[From the bench to the clinic: The challenge of translating platelet production in vitro]. / Du laboratoire à l'usage clinique : le défi de la production de plaquettes in vitro.

Platelet transfusions, which are currently totally dependent on altruistic donations, are absolutely necessary to the treatment of patients with thrombocytopenia following trauma, surgery or other pathologies (especially malignancies). Producing platelets in vitro represent a major technological and scientific breathrough that would address logistical issues (supply chain, stock holding…) and medical concerns (compatibility and biosafety). The translation of this innovation will need to be accompanied by rigorous quality control, harmonised between laboratory when it comes to functionality and biosafety for use in the clinic.

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold.

An engineered three dimensional (3D) in vitro cell culture system was designed with the goal of inducing and controlling in vitro osteogenesis in a reproducible manner under conditions more similar to the in vivo bone microenvironment than traditional two-dimensional (2D) models. This bioreactor allows efficient mechanical loading and perfusion of an original cubic calcium phosphate bioceramic of highly controlled composition and structure. This bioceramic comprises an internal portion containing homogeneously interconnected macropores surrounded by a dense layer, which minimises fluid flow bypass around the scaffold. This dense and flat layer permits the application of a homogeneous loading on the bioceramic while also enhancing its mechanical strength. Numerical modelling of constraints shows that the system provides direct mechanical stimulation of cells within the scaffold. Experimental results establish that under perfusion at a steady flow of 2 µL/min, corresponding to 3 ≤ Medium velocity ≤ 23 µm/s, mouse calvarial cells grow and differentiate as osteoblasts in a reproducible manner, and lay down a mineralised matrix. Moreover, cells respond to mechanical loading by increasing C-fos expression, which demonstrates the effective mechanical stimulation of the culture within the scaffold. In summary, we provide a "proof-of-concept" for osteoblastic cell culture in a controlled 3D culture system under perfusion and mechanical loading. This model will be a tool to analyse bone cell functions in vivo, and will provide a bench testing system for the clinical assessment of bioactive bone-targeting molecules under load.

Synthesis, structure and biological activity of nickel(II) complexes of 5-methyl 2-furfural thiosemicarbazone.

5-Methyl 2-furfuraldehyde thiosemicarbazone (M5HFTSC) with nickel(II) leads to three types of complexes: [Ni(M5HFTSC)(2)X(2)], [Ni(M5FTSC)(2)] and [Ni(M5FTSC)(2)] x 2DMF. In the first type the ligand remains in thione form, while in the two other, the anionic thiolato form is involved. The species [Ni(M5HFTSC)(2)X(2)] has been characterized spectroscopically. The structures of [Ni(M5FTSC)(2)] x 2DMF and [Ni(M5FTSC)(2)] have been solved using X-ray diffraction. Biological studies of [Ni(M5HFTSC)(2)Cl(2)] have been carried out in vitro for antifungal activity on human pathogenic fungi, Aspergillus fumigatus and Candida albicans, and in vivo for toxicity on mice. The results are compared to those of the ligand, the metal salt and a similar copper complex [Cu(M5HFTSC)Cl(2)].

Stability constants of potent cytotoxic copper(II) complexes with furan semicarbazones in ethanolic solutions.

Potent cytotoxic copper(II) complexes of furannic semicarbazones were studied in ethanolic solutions at 25 degrees C. The four ligands used were synthesized in our laboratory, i.e. 2-furfural semicarbazone (FSC), 5-methyl 2-furfural semicarbazone (MFSC), 2-furfural 4-phenyl semicarbazone (FPSC) and 3-(2-furyl) prop-2-enal semicarbazone (FASC). The mathematical analysis was carried out with a recent computer program SIRKO which indicated the formation of one metal-ligand complex in each case and the logarithm of their stability constants are: log beta=2.02, 3.84, 4.58 and 4.52 for ligands FSC, MFSC, FASC and FPSC, respectively. A relation between stability and molecular weight of the ligands is proposed which may prove to be interesting as these complexes are being investigated for their cytotoxic activities.

Cytotoxicity of copper and cobalt complexes of furfural semicarbazone and thiosemicarbazone derivatives in murine and human tumor cell lines.

The 2-furfural semicarbazone and thiosemicarbazone copper and cobalt complexes demonstrated potent cytotoxicity against the growth of suspended leukemias and lymphomas as well as human lung MB9812, colon SW480, ovary 1-A9 and HeLa-S3 uterine carcinoma. In L1210 lymphoid leukemia cell the complexes inhibited preferentially DNA synthesis over 60 min at 25 to 100 microM. The copper and cobalt complexes functioned by multiple mechanisms to suppress synthetic steps in nucleic acid metabolism to reduce deoxynucleotide pools for incorporation into DNA. At high concentrations the complexes suppressed human DNA topoisomerase II activity with DNA nicks and DNA fragmentation but they did not alkylate the bases of DNA, cause intercalation between base pairs or cause cross-linking of DNA strands.

Stability, toxicity and cytotoxicity of a cupric complex towards cultured CaCo-2 cells.

The human colon adenocarcinoma-derived cell line CaCo-2 was used as a model system to study the effects of copper (II), tetradentate N-alkyl ligand (H2L) and their complex. The stimulatory effect of the complex was obtained with lower concentrations from 10(-7) to 10(-17) mol.L-1, while inhibiting effects occur from 10(-4) to 10(-6) mol.L-1. According to this study, copper (II) and free ligand were toxic for cultured cells. Our data suggested that the complex, according to our toxicity assays in mice, could be used as an antimitotic agent.

Stability constants of thiocyanato complexes of cobalt(II), nickel(II) and copper(II) in methanol.

A spectrophotometric study of cobalt(II), nickel(II) and copper(II) thiocyanato complexes was carried out in methanol at 25 degrees C and at a constant ionic strength of 1 M. Under the experimental conditions, two mononuclear complexes are identified with each of the three metal ions. Their stability constants are determined with a recent PC program SIRKO and the calculated values are: for cobalt, log beta 1 = 1.6, log beta 2 = 2.7; for nickel, log beta 1 = 1.8, log beta 2 = 3.0; and for copper, log beta 1 = 3.0, log beta 2 = 3.6.

Potentiometric study of vitamin D3 complexes with manganese(II), iron(II), iron(III) and zinc(II) in water-ethanol medium.

Vitamin D3 (LH) complexes with manganese(II), iron(II), iron(III) and zinc(II) were identified in water-ethanol medium (30/70). Their stability constants were determined at 298 K and at a constant ionic strength of 0.100 M using potentiometric methods. The computerisation of the experimental data showed the presence of ML (M = metal, L = deprotonated vitamin D3) and ML2 species in all cases; in addition, the ML3 iron(III) complex was detected. The calculated overall stability constants beta for MnIIL, FeIIL, FeIIIL and ZnIIL are, respectively, in logarithms, 12.4, 16.5, 28.5 and 16.5. Under the experimental conditions, the only protonated species MLH detected was with iron(III).