Julianne L. Holloway

Guest–host chemistry utilizing adamantane (guest) and cyclodextrin (host) groups on HA crosslink to form hydrogels. Guest–host hydrogels exhibit shear-thinning and self-healing behavior that allow the material to be injected for neural applications.

Magnetically-responsive fibers embedded within hydrogels enable spatial and temporal control over fiber alignment via the application of a magnetic field. These materials could mimic the complex spatiotemporal organization of fibers within tissues.

Delivery of therapeutic compounds via biomaterial systems has shown promise for tissue regeneration following central nervous system (CNS) injuries. Stromal cell‐derived factor‐1a (SDF‐1a) modulates progenitor cell recruitment to neural injury sites and may contribute to neural repair. However, SDF‐1a has a short half‐life and requires a delivery system to both protect and sustain its release. Here, we sought to develop a drug delivery platform capable of releasing SDF‐1a in a controlled fashion while minimizing inflammation. We used modified hyaluronic acid and microfluidics to generate monodisperse microgels. Characterization of these microgels included size, tunability, degradation, and controlled release properties. Finally, we delivered SDF‐1a‐loaded microgels to a mouse model of traumatic brain injury at 7 days post‐injury and assessed their impact on neural progenitor cell recruitment and astrogliosis. The microfluidic system generated highly monodisperse microgels that successfully encapsulated a matrix metalloproteinase (MMP)‐cleavable SDF‐1a peptide and retained sensitivity to collagenase. Following intracortical injections, the microgels did not exacerbate the astrocytic response compared to saline injections; no significant difference was observed in neural progenitor cell migration patterns compared to controls. Therefore, we developed a biocompatible microgel system that is highly adaptable for biological delivery and may be utilized in brain/neural applications without exacerbating neuroinflammation.

Vaccine development requires innovative approaches to improve immune responses while reducing the number of immunizations. In this study, we explore the impact of controlled antigen release on immune activation and regulation using programmable infusion pumps and biodegradable biomaterials in OT‐II and wild‐type mice to understand the adaptive immune response through controlled antigen delivery in the absence of adjuvant. Ovalbumin (OVA) was delivered via an exponentially decreasing profile, mimicking clearance of infection, and an exponentially increasing profile, mimicking induction of infection. Exponentially decreasing OVA delivery through infusion pumps promoted regulatory T‐cell (Treg) activation in secondary lymphoid organs and suppressed pro‐inflammatory T‐helper type 17 (Th17) responses in blood. An exponentially increasing OVA profile enhanced central memory T‐cell (TCM) populations in submandibular blood and humoral immune responses in cardiac blood serum, demonstrating distinct immune modulation based on release kinetics. OVA was also delivered using a biodegradable PLGA‐PEG‐PLGA (PPP) depot, which provided controlled OVA release in an exponentially decreasing pattern. PPP‐OVA treatment significantly reduced the frequency of pro‐inflammatory T‐helper type 1 (Th1) cells while increasing CD25 ⁺ FOXP3 ⁺ Treg cells in the spleen. Moreover, to identify T‐cell populations that most accurately characterize the divergence in Treg and T‐helper response to OVA kinetics, a Sequential Feature Selection (SFS) algorithm with Machine Learning (ML) models was used. ML algorithms identified gMFI of RORγt ⁺ as a key feature in submandibular blood and the ratio of gMFI of FOXP3 ⁺ to GATA3 ⁺ as the marker that was significantly changed by treatments in inguinal lymph nodes (iLN) when infusion pumps were used to deliver OVA. In addition, ML‐based SFS identified CD25 ⁺ FOXP3 ⁺ regulatory T cells as the most important feature, influencing the expression of other cell types in both inguinal lymph nodes (iLN) and spleen when PPP was used to deliver OVA. This finding suggests that the exponentially decreasing profile may generate anti‐inflammatory responses. Overall, these findings suggest that controlled antigen delivery enhances immune regulation and memory T cells, providing new insights into immune responses mediated by the release kinetics.

Wound healing is tightly regulated in both space and time. To mimic this behavior, we designed magnetically-responsive, fiber-hydrogel composites. When covalently crosslinked hydrogels were combined with layer-by-layer stacking, we demonstrated precise spatial control over fiber orientation. For temporal control, we used non-covalently crosslinked hydrogels to enable in situ fiber alignment.

The extracellular matrix is a highly dynamic environment, and the precise temporal presentation of biochemical signals is critical for regulating cell behavior during development, healing, and disease progression. To mimic this behavior, we developed a modular DNA-based hydrogel platform to enable independent and reversible control over the immobilization of multiple biomolecules during in vitro cell culture. We combined reversible DNA handles with a norbornene-modified hyaluronic acid hydrogel to orthogonally add and remove multiple biomolecule-DNA conjugates at user-defined timepoints. We demonstrated that the persistent presentation of the cell adhesion peptide RGD was required to maintain cell spreading on hyaluronic acid hydrogels. Further, we discovered the delayed presentation of osteogenic growth peptide (OGP) increased alkaline phosphatase activity compared to other temporal variations. This finding is critically important when considering the design of OGP delivery approaches for bone repair. More broadly, this platform provides a unique approach to tease apart the temporal role of multiple biomolecules during development, regeneration, and disease progression.

Musculoskeletal interfacial tissues consist of complex gradients in structure, cell phenotype, and biochemical signaling that are important for function. Designing tissue engineering strategies to mimic these types of gradients is an ongoing challenge. In particular, new fabrication techniques that enable precise spatial control over fiber alignment are needed to better mimic the structural gradients present in interfacial tissues, such as the tendon‐bone interface. Here, we report a modular approach to spatially controlling fiber alignment using magnetically‐assisted electrospinning. Electrospun fibers were highly aligned in the presence of a magnetic field and smoothly transitioned to randomly aligned fibers away from the magnetic field. Importantly, magnetically‐assisted electrospinning allows for spatial control over fiber alignment at sub‐millimeter resolution along the length of the fibrous scaffold similar to the native structural gradient present in many interfacial tissues. The versatility of this approach was further demonstrated using multiple electrospinning polymers and different magnet configurations to fabricate complex fiber alignment gradients. As expected, cells seeded onto gradient fibrous scaffolds were elongated and aligned on the aligned fibers and did not show a preferential alignment on the randomly aligned fibers. Overall, this fabrication approach represents an important step forward in creating gradient fibrous materials, where such materials are promising as tissue‐engineered scaffolds for regenerating functional musculoskeletal interfacial tissues.

The lamina propria within the vocal fold (VF) is a complex multilayered tissue that increases in stiffness from the superficial to deep layer, where this characteristic is crucial for VF sound production. Tissue-engineered scaffolds designed for VF repair must mimic the biophysical nature of the native vocal fold and promote cell viability, cell spreading, and vibration with air flow. In this study, we present a unique trilayered, partially degradable hydrogel scaffold that mimics the multilayered structure of the VF lamina propria. Using thiol-norbornene photochemistry, trilayered hydrogel scaffolds were fabricated via layer-by-layer stacking with increasing polymer concentration from the top to middle to deep layer. Mechanical analysis confirmed that hydrogel modulus increased with increasing polymer concentration. Partially degradable hydrogels promoted high cell viability and cell spreading in three dimensions as assessed via live/dead and cytoskeleton staining, respectively. Importantly, partially degradable hydrogels maintained some degree of the three dimensional polymer network following protease exposure, while still enabling encapsulated cells to remodel their local environment via protease secretion. Finally, the trilayered hydrogel scaffold successfully vibrated and produced sound in proof-of-concept air flow studies. This work represents a critical first step toward the design of a multilayered, hydrogel scaffold for vocal fold tissue engineering.

Musculoskeletal interfacial tissues consist of complex gradients in structure, cell phenotype, and biochemical signaling that are important for function. Designing tissue engineering strategies to mimic these types of gradients is an ongoing challenge. In particular, new fabrication techniques that enable precise spatial control over fiber alignment are needed to better mimic the structural gradients present in interfacial tissues, such as the tendon-bone interface. Here, we report a modular approach to spatially controlling fiber alignment using magnetically-assisted electrospinning. Electrospun fibers were highly aligned in the presence of a magnetic field and smoothly transitioned to randomly aligned fibers away from the magnetic field. Importantly, magnetically-assisted electrospinning allows for spatial control over fiber alignment at sub-millimeter resolution along the length of the fibrous scaffold similar to the native structural gradient present in many interfacial tissues. The versatility of this approach was further demonstrated using multiple electrospinning polymers and different magnet configurations to fabricate complex fiber alignment gradients. As expected, cells seeded onto gradient fibrous scaffolds were elongated and aligned on the aligned fibers and did not show a preferential alignment on the randomly aligned fibers. Overall, this fabrication approach represents an important step forward in creating gradient fibrous materials, where such materials are promising as tissue-engineered scaffolds for regenerating functional musculoskeletal interfacial tissues.