International audienceCarbonated apatites represent an important class of compounds encountered in many fields including anthropology, archeology, geology, medicine and biomaterials engineering. They constitute, in particular, the mineral part of bones and teeth, are found in sedimentary settings, and are used as biomimetic compounds for the development of bone tissue engineering scaffolds. Whether for assessing the degree of biomimetism of synthetic apatites or for better understanding diagenetic events, their thorough physico-chemical characterization is essential, and includes, in particular, the evaluation of their carbonate content. FTIR is especially well-suited for such a goal, as this spectroscopy technique requires only a low amount of specimen to analyze, and carbonate ions exhibit a clear vibrational signature. In this contribution, we critically discuss several FTIR-approaches that may be (or have been) considered in view of carbonation quantification. The best methodology appears to be based on the analysis of the n3(CO3)and n1n3(PO4) modes. The area ratio rc/p between these two contributions was found to be directly correlated to the carbonate content of the samples (R2 ¼ 0.985), with the relation wt.% CO3 ¼ 28.62*rc/ p þ 0.0843. The method was validated thanks to titrations by coulometry assays for various synthetic reference samples exhibiting carbonate contents between 3 and 7 wt.%. The FTIR carbonate quantification methodology that we propose here was also tested with success on three skeletal specimens (two bones/one tooth), after elimination of the collagen contribution. Comparative data analysis is also presented,showing that the use of other vibration bands, or only peak heights (instead of peak areas), leads to significantly lower correlation agreement. This FTIR data treatment methodology is recommended so as to limit errors on the evaluation of carbonate contents in apatite substrates

International audienceIn order to shed some light on DNA preservation over time in skeletal remains from a physicochemicalviewpoint, adsorption and desorption of DNA on a well characterized synthetic apatite mimicking boneand dentin biominerals were studied. Batch adsorption experiments have been carried out to determinethe effect of contact time (kinetics), DNA concentration (isotherms) and environmentally relevant factorssuch as temperature, ionic strength and pH on the adsorption behavior. The analogy of the nanocrystallinecarbonated apatite used in this work with biological apatite was first demonstrated by XRD, FTIR, andchemical analyses. Then, DNA adsorption kinetics was fitted with the pseudo-first order, pseudo-secondorder, Elovich, Ritchie and double exponential models. The best results were achieved with the Elovichkinetic model. The adsorption isotherms of partially sheared calf thymus DNA conformed satisfacto-rily to Temkin's equation which is often used to describe heterogeneous adsorption behavior involvingpolyelectrolytes. For the first time, the irreversibility of DNA adsorption toward dilution and significantphosphate-promoted DNA desorption were evidenced, suggesting that a concomitant ion exchange pro-cess between phosphate anionic groups of DNA backbone and labile non-apatitic hydrogenphosphateions potentially released from the hydrated layer of apatite crystals. This work should prove helpful fora better understanding of diagenetic processes related to DNA preservation in calcified tissues

Biomimetic calcium phosphate apatites are particularly adapted to bio‐medical applications due to their biocompatibility and high surface reactivity. In this contribution we report three selected examples dealing with mineral/organic interactions devoted to convey new functionalities to apatite materials, either in the form of dry bioceramics or of aqueous colloids. We first studied the adsorption of risedronate (bisphosphonate) molecules, which present potential therapeutic properties for the treatment of osteoporosis. We then addressed the preparation of luminescent Eu‐doped apatites for exploring apatite/collagen interfaces through the FRET technique, in view of preparing “advanced” biocomposites exhibiting close spatial interaction between apatite crystals and collagen fibers. Finally, we showed the possibility to obtain nanometer‐scaled apatite‐based colloids, with particle size tailorable in the range 30–100 nm by controlling the agglomeration state of apatite nanocrystals by way of surface functionalization with a phospholipid moiety. This paper is aimed at illustrating some of the numerous potentialities of calcium phosphate apatites in the bio‐medical field, allowing one to foresee perspectives lying well beyond bone‐related applications.

Viscotoxins A2 (VA2) and B (VB) are, together with viscotoxin A3 (VA3), among the most abundant viscotoxin isoforms that occur in mistletoe-derived medicines used in anti-cancer therapy. Although these isoforms have a high degree of amino-acid-sequence similarity, they are strikingly different from each other in their in vitro cytotoxic potency towards tumour cells. First, as VA3 is the only viscotoxin whose three-dimensional (3D) structure has been solved to date, we report the NMR determination of the 3D structures of VA2 and VB. Secondly, to account for the in vitro cytotoxicity discrepancy, we carried out a comparative study of the interaction of the three viscotoxins with model membranes. Although the overall 3D structure is highly conserved among the three isoforms, some discrete structural features and associated surface properties readily account for the different affinity and perturbation of model membranes. VA3 and VA2 interact in a similar way, but the weaker hydrophobic character of VA2 is thought to be mainly responsible for the apparent different affinity towards membranes. VB is much less active than the other two viscotoxins and does not insert into model membranes. This could be related to the occurrence of a single residue (Arg25) protruding outside the hydrophobic plane formed by the two amphipathic alpha-helices, through which viscotoxins are supposed to interact with plasma membranes.

The interaction of adriamycin with lipids was studied in model (monolayers, small unilamellar vesicles, large multilamellar vesicles) and natural (chinese hamster ovary cell) membranes by measurement of fluorescence energy transfer and fluorescence quenching. 2-APam, 7-ASte, 12-ASte and anthracene-phosphatidylcholine were used as fluorescent probes in which the anthracene group is well located at graded depths in the membrane. Egg-yolk phosphatidylcholine and a 1/1 mixture of it with bovine brain phosphatidylserine were used in model membrane systems. Large fluorescence energy transfer was observed between these molecules as donors and the drug as acceptor. With liposomes, at pH 7.4 and over an adriamycin concentration range of 0-100 microM, the efficiency of energy transfer was 12-ASte greater than 7-ASte greater than 2-APam, with 100% energy transfer for 12-ASte above a drug concentration of 30 microM. At pH 5, where the fatty acids are buried deeper (0.45 nm) in the lipid bilayer due to protonation of the carboxyl group, the order of energy transfer 7-ASTe greater than 12-ASte = 2-APam was observed. Measurements of fluorescence quenching using the non-permeant Cu2+ ion as quencher and spectrophotometric assays indicated that around 40% of the adriamycin molecules were deeply embedded in the lipid bilayer. Adriamycin molecules thus appear to penetrate the lipid bilayer, with the aminoglycosyl group interacting with the lipid phosphate groups and the dihydroanthraquinone residue in contact with the lipid fatty acid chains. In contrast, fluorescence energy transfer and quenching studies on CHO cells showed that adriamycin penetrated the plasma membrane of these cells to a much more limited extent than in the model membrane systems. This can be related to the squeezing out of the drug from a film of phosphatidylcholine which was observed in monolayers by means of surface pressure, potential and fluorescence experiments. These observations indicated that the penetration of adriamycin into lipid bilayers strongly depends on the molecular packing of the lipid.

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The extraction of DNA from skeletal remains is a major step in archeological or forensic contexts. However, diagenesis of mineralized tissues often compromises this task although bones and teeth may represent preservation niches allowing DNA to persist over a wide timescale. This exceptional persistence is not only explained on the basis of complex organo-mineral interactions through DNA adsorption on apatite crystals composing the mineral part of bones and teeth but is also linked to environmental factors such as low temperatures and/or a dry environment. The preservation of the apatite phase itself, as an adsorption substrate, is another crucial factor susceptible to significantly impact the retrieval of DNA. With the view to bring physicochemical evidence of the preservation or alteration of diagenetic biominerals, we developed here an analytical approach on various skeletal specimens (ranging from ancient archeological samples to recent forensic specimens), allowing us to highlight several diagenetic indices so as to better apprehend the complexity of bone diagenesis. Based on complementary techniques (X-ray diffraction (XRD), Fourier transform infrared (FTIR), calcium and phosphate titrations, SEM-EDX, and gravimetry), we have identified specific indices that allow differentiating 11 biological samples, primarily according to the crystallinity and maturation state of the apatite phase. A good correlation was found between FTIR results from the analysis of the v3(PO4) and v4(PO4) vibrational domains and XRD-based crystallinity features. A maximal amount of information has been sought from this analytical approach, by way of optimized posttreatment of the data (spectral subtraction and enhancement of curve-fitting parameters). The good overall agreement found between all techniques leads to a rather complete picture of the diagenetic changes undergone by these 11 skeletal specimens. Although the heterogeneity and scarcity of the studied samples did not allow us to seek direct correlations with DNA persistence, the physicochemical parameters described in this work permit a fine differentiation of key properties of apatite crystals among post mortem samples. As a perspective, this analytical approach could be extended to more numerous sets of specimens so as to draw statistical relationships between mineral and molecular conservation.

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