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 in vitro 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., T > T 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 (R). 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 R 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 R by 1 order of magnitude, achieving elastic modulus E 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 (E < 1 kPa). Hydrogels prepared with an excess of Tz or Nb were successfully adhered and remained in contact during in vitro 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 in vitro modeling applications.

In vitro and in vivo assessment of a 3D printable gelatin methacrylate hydrogel for bone regeneration applications

“…Up to date, ceramic‐based scaffolds, 2–6 synthetic thermoplastic polymeric scaffolds, 7–10 or their composites 8,11–13 have widely been employed to satisfy the mechanical characteristics of the native bone tissue. In recent years hydrogels have become a prominent choice of material thanks to their significant intrinsic resemblances to a natural extracellular matrix 14–22 . Specifically, hydrogels driven from natural sources such as collagen, 15,20 fibrinogen, 15–18 hyaluronic acid 23,24 possess integrins that do not only promote cell attachment but also play an important role in extracellular matrix deposition and differentiation.…”

Editorial: Special Issue on Advanced Biomedical Hydrogels

“…Continuing with new molecules, Celiz and co-workers report mechanically tunable and adhesive gelatin hydrogels via bio-orthogonal click chemistry reactions of tetrazine and norbornene . Varying the degree of tetrazine or norbornene modification on gelatin and the molar ratio of the tethering groups in the hydrogel, the elastic moduli ranged from 0.5 to 5 kPa.…”

Mimicking the Natural Basement Membrane for Advanced Tissue Engineering

“…By tuning the ratio of tetrazine to norbornene and their degree of modification, hydrogels ranging from 1 to 5 kPa were achieved that were used to embed human dental pulp stem cells. 238 …”