Engineering Protein Based Nanoparticles for Applications in Tissue Engineering

Inclusion bodies (IBs) are mechanically stable, submicron protein particles of 50-500nm that dramatically favor mammalian cell spread when used for substrate surface decoration. IBs are protein deposits produced by recombinant bacteria that can retain the biological activities of the embedded polypeptides. Here we report on the physicochemical characteristics of these protein nanoparticles, such as size, wettability, stiffness, and so on, and their relevant nanoscale properties as particulate bionanomaterials using SEM, DLS, cryoTEM, force spectroscopy, AFM, and confocal microscopy. Notably, the produced nanoparticles can be tailored (stiffness, size, shape, etc.) by simple approaches like the proper choice of the producing bacteria genetic background or the tuning of process variables such as culture temperature, harvesting time, and media composition among others. IBs deposited on surfaces of self-assembled molecular monolayers promote the adhesion, proliferation, and guidance of different kinds of mammalian cells, owing to the biofunctional nature and topochemical characteristics. Moreover, macroscopic responses of cultured mammalian cells are different for the different IB variants when used as particulate materials to engineer the nanoscale topography, proving that the actual range of referred mechanical and physicochemical properties are sensed and discriminated by the cells. This chapter also shows the recent advances made in the engineering of supports at the microscale by decorating them with this novel protein-based nanomaterial. This procedure consists of a modification of the μ-CP technique that allows an increase of the mass transfer of IBs from the stamps to the substrates. In addition, a methodology for the statistical analysis of images of fibroblast cells cultured at different times over IBs patterns, with various geometries and densities, to study the cell response, is presented. These analyses enabled the study of the influence of different microscale structuring (dots and stripes of different sizes) of IBs on the orientation, morphology, and positioning of cells, demonstrating the importance of 2D microscale engineering and the usefulness of the IB nanoscale profiling for cell guidance. It has been shown that cells preferentially adhere to IB-rich areas, aligning and elongating according to the IB pattern and choosing the shortest way to reach new adhesion spots on the IBs. The 2D engineering technique based on IBs presented in this chapter fills the gap between existing techniques based on the local modification of the chemical nature of the surface and those based on modification of the topography at the nanoscale level by physical methods, since IBs combine at the same time biofunctionalization and topographical modification of the roughness. Therefore, IBs are interesting and useful nanomaterials with application in the control of cell culture as well as promising biomaterials for tissue engineering.