Tunable Cross-Linking and Adhesion of Gelatin Hydrogels via Bioorthogonal Click Chemistry

Abstract

Engineering cytocompatible hydrogels with tunable physico-mechanical properties as a biomimetic three-dimensional extracellular matrix (ECM) is fundamental to guide cell response and target tissue regeneration or development of <i>in vitro</i> models. Gelatin represents an optimal choice given its ECM biomimetic properties; however, gelatin cross-linking is required to ensure structural stability at physiological temperature (i.e., <i>T</i> > <i>T</i>_{sol-gel gelatin}). Here, we use a previously developed cross-linking reaction between tetrazine (Tz)- and norbornene (Nb) modified gelatin derivatives to prepare gelatin hydrogels and we demonstrate the possible tuning of their properties by varying their degree of modification (DOM) and the Tz/Nb ratio (<i>R</i>). The percentage DOM of the gelatin derivatives was tuned between 5 and 15%. Hydrogels prepared with higher DOM cross-linked faster (i.e., 10-20 min) compared to hydrogels prepared with lower DOM (i.e., 60-70 min). A higher DOM and equimolar Tz/Nb ratio <i>R</i> resulted in hydrogels with lower weight variation after immersion in PBS at 37 °C. The mechanical properties of the hydrogels were tuned by varying DOM and <i>R</i> by 1 order of magnitude, achieving elastic modulus <i>E</i> values ranging from 0.5 (low DOM and nonequimolar Tz/Nb ratio) to 5 kPa (high DOM and equimolar Tz/Nb ratio). Human dental pulp stem cells were embedded in the hydrogels and successfully 3D cultured in the hydrogels (percentage viable cells >85%). An increase in metabolic activity and a more elongated cell morphology was detected for cells cultured in hydrogels with lower mechanical properties (<i>E</i> < 1 kPa). Hydrogels prepared with an excess of Tz or Nb were successfully adhered and remained in contact during <i>in vitro</i> cultures, highlighting the potential use of these hydrogels as compartmentalized coculture systems. The successful tuning of the gelatin hydrogel properties here developed by controlling their bioorthogonal cross-linking is promising for tissue engineering and <i>in vitro</i> modeling applications.