthesis of non-articular chondrocyte extracellular matrix.

Cartilage engineering consists of re-constructing functional cartilage by seeding chondrocytes in suitable biomaterials in vitro. The characteristics of neocartilage differ upon the type of biomaterial chosen. This study aims at determining the appropriate scaffold material for articular cartilage reconstruction using non articular chondrocytes harvested from rat sternum. For this purpose, the use of polysaccharide hydrogels such as alginate (AA) and hyaluronic acid (HA) was investigated. Several ratios of AA/HA were used as well as three derivatives obtained by chemical modification of HA (HA-C18, HA-C12(2.3), HA-C12(2.5)-TEG0.5). Sternal chondrocytes were successfully cultured in 3D alginate and alginate/HA scaffolds. HA retention in alginate beads was found to be higher in beads seeded with cells than in beads without cells. HA-C18 improved HA retention in beads but inhibited the chondrocyte synthesis process. Cell proliferation and metabolism were enhanced in all biomaterials when beads were mechanically agitated. Preliminary results have shown that the chondrocyte neo-synthesised matrix had acquired articular characteristics after 21 days culture.

Hyaluronidase degradation of hyaluronic acid from different sources: influence of the hydrolysis conditions on the production and the relative proportions of tetra- and hexasaccharide produced.

1. Hyaluronic acid (HA) can be digested with a Streptomyces hyaluronidase. 2. The rate of production and the ratio of tetrasaccharide (T) and hexasaccharide (H), studied by HPLC, varied with the temperature and duration of hydrolysis. 3. The rates of production and the respective amounts of the two oligosaccharides depended on the rheological properties of the HA from different sources. 4. A close relationship was found between the initial rate of hydrolysis and the intrinsic viscosity of the HA (eta i). 5. Our data suggest that enzymatic degradation at a given pH value, temperature, and duration of hydrolysis is dependent on the conformation of HA. 6. Moreover, under given conditions, the relative proportions of the two oligosaccharides depend on the eta i and may also reflect the degree of hydrolysis of the substrate.

Amphiphilic derivatives of sodium alginate and hyaluronate for cartilage repair: rheological properties.

Various amphiphilic derivatives of sodium alginate and hyaluronate were prepared by covalent fixation of long alkyl chains (dodecyl and octadecyl) with various ratios on the polysaccharide backbones via ester functions. In the semidilute regime, aqueous solutions of the resulting compounds exhibited the typical rheological properties of hydrophobically associating polymers: tremendous enhancement of zero shear rate Newtonian viscosity, steep shear-thinning behavior, and formation of physically cross-linked gel-like networks. The influence of the alkyl chain length, its content on the polysaccharide and of the polymer concentration in the solution was well identified. All obtained results are discussed with respect to the schedule of conditions related to materials, which could be used for cartilage repair, such as in synovial fluid viscosupplementation as well as in cartilage replacement. In particular, it is seen that HA-C(12)-5 (hyaluronate substituted with 5% of dodecyl chains) and HA-C(18)-1 (hyaluronate substituted with 1% of octadecyl chains) in a 0.15N NaCl solution at 8 g/L have rheological properties quite similar to those of healthy synovial fluid. On the other hand, the rheological parameters of solutions at 8 g/L in 0.15N NaCl of some of derivatives, such as, for example, AA-C(12)-8 (alginate substituted with 8% of dodecyl chains) or HA-C(18)-2, are well fitted for a use in cartilage repair.

Stereoselective binding of etodolac to human serum albumin.

The protein binding of etodolac enantiomers was studied in vitro by equilibrium dialysis in human serum albumin (HSA) of various concentrations varying from 1 to 40 g/liter, by addition of each enantiomer at increasing concentrations. In the 1 g/liter solution, at the lowest drug levels, the (R)-form is more bound than its antipode, the contrary being observed at the highest drug levels. For higher albumin concentrations, S was bound in a larger extent than R. Using the displacement of specific markers of HSA sites I and II, studied by spectrofluorimetry, it was suggested that R and S are both bound to site I, while only S is strongly bound to site II.

Exercise-induced hypoxaemia in master athletes: effects of a polyunsaturated fatty acid diet.

Exercise-induced hypoxaemia (EIH) has been associated with an oxygen diffusion limitation. Because polyunsaturated fatty acids (PUFA) administration can modify cell membrane fluidity, we hypothesized that the importance of EIH could be reduced after a 6-week PUFA diet. Resting pulmonary functions and a maximal cycling test were performed before and after the diet, in eight master athletes -48 (SD 6 years)-. The partial pressure of O2 in arterial blood (PaO2), alveolar ventilation (VA) and ideal alveolar-arterial oxygen partial pressure difference (P(Ai-a) O2) were obtained at each exercise intensity. The extent of EIH at maximal exercise was significantly lower after PUFA [PaO2-17.2 (SEM 1.9) vs -12.9 (SEM 2.2)]. Before PUFA, VA accounted for 50% of the variance in the fall in P (Ai-a) for intensities below 80% maximal oxygen uptake (VO2max) and P(Ai-a)O2 for 60% between 70% and 100% VO2max. After PUFA, the reduction in EIH was highly correlated (r2 = 0.85; P < 0.001) to resulting changes in P(Ai-a)O2 and resting pulmonary diffusing capacity (DLCO)/VA but not with changes in ideal alveolar partial pressure of oxygen. The improvement in EIH following PUFA could be related to an increase in alveolar-arterial oxygen conductance following improved pulmonary diffusion.