The formation ofpolysaccharide films based on the alternate deposition of chitosan (CHI) and hyaluronan (HA) was investigated by several techniques. The multilayer buildup takes place in two stages: during the first stage, the surface is covered by isolated islets that grow and coalesce as the construction goes on. After several deposition steps, a continuous film is formed and the second stage of the buildup process takes place. The whole process is characterized by an exponential increase of the mass and thickness of the film with the number of deposition steps. This exponential growth mechanism is related to the ability of the polycation to diffuse "in" and "out" of the whole film at each deposition step. Using confocal laser microscopy and fluorescently labeled CHI, we show that such a diffusion behavior, already observed with poly(L-lysine) as a polycation, is also found with CHI, a polycation presenting a large persistence length. We also analyze the effect of the molecular weight (MW) of the diffusing polyelectrolyte (CHI) on the buildup process and observe a faster growth for low MW chitosan. The influence of the salt concentration during buildup is also investigated. Whereas the CHI/HA films grow rapidly at high salt concentration (0.15 M NaCl) with the formation of a uniform film after only a few deposition steps, it is very difficult to build the film at 10(-4) M NaCl. In this latter case, the deposited mass increases linearly with the number of deposition steps and the first deposition stage, where the surface is covered by islets, lasts at least up to 50 bilayer deposition steps. However, even at these low salt concentrations and in the islet configuration, CHI chains seem to diffuse in and out of the CHI/HA complexes. The linear mass increase of the film with the number of deposition steps despite the CHI diffusion is explained by a partial redissolution of the CHI/HA complexes forming the film during different steps of the buildup process. Finally, the uniform films built at high salt concentrations were also found to be chondrocyte resistant and, more interestingly, bacterial resistant. Therefore, the (CHI/HA) films may be used as an antimicrobial coating.

The mechanisms underlying the immunomodulatory functions of mesenchymal stem cells (MSC) on dendritic cells (DC) have been shown to involve soluble factors, such as IL-6 or TGF-β, or cell-cell contact, or both depending on the report referenced. In this study, we intend to clarify these mechanisms by examining the immunosuppressive effect of human adult MSC on adult DC differentiated from CD34+ hemopoietic progenitor cells (HPC). MSC have been shown to inhibit interstitial DC differentiation from monocytes and umbilical CD34+ HPC. In this study, we confirm that MSC not only halt interstitial DC but also Langerhans cell differentiation from adult CD34+ HPC, as assessed by the decreased expression of CD1a, CD14, CD86, CD80, and CD83 Ags on their cell surface. Accordingly, the functional capacity of CD34+ HPC-derived DC (CD34-DC) to stimulate alloreactive T cells was impaired. Furthermore, we showed that 1) MSC inhibited commitment of CD34+ HPC into immature DC, but not maturation of CD34-DC, 2) this inhibitory effect was reversible, and 3) DC generated in coculture with MSC (MSC-DC) induced the generation of alloantigen-specific regulatory T cells following secondary allostimulation. Conditioned medium from MSC cultures showed some inhibitory effect independent of IL-6, M-CSF, and TGF-β. In comparison, direct coculture of MSC with CD34+ HPC resulted in much stronger immunosuppressive effect and led to an activation of the Notch pathway as assessed by the overexpression of Hes1 in MSC-DC. Finally, DAPT, a γ-secretase inhibitor that inhibits Notch signaling, was able to overcome MSC-DC defects. In conclusion, our data suggest that MSC license adult CD34+ HPC to differentiate into regulatory DC through activation of the Notch pathway.

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Osteoarthritis is characterized by a gradual degradation of extracellular matrix, resulting from an excess of chondrocyte cell death, mainly due to an increase in apoptotis. Recent studies have revealed the essential role of HSP70 in protecting cells from stressful stimuli. Therefore, overexpressing HSP70 in chondrocytes could represent a good strategy to prevent extracellular matrix destruction. To this end, we have developed a vector carrying HSP70/GFP, and transduced chondrocytes were thus more resistant to cell death induced by mono-iodoacetate (MIA). To overcome the barrier-effect of matrix, we investigated the efficacy of plasmid delivery by electroporation (EP) in rat patellar cartilage. Two days after EP, 50% of patellar chondrocytes were HSP/GFP+. After 3 months, long-term expression of transgene was only depicted in the deep layer (20-30% positive cells). HSP70 overexpression inhibited the natural endochondral ossification in the deep layer, thus leading to a lesser decrease in chondrocyte distribution. Moreover, overexpression of HSP70, after a preventive EP transfer in rat patella, was sufficient to decrease the severity of osteoarthritis-induced lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of HSP70, through EP delivery, could protect chondrocytes from cellular injuries and thus might be a novel chondroprotective modality in rat OA.