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 > Tsol-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.
Highlights
Hydrogels, water-swollen polymeric networks, have emerged as unique, powerful platforms for the three-dimensional (3D) culture of cells for tissue engineering, regenerative medicine, and in vitro modeling applications.[1,2] Compared to traditional two-dimensional (2D) culture systems which are not representative of the in vivo cell environment, hydrogels can be designed to recreate a biomimetic 3D extracellular matrix (ECM) by tuning their physical,chemical, and mechanical properties.[3]
We investigate the possibility of finely tuning the physico-mechanical hydrogel properties by fixing the polymer concentration and varying (1) the degree of modification of the gelatin derivatives and (2) the Tz/Nb ratio used to prepare the hydrogel
We demonstrate that rheological properties, swelling, and mechanical properties of the hydrogels can be modulated by varying these hydrogel preparation parameters, and we demonstrate how cell response varies during 3D in vitro culture in different cross-linked gelatin hydrogel formulations
Summary
Water-swollen polymeric networks, have emerged as unique, powerful platforms for the three-dimensional (3D) culture of cells for tissue engineering, regenerative medicine, and in vitro modeling applications.[1,2] Compared to traditional two-dimensional (2D) culture systems which are not representative of the in vivo cell environment, hydrogels can be designed to recreate a biomimetic 3D extracellular matrix (ECM) by tuning their physical, (bio)chemical, and mechanical properties.[3]. We considered the time required to reach 50% of the G′ values at the end of the tests (t50% plateau) as an indicator of the speed of the cross-linking reaction and hydrogel formation.[44] The t50% plateau varies between 10 and 70 min from the onset of cross-linking (Figure 3Aii), depending on the DOM of the gelatin derivatives used. For all the considered ratios R, hydrogels prepared with a low DOM L were characterized by a higher weight variation, compared to hydrogels prepared with medium M and high H DOM (p < 0.05) These differences in hydrogel swelling were consistent with the differences between the calculated swelling ratio values (Figure S2A). If the same DOM is considered, hydrogels prepared with R05 were characterized by lower gel fraction values compared to R1 and R2 hydrogels (p < 0.05), indicating a higher loss of gelatin during swelling. R05_R2 hydrogels were characterized approximately by double the adhesion tensile strength compared to R1_R1 and GTA_GTA hydrogels (Figure 7B, inset graph)
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