Denitrification Nitrification abundance KEY WORDSNitrogen isotope fractionation Nitrogen-15 natural SUMMARY A few principles relative to the presentation and use of nitrogen stable isotopic data are briefly reviewed. Some classical relationships between the isotope composition of a substrate undergoing a single-step unidirectional reaction, are introduced.They are illustrated through controlled experiments on denitrification in a soil, and through nitrification by pure cultures of Nitrosomonas europaea. In the latter case, the isotope fractionation is calculated from the isotopic composition of the residual substrate, then of the product and the result is shown to be statistically the same for the two procedures.The isotopic enrichment factor for denitrification is -29.4 + 2.49'0o at 20~ and -24.6 + 0.9%o at 30~ for nitrification this factor is -34.7 • 2.5%0 under the experimental conditions employed.

The formation of a new kind of biocompatible film based on poly(L-lysine) and hyaluronic acid (PLL/HA) by alternate deposition of PLL and HA was investigated. Optical waveguide lightmode spectroscopy, streaming potential measurements, atomic force microscopy, and quartz crystal microbalance (QCM) were used to analyze the different aspects of the buildup process such as the deposited mass after each new polyelectrolyte adsorption, the overall surface charge of the film, and its morphology. As for "conventional" polyelectrolyte multilayer systems, the driving force of the buildup process is the alternate overcompensation of the surface charge after each PLL and HA deposition. The construction of (PLL/HA)n films takes place over two buildup regimes. The first one is characterized by the formation of isolated islands that grow both by addition of new polyelectrolytes on their top and by mutual coalescence of the islands. The second regime sets in once a continuous film is formed after the eighth layer pair deposition in our working conditions and is characterized by an exponential increase of the mass. QCM measurements at different frequencies evidenced a viscoelastic behavior of the films with a shear viscosity on the order of 0.1 Pa‚s. This new kind of biocompatible film incorporating a natural polymer of the cartilage and a widely used polypeptide is of potential use for cell-targeted action.

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Studies are underway to design biosystems containing embedded chondrocytes to fill osteochondral defects and to produce a tissue close to native cartilage. In the present report, a new alginate three-dimensional support for chondrocyte culture is described. A sodium alginate solution, with or without hyaluronic acid (HA), was freeze-dried to obtain large-porosity sponges. This formulation was compared with a hydrogel of the same composition. In the sponge formulation, macroscopic and microscopic studies demonstrated the formation of a macroporous network (average pore size, 174 microm) associated with a microporous one (average pore size, 250 nm). Histological and biochemical studies showed that, when loaded with HA, the sponge provides an adapted environment for proteoglycan and collagen synthesis by chondrocytes. Cytoskeleton organization was studied by three-dimensional fluorescence microscopy (CellScan EPR). Chondrocytes exhibit a marked spherical shape with a nonoriented and sparse actin microfilament network. Type II collagen was detected in both types of sponges (with or without HA) using immunohistochemistry. In conclusion, the sponge formulation affords new perspectives with respect to the in vitro production of "artificial" cartilage. Furthermore, the presence of hyaluronate within the alginate sponge mimics a functional environment, suitable for the production by embedded chondrocytes of an extracellular matrix.

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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.

Hydrogels or sponges proved to be colonized by cells synthesizing a matrix with a high HA content. The matrix obtained eventually turns hyaline and takes over the scaffold. The addition of HA and/or chondrocytes to Asp significantly improves the macroscopic and histological scores (P< 0.05 and P< 0.02 respectively). However, biochemical parameters are significantly different of those evaluated in native cartilage. The present study shows that only biochemical parameters allow to discriminate between various biomaterials in tissue engineering and are essential informations which should be taken into account in addition to macroscopic and histological observations.