Advanced Hydrogel Design for Soft Tissue Culture and Regeneration

Soft tissues, including neural, adipose, and some vascular tissues, perform processes critical to survival and function such as circulation, cognition, and thermal regulation. Many soft tissue-specific pathologies cause damage to soft tissues that may result in significantly reduced patient health and quality of life. Larger-scale damage to functional soft tissue, as in cases of stroke, traumatic injury, or therapeutic removal such as mastectomy, results in loss of functional tissue on a volume scale that cannot be endogenously regenerated. Adult mammalian brain tissue is particularly limited in its potential to regenerate; thus, cell therapy is a favorable avenue for functional neural tissue regeneration. Transplantation of cells within a biomaterial delivery vehicle improves implanted cell viability and promotes integration of the transplanted cells with the host tissue. Neural tissue cell therapy outcomes may be further improved adding instructional cues to the protective delivery matrix that promote tissue regeneration; however, soft tissue regeneration processes are complex, consist of multiple stages, and in the case of neurogenesis, are not fully understood. Thus, in vitro model systems with tunable control over material properties are needed for modeling soft tissue interactions towards better understanding of their regeneration and design of therapeutic constructs.Current hydrogel models are largely inadequate in achieving soft-tissue mimetic stiffnesses while also providing independent and spatiotemporal control over biochemical cue presentation to cells. Soft tissue culture models made from naturally-derived polymers provide appropriate mechanical properties for soft tissue but sacrifice control over biological signaling. In contrast, synthetic matrix systems offer greater independent control over biochemical signal presentation and mechanical properties; however, they generally do not form stable networks within the mechanical range of soft tissue (E<1 kPa). Further, even the most advanced synthetic tissue culture constructs do not optimally model the dynamic biochemical signaling that occurs in vivo; while molecule-addition and molecule-removal methods do exist separately, current systems are limited in their capability to sequentially add and then remove the same molecule to model transient signaling. Thus, there exists a need for improved soft tissue engineered constructs that accurately recapitulate the stiffness and highly controlled dynamic signaling of natural soft tissue such as neural and fat tissues. Here we present two developments in synthetic hydrogel design for improved accuracy of modeling soft tissue regeneration within a controlled, reductionist environment. We first implemented a novel method of precisely and independently tuning poly(ethylene glycol) (PEG)-based hydrogel stiffness to within the regime of soft tissue by incorporating soluble, allyl-presenting monomers in the hydrogel precursor solution before crosslinking, resulting in allyl-acrylate competition that alters crosslinking mechanics to decrease hydrogel bulk stiffness. PC12 neural cells displayed enhanced neurite outgrowth within this neural stiffness-mimetic environment, both in 2D culture on the surface of PEG-based gels and within a more physiologically relevant degradable PEG-based hydrogel. We then implemented these hydrogels as a platform for investigating the effects of multiple environmental factors on neural stem cell behavior, observing that NSC behavior was influenced by interplay between matrix stiffness, adhesive peptide signaling, and soluble growth factor stimulation. These results indicate that this compliant reductionist hydrogel is an appropriate system for evaluating the influence of single and multiple factors on cell behavior individually and in concert within a controlled environment. This controlled investigation of soft tissue behavior is a promising approach for improved understanding of soft tissue regeneration. We next developed a genetically-encoded method for reversible biochemical signaling within PEG-based hydrogels. We designed and implemented a recombinant protein with a SpyCatcher domain (capable of linking to a SpyTag-functionalized PEG matrix) connected to a cell adhesive RGDS peptide domain by a long-range UV light-cleavable domain known as PhoCl. This protein was shown to bind to SpyTag-functionalized PEG-matrices via isopeptide bonding to present RGDS adhesive ligands to cells, then upon 405 nm light exposure, the PhoCl domain was cleaved to subsequently release the RGDS domain, which diffused out of the matrix. This system was implemented to confer reversible adhesion of cells to the PEG-based hydrogel surface in 2D culture and differential cell spreading over time in 3D culture within cell-degradable PEG-based hydrogels, demonstrating the capability of this system to presenting dynamic signaling events to cells towards modeling soft tissue regeneration processes within in a controlled, ECM-mimetic environment. To address limitations of current in vitro soft tissue culture models, we developed methods for mimicking neural tissue stiffness and presenting reversible biochemical signaling within modularly tunable PEG-based hydrogels. These two synthetic hydrogel design technologies established in this work advance the capabilities for modeling soft tissue behaviors towards designing therapeutic tissue engineered constructs for directing therapeutic regeneration.