Protein-based biomaterials with innovative and controlled performance are being sought due to their unique characteristics for use in biomedical fields such as neural implants, drug delivery systems, cellbased therapies and soft tissue engineering. Here, we present a versatile approach for the synthesis of photo-crosslinkable fibroin silk biomaterial with highly tunable mechanical, chemical and biodegradation properties. Unlike the crystalline rich silk fibroin reported previously, the covalently crosslinked fibroin protein photoresist (FPP) via controlled light-induced radical grafting, allows generating a new amorphous biomaterial with tunable properties. It appears that the use of photoreactive acrylate groups to cross-link FPP induces plasticity that can be tuned by changing the photoinitiator concentration of the film. Tensile strength measurements revealed that elasticity was higher for FPP UV-cross-linked materials with higher concentration of photoinitiator. FTIR and relative humidity measurements showed that hydrophilicity was higher for UV-cross-linked FPP. These materials display stiffness between 0.01-1.5 GPa and tensile strains up to 60%, covering a significant portion of the properties of native soft biomaterials. Besides, in vitro proteolytic degradation of the photocrosslinked FPP films demonstrate a tunable degradation rate over a period ranging from hours to weeks. Those biomaterials have been successfully micropatterned by photolithography techniques across several orders of magnitude (μm to cm) and a systematic study of direct patterning of the fibroin protein to form high fidelity and high-resolution structures has been reported. It was also shown that the fabricated protein features are well suited to cell adhesion. The development of protein-based material with controlled and tunable elasticity that can be easily photopatterned into centimeter, micrometer and nanometer components will allow a wide range of applications in biomedical fields requesting a natural functional tissue.
The development of advanced techniques of fabrication of three-dimensional (3D) microenvironments for the study of cell growth and proliferation has become one of the major motivations of material scientists and bioengineers in the past decade. Here, we present a novel residueless 3D structuration technique of poly(dimethylsiloxane) (PDMS) by water-in-PDMS emulsion casting and subsequent curing process in temperature-/pressure-controlled environment. Scanning electron microscopy and X-ray microcomputed tomography allowed us to investigate the impact of those parameters on the microarchitecture of the porous structure. We demonstrated that the optimized emulsion casting process gives rise to large-scale and highly interconnected network with pore size ranging from 500 μm to 1.5 mm that turned out to be nicely adapted to 3D cell culture. Experimental cell culture validations were performed using SaOS-2 (osteosarcoma) cell lines. Epifluorescence and deep penetration imaging techniques as two-photon confocal microscopy unveiled information about cell morphology and confirmed a homogeneous cell proliferation and spatial distribution in the 3D porous structure within an available volume larger than 1 cm3. These results open alternative scenarios for the fabrication and integration of porous scaffolds for the development of 3D cell culture platforms.
In nature, cells exist in three-dimensional (3D) microenvironments with topography, stiffness, surface chemistry, and biological factors that strongly dictate their phenotype and behavior. The cellular microenvironment is an organized structure or scaffold that, together with the cells that live within it, make up living tissue. To mimic these systems and understand how the different properties of a scaffold, such as adhesion, proliferation, or function, influence cell behavior, we need to be able to fabricate cellular microenvironments with tunable properties. In this work, the nanotopography and functionality of scaffolds for cell culture were modified by coating 3D printed materials (DS3000 and poly(ethylene glycol)diacrylate, PEG-DA) with cellulose nanocrystals (CNCs). This general approach was demonstrated on a variety of structures designed to incorporate macro- and microscale features fabricated using photopolymerization and 3D printing techniques. Atomic force microscopy was used to characterize the CNC coatings and showed the ability to tune their density and in turn the surface nanoroughness from isolated nanoparticles to dense surface coverage. The ability to tune the density of CNCs on 3D printed structures could be leveraged to control the attachment and morphology of prostate cancer cells. It was also possible to introduce functionalization onto the surface of these scaffolds, either by directly coating them with CNCs grafted with the functionality of interest or with a more general approach of functionalizing the CNCs after coating using biotin–streptavidin coupling. The ability to carefully tune the nanostructure and functionalization of different 3D-printable materials is a step forward to creating in vitro scaffolds that mimic the nanoscale features of natural microenvironments, which are key to understanding their impact on cells and developing artificial tissues.
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