3D cell entrapment in crosslinked thiolated gelatin-poly(ethylene glycol) diacrylate hydrogels

Abstract

The combined use of natural ECM components and synthetic materials offers an attractive alternative to fabricate hydrogel-based tissue engineering scaffolds to study cell-matrix interactions in three-dimensions (3D). A facile method was developed to modify gelatin with cysteine via a bifunctional PEG linker, thus introducing free thiol groups to gelatin chains. A covalently crosslinked gelatin hydrogel was fabricated using thiolated gelatin and poly(ethylene glycol) diacrylate (PEGdA) via thiol-ene reaction. Unmodified gelatin was physically incorporated in a PEGdA-only matrix for comparison. We sought to understand the effect of crosslinking modality on hydrogel physicochemical properties and the impact on 3D cell entrapment. Compared to physically incorporated gelatin hydrogels, covalently crosslinked gelatin hydrogels displayed higher maximum weight swelling ratio (Qmax), higher water content, significantly lower cumulative gelatin dissolution up to 7 days, and lower gel stiffness. Furthermore, fibroblasts encapsulated within covalently crosslinked gelatin hydrogels showed extensive cytoplasmic spreading and the formation of cellular networks over 28 days. In contrast, fibroblasts encapsulated in the physically incorporated gelatin hydrogels remained spheroidal. Hence, crosslinking ECM protein with synthetic matrix creates a stable scaffold with tunable mechanical properties and with long-term cell anchorage points, thus supporting cell attachment and growth in the 3D environment.

3-D Non-UV Digital Printing of Hydrogel Microstructures by Optically Controlled Digital Electropolymerization

“…T ECHNIQUES that could precisely pattern hydrogels with tunable geometric shape and size in micro/nano scale are desired for developing hydrogels' applications in drug delivery [1], cell encapsulation [2], [3], cell-environment interaction research [4], [5] and tissue engineering [6]- [8]. To date, three-dimensional micro hydrogel structures have been created by a variety of methods, including photolithography, softlithography (e.g., micro-contact printing, microfluidic patterning, and micro molding) [9]- [11].…”

Protein-based injectable hydrogels towards the regeneration of articular cartilage

“…The effective cellular encapsulation and growth in the two types of hydrogels confirm their viability and prove that the PHEMA grafting onto the fibers does not compromise the use of the composites as injectable materials for tissue engineering, as previously reported for pure injectable gelatin [129], [186].…”

“…Figure 3-7 also illustrates the actin cytoskeleton of L929 cells encapsulated in GEL-TA and composites at the culture time of 1 and 21 days. At day 1, cells are proliferating in the hydrogels, and developed a narrow actin cytoskeleton showing a fairly round shape as typically occurs in cells encapsulated in low stiffness hydrogels for this short period of time [186]- [188]. The cellular growth indicates low toxicity of enzymatically cross-linked hydrogels as reported by previous studies where the compatibility, spreading and cell attachment onto the surface of enzymatically crosslinked GEL-TA was evaluated [129], [189].…”

“…Thus, in the case of the composite, proliferation stops at 14 days of culture. for GEL and GEL+1PLLA-PHEMA, respectively) the density of cells inside the hydrogels is low and their shape is round, as usually reported in low stiffness hydrogels cultured for short periods of time [186]- [188]. The first differences between the two types of hydrogels can be seen in Figure 3-21c and Figure 3-21d.…”

“…In fact, we can consider this relatively unusual compared with other bibliographic references where the cells are able to contract the hydrogel molecules. This is the case of collagen hydrogels seeded with 5 x 10 4 to 5 x 10 7 cells/mL of mesenchymal stem cells (MSCs) [180]- [182] or with 1 x 10 6 cells/mL of porcine smooth muscle cells [165], collagen-methacrylated hyaluronic acid hydrogel seeded with 2 x 10 6 cells/mL of NIH-3T3 cells [183] and fibroblasts seeded in collagen hydrogels [184]- [186]. Figure 3-7 also illustrates the actin cytoskeleton of L929 cells encapsulated in GEL-TA and composites at the culture time of 1 and 21 days.…”