The extracellular matrix (ECM) regulates many aspects of cellular behavior. Although a variety of substrates have been engineered as ligament models to investigate ECM-cell interactions, they do not exhibit the topographical features of native healthy tissues nor possess chemical cues necessary to stimulate cellular proliferation and growth. Hence, starting from the design of a microfluidic device capable to anisotropically reconstitute collagen gels, fiber – like substrates were obtained with tunable stiffness by incorporating multiwalled carbon nanotubes (MWCNTs) and gold nanoparticles (AuNPs) to promote cellular proliferation. Gels’ stiffness was characterized by rheology. Scanning electron microscopy (SEM) was used to assess fibers orientation pattern of microfluidic fabricated gels. Hyperspectral imaging and atomic force microscopy analysis were carried to verify the embedding of nanofillers within the anisotropic collagen gels. Mice fibroblasts were used to address the biocompatibility of the substrate by lactate dehydrogenase (LDH) assay. Confocal and electron microscopies were employed to study the cellular morphology. To evaluate cellular proliferation behavior, cell nuclei were counted by employing imaging analysis and after trypsinization from substrates. Transforming growth factor beta1 (TGF-β1) values excreted by cells were evaluated by Enzyme-linked Immunosorbent Assay (ELISA). Rheological analysis showed a substantial increase in storage moduli for the MWCNTs embedded gels (109.00±10.11 Pa) compared to AuNPs sample (44.20±11.17 Pa) each loaded with 15% in weight of nanofillers. SEM images analysis showed a preferred orientation of collagen fibers arranged in bundles for all the microfluidic fabricated samples compared to the isotropic gel. Hyperspectral imaging and atomic force microscopy confirm the embedding of both the MWCNTs and the AuNPs along the preferred fibers orientation. Confocal pictures demonstrate fibroblast adhesion and an increased cellular spreading when cultured along the aligned collagen bundle. Fibroblasts show a higher proliferation and execration of TGF-β1 behavior on all the aligned gels compared to the isotropic sample. A novel anisotropic, microfluidic fabricated substrate was successfully designed as a platform to study cellular behavior. We demonstrated alignment of reconstituted collagen gels dictated by the microfluidic device as well as oriented cellular cytoskeleton spreading and higher growth factor secretion

Microfluidic spinning as a facile method to fabricate ligament like extracellular matrix model

ACS Spring Meeting 2021

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