Engineering Vessel Walls of Increased Stiffness in Microvascular Channels Within Hydrogels

Vascular networks extend throughout most tissues in the human body and are heavily involved in biological functions in both healthy and diseased states. The inclusion of perfusable vasculature is therefore key to producing engineered tissue constructs either for implantation or as <i>in vitro</i> disease models. Many approaches have been employed to pattern hollow channels within hydrogels, and these channels are often then lined with cells to mimic the architecture of the native vessel wall. While these methods do an excellent job of producing vessels that support nutrient and oxygen exchange (i.e. capillary-like function) and therefore support cell survival, when applied to resistance vessel-sized channels this approach does not properly recapitulate native vessel walls in several key ways. <i>In vivo</i>, in such vessels (the non-capillary microvasculature) there is a distinct difference in the structure and mechanical properties of the vessel wall and those of the tissue through which it extends. Soft tissues such as brain, kidney and lung, have elastic moduli of 5kPa or less, while the walls of small resistance arteries have moduli on the order of 10s to 100s of kPa. For such structures, instead of the “channels in a gel” architecture formed by traditional approaches, a better model of the natural tissue stiffness heterogeneity would be a relatively stiff, thin-walled tube through a relatively soft hydrogel (“tubes in a gel”). This configuration would allow the vessels to distend in a way similar to those<i> in vivo</i> and provide cells at the vessel wall and cells deeper within the surrounding hydrogel distinct environments with appropriate local stiffness. To mimic this stiffness heterogeneity, we seek to create a tube-like channel through a soft hydrogel by perfusing the channel lumen with a large molecule crosslinker which can slowly diffuse into the hydrogel and locally enhance crosslinking density. Experiments were performed using 10% w/v gelatin dissolved in DI water, enzymatically crosslinked with 0.1% w/v microbial transglutaminase (mTg) at 37°C as the primary crosslinking step (to yield a soft gel). Secondary crosslinking steps used a 1% w/v mTg solution. To confirm that secondary crosslinking could be achieved, 2 mm-thick slabs of gelatin were cast; after primary crosslinking, 1% mTg was added on top and allowed to incubate for 8 hours. After incubation, the gelatin was bisected, creating two 1-mm slabs - one proximal and one distal to the mTg/gelatin interface on the top surface. A rheometer was used to measure the elastic modulus of each disc. In a second series of experiments, straight channels with a diameter of 350 µm were formed in a gelatin hydrogel using nylon monofilament as a removable template. MTg conjugated with AF488 fluorophore was perfused into the channels, and diffusion of the fluorescent mTg normal to the channel wall into the hydrogel was observed using a confocal microscope. Enzymatically-crosslinked gelatin exhibits increased elastic modulus after a secondary mTg crosslinking step, in a manner dependent on the distance from the surface where the crosslinker is applied. Fluorescent mTg injected into a microchannel patterned through gelatin was observed to diffuse radially outward tens of microns into the gelatin hydrogel bulk, defining a fluorescent “cylinder” around the channel lumen. These preliminary results show that enzymatically-crosslinked gelatin can be further crosslinked by a second application of crosslinker, and that crosslinker can diffuse radially out of a patterned channel into the bulk hydrogel. This suggests it will be feasible to leverage large molecule crosslinker diffusion to create a tube-like region of enhanced stiffness surrounding a channel through a soft hydrogel, thereby forming a vessel wall with mechanical properties distinct from the bulk hydrogel. Importantly, this approach could be applied to branching channel networks that mimic the architecture of the natural microcirculation.