Hydrogels can have physical properties similar to native extracellular matrix and can promote tissue growth as a 3D environment for cellular encapsulation; however, finding conditions that allow gel formation while maintaining cellular viability is challenging. In this report we present that the functionality of the precursors can significantly enhance network formation, resulting in fewer defects, which is particularly critical for cross-linking conditions that maintain cell viability during encapsulation.
Ideal end-linked cross-linking during hydrogel formation () is described by the Flory-Rehner equation, which predicts mass-equilibrium swelling (Qm
) based on the average molecular weight between cross-links, Mc,ideal
]. PEG hydrogels, however, do not exhibit behavior that is predicted by the Flory-Rehner equation below a 50% PEG content [2
], and the gel swelling increased monotonically as the polymer solids content decreased () and as the cross-linking time decreased (). This trend in swelling is indicative of network defects, such as primary loops, because a decline in cross-linking efficiency induces swelling due to a reduction in network elasticity. Decreasing the solids content would be expected to increase the number of elastically inactive
primary loops (), which results from the decreasing global concentration of reactive groups, yet a stable local concentration of intra
molecular reactive groups () [2
]. Incorporation of a third cross-linking site on the peptide cross-linker introduced an additional branch point for the cross-linking reaction, rather than a network “dead end”, resulting in decreased swelling. Uncontrolled swelling that results from the defects in the network formation can affect the concentration of the incorporated biological cues, and influence the physical environment of the encapsulated cells. Taken together, the differential swelling between the hydrogel formulations and the theory illustrates the opportunity for network design in non-ideal cross-linking conditions.
Ovarian follicle culture is being investigated as a means to preserve fertility for females facing premature ovarian failure as a consequence of chemotherapy treatment or other reproductive pathology [9
]. Previous culture systems have successfully utilized natural biomaterials, such as alginate, to encapsulate and culture immature mouse ovarian follicles to yield live, fertile offspring [23
]. Alginate, however, is not degradable on the time scale of follicle culture [24
]; thus, as follicle expansion displaces the surrounding hydrogel, the material will exert an elastic, compressive force on the follicle that may restrict its expansion [15
]. During mouse folliculogenesis, the volumetric expansion of the follicle is approximately 300-fold, starting at 120 μm in diameter and reaching 400 μm in diameter at its mature stage. Human follicles start at a similar initial diameter, yet reach 5–18 mm in diameter, a volumetric increase 105
-fold. Presently, human follicles are unable to expand larger than 700 μm in vitro
], potentially due to the compressive force imparted by the non-degradable hydrogel as the follicle expands. New materials are required to accommodate the significant increase in follicle volume, while preserving the 3D architecture. In contrast to alginate, the PEG hydrogels degrade in response to proteases [19
] secreted by the follicle during culture, thereby creating the space for follicular expansion without producing the compressive force that can limit growth. Synthetic environments with tunable properties provide a tool to shed light on the basic biology of follicle development and may enable the development of a culture system that can be translated between species.
Matrices for follicle culture must enable easy encapsulation with retention of viability, pore size that allows transport of nutrients and hormones toward and away from the follicle, permit follicle expansion, and enable follicle collection at the end of culture. Naturally occurring biomaterials, such as collagen and fibrin have the advantage of intrinsic biological activity, however, these materials are complex and difficult to modify for desired physical properties, such as degradation. UV crosslinked hydrogels have the advantage of fast and efficient network formation. However, the necessity of using photoinitiator and light source raises the question of germline mutagenesis and the difficulties in human epidemiological studies involving germ cells and animal data extrapolation [26
]. PEG hydrogels formed by MTA at 4:2 functionality ratios presented several limitations, such as the PEG dehydration effect [20
] during the extended time required for network formation, as well as the potentially harmful buffer conditions. Introduction of an increased functionality in the network (4:3) resulted in rapid gelation that reduced the time follicles were exposed to the unreacted PEG in solution, and enabled the use of a more cell compatible buffering condition.
Tunable degradation of the plasmin sensitive PEG gels was achieved by using peptide sequences with different plasmin sensitivity. The hydrogel degradation occurred only around the follicles forming a soft pocket inside otherwise rigid matrix, suggesting that the cell-mediated proteolysis was localized to the follicle-material interface. A tightly regulated balance between plasminogen that originated from media components and the activated plasmin could be responsible for the localized degradation. Building a library of peptides with varying plasmin sensitivity allows matrix design that can be adjusted not only to the follicle stage and culture duration, but also to different species with varying plasmin activity. Furthermore, peptide sequences sensitive to other proteases, such as MMP-1 or MMP-13 [27
] can be utilized with the 3-arm peptide chemistry presented in this work.