Incorporation of modified cellulose nanocrystals in platelet lysate-based hydrogels for 3D microenvironment sequestration of platelet lysate bioactive molecules

Platelet-rich blood derivatives have consistently shown to modulate stem cell behavior, including adhesion, proliferation and differentiation by in vitro and animal studies [1]. Among those, platelet lysate (PL) is easily obtained from plasma by releasing platelets protein content composed of growth factors, clotting factors and cytokines. Currently explored PL-based scaffolds (gels, sponges) have limited mechanical properties, fast degradation of the biologically active substances and in vitro/in vivo stability which restrains their application in Tissue Engineering strategies. In order to overcome these limitations, we propose the use of modified cellulose nanocrystals (CNC). The superior strength of CNC and reactive surface of –OH side groups makes it ideal nanofillers to reinforce low strength hydrogels matrix without compromising the biological performance [2]. Modified CNC can act as 1) reinforcing nanofillers and crosslinkers of the protein matrix and as 2) sulfated glycosaminoglycan mimetic entities to reversibly sequester PL-derived growth factors in a 3D microenvironment.

Here, we explored the in situ PL-clotting via thrombin and calcium activation along with the CNC/protein covalent crosslinking, which can improve hydrogel structural integrity and mechanical properties.  In optimized conditions for PL gelation (1 U.mL-1 thrombin and 5 mM CaCl2), incorporation of up to 0.61 wt% CNC considerably improved the microstructural organization, degradation rate, nanomechanics of fibrin fibers and bulk rheological properties of the hydrogels. Concerning cellular behaviour characterization, incorporation of CNC correlates with improved cellular viability, proliferation and spreading in cell-landen hydrogels. The potential selective PL bioactive factors sequestration within PL-CNC hydrogels will be explore by tailoring CNC surface sulfation degrees.