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Optimal scaffold characteristics are essential for the therapeutic application of engineered tissues. Hydraulic permeability ( k ) affects many properties of collagen gels, such as mechanical properties, cell–scaffold interactions within three dimensions (3D), oxygen flow and nutrient diffusion. However, the cellular response to 3D gel scaffolds of defined k values has not been investigated. In this study, unconfined plastic compression under increasing load was used to produce collagen gels with increasing solid volume fractions. The Happel model was used to calculate the resulting permeability values in order to study the interaction of k with gel mechanical properties and mesenchymal stem cell (MSC)-induced gel contraction, metabolism and differentiation in both non-osteogenic (basal medium) and osteogenic medium for up to 3 weeks. Collagen gels of fibrillar densities ranging from 0.3 to >4.1 wt.% gave corresponding k values that ranged from 1.00 to 0.03 μm 2 . Mechanical testing under compression showed that the collagen scaffold modulus increased with collagen fibrillar density and a decrease in k value. MSC-induced gel contraction decreased as a direct function of decreasing k value. Relative to osteogenic conditions, non-osteogenic MSC cultures exhibited a more than 2-fold increase in gel contraction. MSC metabolic activity increased similarly under both osteogenic and non-osteogenic culture conditions for all levels of plastic compression. Under osteogenic conditions MSC differentiation and mineralization, as indicated by alkaline phosphatase activity and von Kossa staining, respectively, increased in response to an elevation in collagen fibrillar density and decreased gel permeability. In this study, gel scaffolds with higher collagen fibrillar densities and corresponding lower k values provided a greater potential for MSC differentiation and appear most promising for bone grafting purposes. Thus, cell–scaffold interactions can be optimized by defining the 3D properties of collagen scaffolds through k adjustment.

Leishmania parasites have evolved sophisticated mechanisms to subvert macrophage immune responses by altering the host cell signal transduction machinery, including inhibition of JAK/STAT signalling and other transcription factors such as AP-1, CREB and NF-κB. AP-1 regulates pro-inflammatory cytokines, chemokines and nitric oxide production. Herein we show that upon Leishmania infection, AP-1 activity within host cells is abolished and correlates with lower expression of 5 of the 7 AP-1 subunits. Of interest, c-Jun, the central component of AP-1, is cleaved by Leishmania. Furthermore, the cleavage of c-Jun is dependent on the expression and activity of the major Leishmania surface protease GP63. Immunoprecipitation of c-Jun from nuclear extracts showed that GP63 interacts, and cleaves c-Jun at the perinuclear area shortly after infection. Phagocytosis inhibition by cytochalasin D did not block c-Jun down-regulation, suggesting that internalization of the parasite might not be necessary to deliver GP63 molecules inside the host cell. This observation was corroborated by the maintenance of c-Jun cleavage upon incubation with L. mexicana culture supernatant, suggesting that secreted, soluble GP63 could use a phagocytosis-independent mechanism to enter the host cell. In support of this, disruption of macrophage lipid raft microdomains by Methyl β-Cyclodextrin (MβCD) partially inhibits the degradation of full length c-Jun. Together our results indicate a novel role of the surface protease GP63 in the Leishmania-mediated subversion of host AP-1 activity., Author Summary Leishmaniasis is a tropical disease affecting more than 12 million people around the world. The disease is caused by the Leishmania parasites that are transmitted to the mammalian host by a sandfly vector when it takes a blood meal. The parasites are able to survive and multiply inside of cells that comprise the primary defence of the host, the macrophages. We have extensively studied the mechanism whereby Leishmania escapes from macrophage microbicidal functions. Herein we report that the parasite can inactivate these cells by decreasing the activity of transcription factors such as Activated Protein-1(AP-1) that are involved in transcription of genes coding for antimicrobial functions of macrophages. In this study, we showed that Leishmania parasites use their most abundant surface protein GP63 to inactivate the AP-1 transcription factor. Furthermore, we found that GP63 enter into the macrophages independently of parasite internalization using lipid rich microdomains localized in the cellular membrane. In addition, GP63 was observed to reach the nuclear compartment where it cleaves AP-1 subunit proteins. Collectively, our findings reveal a novel mechanism used by Leishmania to facilitate its survival and propagation within its mammalian host cells. Better knowledge concerning the mechanisms whereby this pathogen can escape the innate immune response may help to develop new anti-Leishmania therapy.