Controlling the Adipose-derived Stem cell 3D-organization on micrometric PLGA regular scaffolds for cardiac tissue regeneration and repair

The deposition of cells at sites of injury is a clinically relevant approach to facilitate local tissue regeneration and repair. However, cell engraftment, retention, and survival are generally modest, requiring the development of novel deposition techniques and biomaterials. Here, a micro-sized polymeric network (microMESH) is investigated as a promising biodegradable scaffold for the engraftment and tissue integration of human Adipose-Derived Stem Cells (hADSCs) to be used for a wide range of injuries, including myocardial infarction. microMESH comprises a regular network of PLGA microfilaments spatially organized to form square openings of 5x5, 10x10 and 20x20 μm2 . microMESH is realized using soft lithographic techniques starting from a master silicon template reproducing the actual geometry of the final PLGA network. After extensive geometrical, physico-chemical, and mechanical characterizations using a broad range of techniques, hADSCs were integrated with microMESH. Cell viability, spatial organization, secretome and stemness were characterized for all three different microMESH configurations and compared to conventional systems, including 2D plastic dishes and collagen layers. Interestingly, when hADSCs were cultured on microMESH they organized in spheroidal-like structures, despite the geometry, maintaining viability over time. This peculiar attitude of the microMESH to form assemblies better represents the human tissue outside the body, compared to 2D monolayer cultures. Additionally, spheroids established an intimate interaction with the microMESH resulting in the scaffold incorporation within the 3D arrangement formed by the cells. On the contrary, hADSCs form only superficial interaction above the flat collagen sheet that is currently used for cell transplantation in animal models of cardiac diseases. Moreover, once the hADSCs are placed on microMESH, the actin cytoskeleton reorganizes to confer a 3D cell shape with multidirectional actin arrangements, forming nonlinear structures and ring structures at the anchorage site to the microMESH, relative to linear filaments when the cells adhered and flattened onto the plastic surface and on top of collagen scaffold. This internal reorganization and the stronger interaction may explain why microMESH scaffold fostered the secretion of biologically active molecules, acting in a paracrine fashion on resident cells, which are expected to accelerate tissue regeneration and repair. Specifically, when hADSCs grew on microMESH we observed a trend for higher production of several factors with specific implications in angiogenesis, stem cell proliferation and expansion, cell survival, inflammation modulation, ECM remodeling, stem cell mobilization, chemotaxis and homing, relative to 2D monolayer conditions. The paracrine effect of hADSCs is scaffold dependent and can be modulated by tailoring the geometrical and mechanical properties of microMESH. Indeed, the 5x5 microMESH showed its contribution in angiogenesis, ECM remodeling and stem cell mobilization from bone marrow into the bloodstream. Indeed, highest amounts of VEGF, TIMP-2 and GCSF, respectively were detected in 5x5 geometry compared to the other conditions. Rather, 10x10 geometry promotes angiogenesis enhancing the VEGF production, stem cell proliferation and survival by raising the Fibroblast Growth Factors family secretion and EGF factor, respectively and favors ECM remodeling increasing the TIMP-2 production compared to other conditions. Lastly, the 20x20 seems to have a more anti-inflammatory role (combination of IL-10 and TGF-β1) and chemotactic function (e.g. RANTES). Finally, in this work, we started to shed new light on the ability of micromMESH geometry to modulate the hADSCs stemness evaluating the expression levels of CD44, CD90 and CD105 markers over time. The proposed microMESH scaffold is expected to provide an effective alternative to more conventional hADSCs transplant techniques.