Search

Different formulations of Chitosan/sulphate and Chitosan/PEGDE were produced by physical and chemical reticulation to obtain hydrogels with better physiochemical properties. The hydrogels were analyzed – in terms of their non-toxicity, proliferative, differentiative, inflammatory and immunology responses. Commercial grade Chitosan (Sigma) was solubilized and purified by progressive filtrations. Then, the polymer was freeze-dried in a water soluble cationic form. A physical hydrogel was prepared by mixing a 3 % w/w water solution of the a.m. polymer with different stoichiometric ratios of (SO4=) 1/0.5;1/0.75;1/1 respectively. The hydrogels were cross-linked in multiwells. The chemical hydrogel was synthesized mixing a 3 % w/w water solution of the a.m. polymer, with PEGDE. The hydrogel was purified by water diffusion, then loaded into multiwells. All gels were freeze-dried and sterilized by EtO. Primary bovine chondrocytes were extracted by collagenases (ABIEL) and Pronase (Sigma) from articular hoof tissue. MTS and Acrydin Orange assays were performed to quantify cell viability and apoptosis. CD11b, CD69 and ROS activation were evaluated in inflammatory response and CD11 expression in immune-response. Biocompatibility analyses showed that formulated hydrogels were not toxic and didn’t induce apoptosis. Chondrocyte were alive but not proliferative, inflammation and immune responses were observed but under certain acceptability threshold. The results of this preliminary work suggest that the formulated Chitosan hydrogels are cyto-compatible and induce chondrocyte differentiation. The here tested synthesis process generated hydrogels, which are potentially useful for cartilage regenerative processes. Further mechanical characterizations are in progress.

This report shows that histone deacetylase inhibitors (HDACIs) induced apoptosis in human hepatoma HepG2 cells in a dose- and time-dependent manner. Trichostatin A (TSA), ITF2357 and suberoylanilide hydroxamic acid (SAHA), which were very effective agents, caused apoptotic effects after a lag phase of 12-16 h. In order to elucidate the mechanism of HDACIs action in HepG2 cells we have studied the effects of TSA, ITF2357 and SAHA on acetylation of p53 and histones H2A, H2B, H3 and H4. It was observed that HDACIs rapidly induced acetylation of these proteins, being the effects clearly visible already at 30 min of treatment at the same doses which caused apoptosis. Analysis of the immunocomplexes, obtained from nuclear extracts using an antibody against p53, revealed the presence of acetylated p53 together with acetylated forms of histones and histone acetyltransferases p300 and PCAF. Experiments performed using pifithrin-alpha, a reversible inhibitor of p53, showed a correlation between acetylation of p53 and induction of apoptosis. In addition treatment with siRNA against p53 indicated that p53 is involved in the acetylation of histones. In conclusion, this report suggests that complexes constituted by acetylated p53, acetylated histones and coactivators can play a central role in HDACI-induced apoptosis in HepG2 cells.

This study describes the molecular mechanism by which treatment with 3-AB, a potent inhibitor of PARP, allows human osteosarcoma MG-63 cells to restrict growth and enter differentiation. Our findings show that in MG-63 cells, aberrant gene expression keeps Rb protein constitutively inactivated through hyperphosphorylation and this promotes uncontrolled proliferation of the cells. After 3-AB-treatment, the poly(ADP-ribosyl)ation of nuclear proteins markedly decreases and this results in an increase in both the hypophosphorylated active form of Rb and pRb/E2F complexes. These effects are accompanied by G1 arrest, downregulation of gene products required for proliferation (cyclin D1, beta-catenin, c-Jun, c-Myc and Id2) and upregulation of those implicated in the osteoblastic differentiation (p21/Waf1, osteopontin, osteocalcin, type I collagen, N-cadherins and alkaline phosphatase). Our study suggests that use of PARP inhibitors may induce a remodeling of chromatin with the reprogramming of gene expression and the activation of differentiation.