Comparison of Bioorthogonally Cross-Linked Hydrogels for in Situ Cell Encapsulation

Hydrogels are water-saturated polymer networks and extensively used in drug delivery, tissue repair engineering, and cell cultures. For encapsulation of drugs or cells, the possibility to form hydrogels in situ is very much desired. This can be achieved in numerous ways, including use of bioorthogonal chemistry to create polymer networks. Here we report a set of bioorthogonally clickable polymers that was designed with the aim to find a combination that could rapidly encapsulate cells in a three-dimensional manner to improve the preparation of hydrogels as tissue mimics. To this end, tetrazine (Tet), trans-cyclooctene (TCO), azide (N₃), dibenzocyclooctyne (DBCO), bicyclo[6.1.0]nonyne (BCN), 3,4-dihydroxyphenylacetic acid (DHPA), and norbornene (Norb) were grafted to four-armed poly(ethylene)glycol (star-PEG) polymers of 10 kDa. Inverted vial tests and rheology demonstrated that hydrogels formed within seconds from combinations of TCO-Tet, BCN-DHPA, and BCN-Tet. Hydrogels from DBCO-N₃, DBCO-DHPA, and BCN-N₃ formed in the range of minutes, whereas the Norb-Tet ligation required multiple hours to form a gel. After this comparison, we chose to prepare hydrogels via DBCO-N₃ and BCN-N₃ and employed them for human mesenchymal stem cell (HMSC) cultures for a period of 5 days. We additionally incorporated RGDS and MMP cleavable peptide (MMPcp) motifs in these gels to stimulate cell adhesion and add degradability. Both DBCO and BCN gel systems including the functional peptide motifs allowed HMSCs to be viable and spread in 5 days. The DBCO-based hydrogel could trap cells at different depths due to its fast gelation process, while the slower gelation of the BCN-based hydrogel led to cell sedimentation.