Biofabrication of Composite Bioink-Nanofiber Constructs: Effect of Rheological Properties of Bioinks on 3D (Bio)Printing and Cells Interaction with Aligned Touch Spun Nanofibers.

This paper reports on a novel approach for the fabrication of composite multilayered bioink-nanofibers construct. This work achieves this by using a hands-free 3D (bio)printing integrated touch-spinning approach. Additionally, this work investigates the interaction of fibroblasts in different bioinks with the highly aligned touch-spun nanofibers. This work conducts a comprehensive characterization of the rheological properties of the inks, starting with low-strain oscillatory rheology to analyze the viscoelastic behavior, when the material structure remains intact. Moreover, this work performs amplitude sweeps to investigate the stability of the inks under large deformations, rotational rheology to examine the shear thinning profile, and a three-step creep experiment to study time-dependent rheological behavior. The obtained rheological results are correlated to visual observation of the flow behavior of inks. These behaviors span from an ink with zero-shear viscosity, very weak shear thinning, and no thixotropic behavior to inks exhibiting flow stress, pronounced shear thinning, and thixotropy. It is demonstrated that inks have an essential effect on cell behavior. While all bioinks allow a preferred directionality of the fibroblasts along the fiber direction, cells tend to form aggregates in bioinks with higher viscosity, and a considerable number of agglomerates are observed in the presence of laponite-RD.

1D micro-nanopatterned integrin ligand surfaces for directed cell movement.

Cell-extracellular matrix (ECM) adhesion mediated by integrins is a highly regulated process involved in many vital cellular functions such as motility, proliferation and survival. However, the influence of lateral integrin clustering in the coordination of cell front and rear dynamics during cell migration remains unresolved. For this purpose, we describe a novel protocol to fabricate 1D micro-nanopatterned stripes by integrating the block copolymer micelle nanolithography (BCMNL) technique and maskless near UV lithography-based photopatterning. The photopatterned 10 µm-wide stripes consist of a quasi-perfect hexagonal arrangement of gold nanoparticles, decorated with the RGD (arginine-glycine-aspartate) motif for single integrin heterodimer binding, and placed at a distance of 50, 80, and 100 nm to regulate integrin clustering and focal adhesion dynamics. By employing time-lapse microscopy and immunostaining, we show that the displacement and speed of fibroblasts changes according to the nanoscale spacing of adhesion sites. We found that as the lateral spacing of adhesive peptides increased, fibroblast morphology was more elongated. This was accompanied by a decreased formation of mature focal adhesions and stress fibers, which increased cell displacement and speed. These results provide new insights into the migratory behavior of fibroblasts in 1D environments and our protocol offers a new platform to design and manufacture confined environments in 1D for integrin-mediated cell adhesion.

Effect of acoustic standing waves on cellular viability and metabolic activity.

Acoustic standing wave devices offer excellent potential applications in biological sciences for drug delivery, cell manipulation and tissue engineering. However, concerns have been raised about possible destructive effects on cells due to the applied acoustic field, in addition to other produced secondary factors. Here, we report a systematic study employing a 1D resonant acoustic trapping device to evaluate the cell viability and cell metabolism for a healthy cell line (Human Dermal Fibroblasts, HDF) and a cervical cancer cell line (HeLa), as a function of time and voltages applied (4-10 Vpp) under temperature-controlled conditions. We demonstrate that high cell viability can be achieved reliably when the device is operated at its minimum trapping voltage and tuned carefully to maximise the acoustic standing wave field at the cavity resonance. We found that cell viability and reductive metabolism for both cell lines are kept close to control levels at room temperature and at 34 °C after 15 minutes of acoustic exposure, while shorter acoustic exposures and small changes on temperature and voltages, had detrimental effects on cells. Our study highlights the importance of developing robust acoustic protocols where the operating mode of the acoustic device is well defined, characterized and its temperature carefully controlled, for the application of acoustic standing waves when using live cells and for potential clinical applications.