Co-culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small-scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold-based constructs generated by combining primary human ACs with human bone marrow MSCs (BM-MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM-MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co-culture of ACs (25%) with BM-MSCs (75%) in scaffolds resulted in up to 1.4-fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8-fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM-MSCs with a relatively low fraction of non-expanded ACs in porous scaffolds. The proposed co-culture strategy is directly relevant towards a single-stage surgical procedure for cartilage repair.

The p73 gene has a complex regulation, which leads to the expression of different isoforms, often with opposite biological effects. We have generated in the human colocarcinoma cell line HCT116, expressing a wild-type p53, an inducible DNp73a expressing system. Two clones (HCT116/DN3 and HCT116/DN14), upon doxycycline addition, show a strong expression of DNp73a. In vitro the two DNp73a overexpressing clones grow at similar rate of the control transfected clone (HCT116/8a) and similarly respond to DNA damage. When injected in mice, HCT116/DN3, HCT116/DN14, and HCT116/8a cells grew similarly in the absence or presence of tetracycline. In HCT116/DN3 and HCT116/DN14 tumors, tetracycline induced a strong expression of DNp73a both as mRNA and protein. These results indicate that in this system the overexpression of the DNp73a does not induce a more aggressive phenotype and does not seem to be associated with a reduced response of the cells to treatment with anticancer agents.

BackgroundDrug resistance is one of the major obstacles limiting the activity of anticancer agents. Activation of DNA repair mechanism often accounts for increase resistance to cancer chemotherapy.ResultsWe present evidence that nemorubicin, a doxorubicin derivative currently in clinical evaluation, acts through a mechanism of action different from classical anthracyclines, requiring an intact nucleotide excision repair (NER) system to exert its activity. Cells made resistant to nemorubicin show increased sensitivity to UV damage. We have analysed the mechanism of resistance and discovered a previously unknown mechanism resulting from methylation-dependent silencing of the XPG gene. Restoration of NER activity through XPG gene transfer or treatment with demethylating agents restored sensitivity to nemorubicin. Furthermore, we found that a significant proportion of ovarian tumors present methylation of the XPG promoter.ConclusionsMethylation of a NER gene, as described here, is a completely new mechanism of drug resistance and this is the first evidence that XPG gene expression can be influenced by an epigenetic mechanism. The reported methylation of XPG gene could be an important determinant of the response to platinum based therapy. In addition, the mechanism of resistance reported opens up the possibility of reverting the resistant phenotype using combinations with demethylating agents, molecules already employed in the clinical setting.

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