3D Printing of Thermo-Responsive Methylcellulose Hydrogels for Cell-Sheet Engineering.

Andrea Cochis,Nicola Contessi Negrini,Silvia Farè,Massimiliano Leigheb,Federico Grassi,Lorenzo Bonetti,Lia Rimondini,Rita Sorrentino

doi:10.3390/ma11040579

Abstract

A possible strategy in regenerative medicine is cell-sheet engineering (CSE), i.e., developing smart cell culture surfaces from which to obtain intact cell sheets (CS). The main goal of this study was to develop 3D printing via extrusion-based bioprinting of methylcellulose (MC)-based hydrogels. Hydrogels were prepared by mixing MC powder in saline solutions (Na2SO4 and PBS). MC-based hydrogels were analyzed to investigate the rheological behavior and thus optimize the printing process parameters. Cells were tested in vitro on ring-shaped printed hydrogels; bulk MC hydrogels were used for comparison. In vitro tests used murine embryonic fibroblasts (NIH/3T3) and endothelial murine cells (MS1), and the resulting cell sheets were characterized analyzing cell viability and immunofluorescence. In terms of CS preparation, 3D printing proved to be an optimal approach to obtain ring-shaped CS. Cell orientation was observed for the ring-shaped CS and was confirmed by the degree of circularity of their nuclei: cell nuclei in ring-shaped CS were more elongated than those in sheets detached from bulk hydrogels. The 3D printing process appears adequate for the preparation of cell sheets of different shapes for the regeneration of complex tissues.

Highlights

Tissue engineering (TE) is one possible approach in regenerative medicine; its main goal is to overcome limited donor availability and the high risk of rejection, which affects the clinical treatments currently used [1,2,3].Among possible TE approaches, cell-sheet engineering (CSE) provides an approach that is innovative compared to the use of biodegradable scaffolds or the injection of cell suspensions at the body site, including by a cell carrier
Among the possible smart hydrogels used for CSE, methylcellulose (MC), a cellulose derivative obtained by partial substitution of the hydroxyl groups (–OH) with methoxy groups (–OCH3 ), can be mixed in aqueous solution to obtain thermo-responsive hydrogels [7,8,9,10]
lower critical solution temperature (LCST) reduction for MC-Na005 and MC-PBS20 solutions is triggered by SO4 − ions and Cl− ions, respectively, inducing water molecule spillage from the polymeric structure [13,22,24]

Summary

Introduction

Tissue engineering (TE) is one possible approach in regenerative medicine; its main goal is to overcome limited donor availability and the high risk of rejection, which affects the clinical treatments currently used [1,2,3].Among possible TE approaches, cell-sheet engineering (CSE) provides an approach that is innovative compared to the use of biodegradable scaffolds or the injection of cell suspensions at the body site, including by a cell carrier. CSE entails developing smart cell culture surfaces, permitting the in vitro culture of cells and the detachment by specific stimuli (i.e., temperature variation) of intact cell sheets (CS), preserving the gap junctions between cells and the extracellular matrix (ECM) proteins [2]. These smart surfaces are usually composed of thermo-responsive polymers, i.e., materials whose chemical-physical properties and rheological behavior changes in response to Materials 2018, 11, 579; doi:10.3390/ma11040579 www.mdpi.com/journal/materials. The sol-gel transition is accompanied by a change in the hydrogel’s water affinity from hydrophilic in the sol state to hydrophobic in the gel state [10]

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