In vitro assessment of a novel composite scaffold for articular cartilage restoration

Articular cartilage (AC) lesions are a particular challenge for regenerative medicine due to cartilage low self-ability repair in case of damage. Hence, a significant goal of musculoskeletal tissue engineering is the development of suitable structures in virtue of their matrix composition and biomechanical properties [1]. The objective of our study was to design in vitro a supporting structure for cartilage chondrocytes to treat focal articular joint defects. We realized a bio-hybrid composite scaffold combining decellularized Wharton’s jelly (W’s J) with the biomechanical properties of the synthetic hydrogel polyvinyl alcohol (PVA). The hydrogel itself and the more specific decellularized cartilage matrix were used as controls. Immunohistochemical analysis highlighted a similar histomorphology for W’s J and AC matrices. Human chondrocytes were isolated from articular cartilage by collagenase II digestion and then characterized by flow-cytometry and RT-PCR to assess the expression of specific markers. CD44+/CD73+/CD151+ chondrocytes were seeded on PVA, PVA/AC and PVA/W’s J scaffolds to test their ability to support cell colonization. According to SEM micrographs and MTT proliferation assay, PVA/W’s J revealed a singular attitude to sustain cell proliferation despite its aspecific origin. Our preliminary evidences highlighted the chance of using Wharton’s jelly in combination with PVA hydrogels as an innovative and easily available scaffold for cartilage restoration

Anticancer gold(III) peptidomimetics: from synthesis to in vitro and ex vivo biological evaluation

Five new Au(III)‐peptidodithiocarbamato complexes of the type [AuIIIBr2(dtc‐AA1‐AA2‐OR] (AA1=Sar, L/D‐Pro; AA2=L/D‐Ala, Aib; R=OtBu, TEG), differing in the amino acidic sequence and/or the chiral amino acid configuration, were designed to enhance the tumor selectivity and bioavailability. The gold(III)‐based moiety was functionalized to exploit the targeting properties of the peptidomimetic ligand towards two peptide transporters (namely, PEPT1 and PEPT2), up‐regulated in several tumor cells. The compounds were synthesized and fully characterized, mainly by means of elemental analysis, mono‐ and bidimensional NMR spectroscopy, FT‐IR and UV‐Vis spectrophotometries. The crystal structures of three compounds were also solved by X‐ray diffraction. In vitro cytotoxicity studies using a panel of human tumor cell lines (A549, MCF‐7, A2780, H1975, H460 and A431) showed that the dtc‐Pro‐Aib‐OtBu derivative is very effective, with GI50 values much lower than those of Cisplatin. It was thus selected for evaluating the stability under physiological conditions and possible interaction with serum albumin as well as for PARP‐1 enzyme inhibition assays and preliminary ex vivo toxicity experiments on healthy rat tissues

Exploring a tissue engineering strategy as a novel approach for haemophilic arthropathy treatment

Among the most disabling complications of Haemophilia, repeated and sponta- neous intra-articular haemorrhages may cause irreversible damage to the joint. This leads to haemophilic arthropathy, a polyarticular disease characterized by joint stiff- ness, chronic pain and a severely limited range of motion. Occurrence of haemophilic arthropathy can be avoided by the prophylactic administration of clotting factors to prevent articular haemorrhages, but it can also be addressed using anti-inflammatory drugs and surgery to alleviate the effects of articular damage, up to arthroplasty as resolute option [1]. However, innovative strategies for the prevention and treatment of this common and serious complication are still required, due to some important limits of current therapies, first of all inhibitor development. In this work, we inves- tigated a tissue engineering approach to regenerate articular focal lesions in Haemo- philic patients by in vitro development of an autologous bio-hybrid prosthesis. For this purpose, we isolated articular chondrocytes from Haemophilic patients (HaeCs) and characterized them for the first time in literature, to verify whether they were altered by blood exposure. Using healthy chondrocytes as control, optical microscope morphological analysis, flow cytometry immunophenotype evaluation and gene expression study by qRT-PCR were performed. After that, an innovative compos- ite scaffold was obtained by combining decellularized Wharton’s Jelly (W’s J) from human umbilical cord with a novel biodegradable polyvinyl alcohol (PVA) hydrogel [2]. Finally, we assessed HaeCs capacity to re-populate biosynthetic scaffolds by Scan- ning Electron Microscopy and MTT assay on cells seeded on supports. Taken togeth- er, our results contributed to define HaeCs phenotype, highlighting the possibility to use these cells for autologous implant. What is more, HaeCs capacity to growth and proliferate on composite scaffolds set the stage for planning the development of autologous tissue substitutes for haemophilic cartilage regeneration

BIOMATERIALE PER DISPOSITIVI MEDICI, IN PARTICOLARE PROTESI

L'invenzione riguarda un biomateriale per dispositivi medici, in particolare protesi, che comprende come base un polimero poliolico. Il biomateriale trova applicazione in dispositivi medici e come portatore di farmaco per essere applicato in sistemi di DDS (drug delivery System, sistema di rilascio di farmaci)

Fluphenazine phototoxicity: a mechanism involving the trifluoromethyl group

Fluphenazine is a neuroleptic drug used for the long-term treatment of mental disorders, in particular in the therapy of various psychoses, including schizophrenia and mania. Occurrence of skin phototoxic and photoallergic reactions is observed when patients expose themselves to sunlight during treatment. In order to identify the mechanism of fluphenazine phototoxicity, the drug was irradiated with UVA in different solvents and in presence of nucleophilic substrates of biological relevance, like serine, lysine and a serine-containing oligopeptide. The photoproducts were characterized by mass spectroscopy and, for three of them, by NMR spectroscopy. A minor product in which N-oxidation occurred was found in all conditions. The major product which formed in water was a carboxylic acid derived from the hydrolytic defluorination of the CF3 group. In methanol and ethanol the main product was the ester of the carboxylic acid. Serine also bound fluphenazine through the same mechanism. Although with low yield, a similar product was identified in a 14-peptide irardiated in presence of the drug. Lysine did not react in the same conditions. These results suggest that photobinding of fluphenazine to serine residues of proteins may occur in vivo, thus initiating the processes leading to photoallergy

Folding of thermolysin fragments. Identification of the minimum size of a carboxyl-terminal fragment that can fold into a stable native-like structure.

The COOH-terminal cyanogen bromide fragment 206-316 of thermolysin has been shown to possess protein domain characteristics that are able to refold into a stable native-like structure (Fontana et al., 1982). We now report the results of limited proteolysis of this fragment with the aim of identifying the minimum size of a COOH-terminal fragment of thermolysin that is able to fold by itself. Proteolysis with subtilisin, chymotrypsin, thermolysin and trypsin allowed us to isolate to homogeneity eight different subfragments, which can be grouped in two sets of peptides, i.e. (218-222)-316 and (252-255)-316. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. In addition, even the smallest fragment isolated (sequence 255-316) shows co-operative and reversible unfolding transitions mediated by heat (tm 65 degrees C) and guanidine hydrochloride (midpoint transition at 2.5 M denaturant), as often observed with globular proteins. From the kinetics of the proteolytic digestion and analysis of the isolated subfragments, it is concluded that proteases lead to a stepwise degradation of fragment 206-316 from its NH2-terminal region, leading to the highly helical fragment (252-255)-316, quite resistant to further proteolytic digestion. The results of this study provide evidence that it is possible to isolate stable supersecondary structures of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain of thermolysin

Limited proteolysis of thermolysin by subtilisin: isolation and characterization of a partially active enzyme derivative.

Incubation of the neutral metalloendopeptidase thermolysin at pH 9-10 in the presence of 10 mM CaCl2 for 2 days at room temperature with subtilisin at a 50:1 molar ratio leads to a derivative possessing lower (approximately 3%) but intrinsic catalytic activity. This derivative, called thermolysin S, was isolated by gel filtration in approximately 80% yield and then separated from some residual intact thermolysin by an affinity chromatographic step on Sepharose-Gly-D-Phe. It was found that thermolysin S results from a tight association of two polypeptide fragments of apparent Mr of 24000 and 10000. Dissociation of the complex was achieved under strong denaturing conditions, such as gel filtration on a column equilibrated and eluted with 5 M guanidine hydrochloride. The positions of the clip sites were defined by amino acid analysis, end-group determination, and amino acid sequencing of the isolated fragments and shown to lie between Thr-4 and Ser-5, between Thr-224 and Gln-225, and also between Gln-225 and Asp-226. Thermolysin S, which is therefore a stable complex of fragments 5-224(225) and 225(226)-316, shows a shift in optimum pH of about 1 unit toward the acid range with respect to intact thermolysin and a Km essentially unchanged, with furylacryloyl-Gly-Leu-NH2 as substrate. Inhibitors of thermolysin such as ethoxyformic anhydride and Zn2+ ions inactivate also the nicked enzyme