Cytokines released by hMSCs have a wide variety of effects on other cells or local tissues, including immunomodulation, angiogenesis, chemoattraction, anti-scarring and supporting local stem cells in tissue regeneration. Hydrogels are widely used as extracellular matrix (ECM) to mimic cellular microenvironment because of their mechanical and structural similarity to native tissues. This motivated us to develop a “cell factory”, where hMSCs encapsulated in hydrogels would be injected in an electrospun scaffold to improve cell survival and retention. Although hydrogels formed by natural biopolymers well recapitulate the architecture and mechanical properties of the ECM, they are difficult to tailor and manipulate. Oppositely, synthetic hydrogels would give rise to uniquely tailorable materials. However, the mechanical properties of these materials are manipulated by typically varying the polymer concentration, which, however changes all other hydrogel properties simultaneously. Strategies to fabricate hydrogels with tailorable mechanical properties but without significantly changing the components are highly challenging.
Here, we started by employing the fully synthetic semi-flexible ethylene glycol-substituted PIC polymers. The thermal-responsive hydrogels are reversibly formed when an aqueous PIC solution is heated above the gelation temperature. The gel is unique as it forms fibrous networks in a non-covalent bundled architecture that closely mimics the architecture of biopolymer networks, such as those based on collagen, fibrin, and actin. PIC gels display the same characteristic strain-stiffening response as the biopolymer hydrogels, meaning the stiffness sharply increases upon deformation. The PIC polymer chains were equipped with azide groups for crosslinking. To introduce dynamic properties in this static fibrous hydrogels, we developed hydrogen bonding crosslinking and ionic crosslinking. The interactions between the polymer chains were achieved by a small spacer with, at one terminus, a dibenzocyclooctyne (DBCO) group that can covalently bind to the azide groups on the PIC chains and on the other terminus a dopamine moiety that forms a dynamic hydrogen bond or metal-coordination bond to the ion. The covalent crosslinking through a di-functional DBCO crosslinkers was introduced as a comparison. To promote cell-matrix interactions, the cell adhesive peptide GRGDS equipped with a DBCO-terminated PEG spacer was conjugated to azide-functionalized PIC polymers. Hence, we developed PIC hydrogels with four crosslinking approaches, including physical crosslinking, non-covalent (hydrogen bonding and ionic) crosslinking, and covalent crosslinking to study the effects on cell behaviors, where crosslinking density was sequentially increasing. As a control, alginate and collagen hydrogels were used to assess the effect of hydrogel types on cell activities. The mechanical properties of PIC hydrogels mixed with magnetic nanoparticles were extremely increased under an external magnetic field, owing to the strain-, or stress-stiffening properties of PIC hydrogels.
hMSCs were encapsulated in PIC hydrogels. We found that a high cell density promoted cell spreading, while a high polymer concentration decreased cell spreading. Live-dead assay indicated that cells had high affinity in RGD-conjugated PIC hydrogels. We also tested the effect of crosslinking density on cell behavior. Cell spreading in physical crosslinked hydrogels was highest, while in covalent crosslinked hydrogels was lowest. Therefore, we could control cell fate by simply varying the crosslinking approaches in hydrogels. hMSCs cytokine secretion was also modulated depending on the hydrogel-electospun cup combination used among the newly generated Hydrocup formulations.
