Biology subjects

Highley, C. B.

Publications and source records attributed to Highley, C. B..

11 recordsLinked to original sources

bioRxiv2026

Fibronectin Tethering as a Shear-thinning Bio-Annealing Strategy for Granular Hydrogels

Granular hydrogel scaffolds (GHS) are injectable, integrative biomaterials built from hydrogel microparticles that permit rapid cell infiltration independent of scaffold degradation. Microporous annealed particle (MAP) scaffolds, a sub-class of GHS, use inter-microparticle crosslinks to anneal adjacent particles, providing greater mechanical stability than purely jammed assemblies. A growing range of inter-particle crosslinking strategies, including covalent photo-initiated radical polymerization and reversible guest-host chemistries, some intended to mimic extracellular matrix (ECM) viscoelasticity, focus on mechanical stabilization, while surface bioactivity is typically added separately via orthogonally tethered cell-adhesion ligands such as RGD, a peptide derived from fibronectin (FN), a key mediator of cell-ECM and ECM-ECM interactions. Here, we present an alternative that unifies these functions: coating MAP microparticle building blocks with covalently bound fibronectin to create fibronectin-modified MAP (FN-MAP). FN-MAP exhibits shear-thinning annealing with complete recovery of scaffold mechanics after physical mixing. Human lung fibroblasts respond favorably, adopting spread morphologies with high viability. Translationally, FN-MAP shows proof-of-principle promise for 3D bioprinting (greater stability and higher print resolution) and wound healing (increased epidermal closure in a diabetic mouse model). FN-MAP thus delivers both increased scaffold strength and bioactivity without exogenous soluble linkers or mixed particle populations.

bioengineering↗

bioRxiv2025

Fiber-based hydrogels for designing viscoelastic responses in particle-based biomaterials that support embedded 3D printing

Viscoelastic biomaterials that exhibit biomimetic responses to applied stresses are important in studying physiology and designing biomaterial scaffolds. Particle-based hydrogels offer potential for engineering viscoelasticity through design of both the component microparticles and their processing into bulk particle-based materials. When particles are not crosslinked to one another, particle movements in response to strain can potentially relieve applied stresses and facilitate the materials use in dynamic processes like bioprinting. In particle-based hydrogels based on spherical hydrogel microparticles (HMPs), particle movement is restricted by contacts with immediately adjacent HMPs. In comparison, fiber-based hydrogel systems leverage high-aspect ratio microfiber components with long-range interactions. Here, microfibers with aspect ratios of [~]15:1 length:diameter are used to form particle-based hydrogels to compare how interparticle interactions at increased length scales alter properties compared to particle-based hydrogels based on spherical HMPs. Like particle-based hydrogels formed from spherical HMPs, those formed from fiber HMPs exhibit viscoelasticity, with shear-thinning and self-healing behaviors. But, fiber-based materials allow enhanced control over bulk stress relaxation times (T1/2 [~] 1-100+ s) across a range of applied strains ({sigma} [~] 2.5-50%) in a packing density-dependent fashion. Fiber-based systems relaxed stresses continually and to a greater degree at low strains in comparison to HMP systems. Dynamic interfiber interactions in fiber-based hydrogels also supported embedded printing where perfusable channels can be printed into fiber-based hydrogels stabilized by physical interfiber interactions. Taken together, fiber-based hydrogels offer opportunities for designing complexity into biomaterial scaffolds, including allowing control over viscoelastic properties through hydrogel design and control over heterogeneous 3D structures through embedded printing.

bioengineering↗

bioRxiv2025

An injectable granular hydrogel stabilized by electrostatic interactions between hyaluronic acid-based microparticles and soluble gelatin exhibits poroelasticity and strain-stiffening

Injectable hydrogels with shear-thinning and self-healing properties are critical for biomedical applications including 3D bioprinting and regenerative medicine. While granular hydrogels inherently exhibit these properties, they often lack post-injection stability. Here, we developed an electrostatically stabilized granular hydrogel system composed of norbornene-modified hyaluronic acid (NorHA) microgels and cationic gelatin ((+) Gel). NorHA microgels (9.91 {+/-} 4.85 m diameter) were synthesized via batch emulsification, while (+) Gel was prepared by modifying gelatin with ethylene diamine to increase zeta potential from 2.08 {+/-} 0.97 mV to 13.76 {+/-} 1.11 mV. The negatively charged NorHA microgels formed stable materials when combined with (+) Gel through electrostatic interactions, confirmed by gel inversion tests and salt sensitivity studies. Rheological characterization revealed that (+) Gel addition produced poroelastic behavior and strain-stiffening properties, with storage modulus and yield onset increasing under compression. Large amplitude oscillatory shear analysis showed strain-stiffening behavior (e3 > 0) that enhanced with both (+) Gel concentration and compression. Confocal microscopy demonstrated tunable porosity through gelatin fraction control, with (+) Gel forming aggregate-like clusters. Extrusion testing showed formulations required low injection pressures (0.47-0.91 kPa) comparable to PBS and significantly lower than Pluronic, while forming robust filaments up to 23 mm in length. The materials exhibited rapid self-healing behavior and maintained structural integrity post-extrusion. This electrostatically stabilized granular hydrogel system offers a promising platform for injectable biomaterials that combine ease of delivery with post-injection stability for wound healing and 3D bioprinting applications.

bioengineering↗

bioRxiv2025

Particle-based hydrogel inks and support matrices for biofabricating structural complexity, soluble gradients, and cell-lined channels in fully granular bioprinted systems

Towards achieving biomimetic complexity in biofabricated systems, an all-granular bioprinting system might use particle-based hydrogel inks to establish structures within a particle-based support matrix. In such a system, the granular support matrix can be designed to persist in the final construct and include cells incorporated prior to printing. To biofabricate complexity, bioprinting can introduce high-resolution heterogeneous structures that guide cell behaviors. The designs of the granular ink and support hydrogels are crucial to achieving complexity. High resolution structures and channels depend on small particles that flow and can be stabilized, and that can be printed and then removed, respectively. Herein, an all-granular system is described that used a granular formulation of an established, tunable hyaluronic acid-based hydrogel as the basis for a support matrix and a small particle gelatin hydrogel as an ink. Towards facilitating stabilization of the printed structure and flow during printing, the support and ink materials included soluble, interstitial components, and all exhibited yield stress behaviors characteristic of granular hydrogel systems. The support matrixs viscoelastic properties were dependent on intraparticle hydrogel network design, and it could be stabilized against flow by photoinitiated crosslinking. The gelatin ink could form fine filaments, as small as 100 {micro}m in testing here, and melted to leave channels within crosslinked support matrices. Channels could support flows introduced by hydrostatic pressure and could be used to rapidly transport soluble factors into the construct, which could be used to establish soluble gradients by diffusion and support cell viability. The all-granular system supported printing of complex, multimaterial structures, with feature resolution on the order of 100 {micro}m and spatial positioning on the order of 10s {micro}m. The process and materials exhibited biocompatibility with respect to cells included within the support matrix during printing or introduced into channels to begin establishing endothelialized bioprinted vessels.

bioengineering↗

bioRxiv2025

Integrating Microchannels and Flows into 3D Printable Granular Hydrogel Matrices

Microfluidic systems incorporating or contained within hydrogels are important in creating microphysiological systems (MPSs). Often naturally derived hydrogels are used, as their inherent bioactivity supports dynamic cellular behaviors. Hydrogel biomaterials that are partly or fully synthetic are desirable in engineering systems with specific, designed properties, though they typically lack bioactive features of natural materials without additional molecular design. In particular, permissive biomaterials enable physiologically relevant dynamic cellular behaviors. Granular hydrogels offer inherent permissiveness, owning to porosity between particles and dynamic behaviors in the absence of interparticle crosslinking. However, applying these in MPS to model tissues requires stable channels to perfuse fluid in these dynamic systems. Here, we establish channels within granular hydrogels to enable perfusion through spatially controlled interparticle crosslinking. Selective crosslinking allowed for the formation of stable channels while allowing the microparticles of a granular hydrogel between two channels to remained uncrosslinked. This allowed spatiotemporal control of signals within an environment established from microparticles without interparticle crosslinking. Fluorescently tagged molecules allowed for the visualization of controlled soluble gradients between two channels within the device. Additionally, embedded 3D printing processes can be used to specify material composition within the system, demonstrating integrated technology for engineering well-defined hydrogel systems. Integrated microfluidic-based control over soluble signals in a system that is compatible with 3D printing processes will establish a basis for building MPSs for broad applications, and the ability to maintain granular systems in culture without interparticle crosslinking will enable design of synthetic hydrogels that access unique dynamic properties within these systems.

bioengineering↗

bioRxiv2025

Focused Ultrasound Crosslinkable Granular Hydrogels

Minimally invasive delivery of biomaterials for tissue regeneration can be achieved using biomaterials delivered by injection that rapidly stabilize at the delivery site. Tissue regeneration has been shown to be dependent on material properties, with porosity strongly supporting revascularization and tissue regrowth. Here, a highly porous granular hydrogel system was designed that is compatible with the unique strengths of highly penetrating, minimal invasive focused ultrasound (FUS), to address this challenge. FUS offers well-defined spatial and temporal control over hydrogel crosslinking. We developed a composite granular hydrogel scaffold composed of two polyethylene glycol (PEG)-based components, microgels and fibers with FUS-responsive chemistry, and a pore-defining gelatin microgel component. Upon applying FUS, the bulk granular hydrogel stabilized through the formation of crosslinks between PEG components, with porosity designed by gelatin microgel melting. FUS-crosslinking parameters were determined that resulted in crosslinking both in vitro and a mouse cadaver model of minimally invasive delivery. The resulting granular hydrogels stability depended on the presence of fibers and exhibited viscoelastic properties comparable to granular hydrogels that were photocrosslinked. Hydrogels were highly porous and cytocompatible. This work defines a FUS-responsive granular system and extends the potential of FUS as a novel, noninvasive method for crosslinking regenerative hydrogel systems.

bioengineering↗

bioRxiv2024

Highly porous granular hydrogels reinforced by electrospun hydrogel fibers for long-term stability

Granular hydrogels (GHs) formed from hydrogel microparticles (HMPs) possess inherent, microscale porosity. In GHs including microporous annealed particle (MAP) hydrogels as well as unannealed GHs, increases in pore size and total porosity correlate with enhanced cellular infiltration and growth. However, increased porosity or pore size are typically achieved by decreasing HMP density, reducing interparticle contacts and weakening hydrogel structure. Here, to achieve a highly porous GH without compromising stability, we developed an approach using sacrificial HMPs to achieve high, mesoscale porosity and high-aspect ratio hydrogel fibers to reinforce the scaffold. The use of sacrificial HMPs circumvented the need to decrease packing density, creating GHs with up to 60% porosity. The hydrogel fibers span particles in the porous space, stabilizing the porous structure by enhancing interactions among GH components. Upon 5-10% v/v fiber incorporation, highly porous GHs retained their structure over 28 days of in vivo culture, compared to 4 days in GHs without fibers. These scaffolds supported and promoted endothelial cell growth as porosity increased. Overall, this study presents a stable, highly porous granular scaffold system with high processability and cytocompatibility.

bioengineering↗

bioRxiv2023

Packed hydrogel microfibers as a granular medium

Particle-based (granular) hydrogels are an attractive class of biomaterials due to their unique properties and array of applications in the biomedical space - serving as platforms for extrusion printing and injecting as well as permissive materials for 3D cell culture. Physical properties of particle-based hydrogels are governed in part by contact forces between particles, which are limited to interactions with neighboring particles. Secondary annealing mechanisms are often used to increase mechanical properties and serve to link particles across the granular material volume. Here, we present a novel particle-based hydrogel where each "particle" is a discrete electrospun hydrogel microfiber that has been segmented to a length of 93 {+/-} 51 m, with a diameter of 1.6 {+/-} 0.3 m. The fibers are flexible and have aspect ratios that are greater than one order of magnitude larger than most traditional hydrogel microparticles. This enables long-range entanglements of discrete fibers following packing into a bulk material, yielding unique properties. Without crosslinking, these packed hydrogel microfiber materials are mechanically robust, they can stretch without breaking when strained, and they exhibit stress relaxation under constant strain. As a cell culture scaffold, shear-induced alignment of the individual fibers within 3D printed filaments confers contact guidance cues to cells and promotes anisotropic cellular morphologies. Packed hydrogel microfibers can also be used as 3D cell culture environments, with cells able to spread due to the permissive nature of the scaffold. Overall, this work introduces a particle-based material system comprised of individual hydrogel microfibers that allows unique properties to be engineered into biomaterials that might be used in extrusion processes and cell cultures and, ultimately, in tissue engineering and regenerative medicine applications.

bioengineering↗

bioRxiv2023

Engineering Particle-based Materials for Vasculogenesis

Vascular networks are critical to the survival of cells within materials designed for regenerative medicine. Developing approaches to vascularize three-dimensional (3D) in vitro models that recreate tissue physiology and 3D tissue constructs for regenerative medicine remain an important focus of tissue engineering. Granular hydrogels are emerging as a promising class of materials for the regeneration of damaged tissues and fabricating tissue constructs. While granular hydrogels have supported vasculature formed by angiogenesis and fabrication processes that establish channels, parameters for designing these materials to support formation of vasculature by vasculogenesis from cells contained within these materials are not fully understood and remain largely unexplored. In this study, vasculogenesis within 3D granular hydrogels formed from polyethylene glycol (PEG) microgels are studied for its potential to establish a microvascular network within this class of materials. Self-organization of endothelial cells into networks within hours is observed in the presence of fibroblasts, and the effects of cell adhesive ligands (RGD) and porosity are measured. Increasing porosity is observed to enhance vasculogenesis while the addition of RGD impairs microvessel network formation. This work establishes parameters that support robust microvasculature formation within granular hydrogels that might be broadly applicable to this class of materials, with implications for other morphogenetic processes in 3D systems.

bioengineering↗

bioRxiv2023

Supramolecular fibrous hydrogel augmentation of uterosacral ligament suspension for treatment of pelvic organ prolapse

Uterosacral ligament suspension (USLS) is a common surgical treatment for pelvic organ prolapse (POP). However, the relatively high failure rate of up to 40% underscores a strong clinical need for complementary treatment strategies, such as biomaterial augmentation. Herein, we describe the first hydrogel biomaterial augmentation of USLS in a recently established rat model using an injectable fibrous hydrogel composite. Supramolecularly-assembled hyaluronic acid (HA) hydrogel nanofibers encapsulated in a matrix metalloproteinase (MMP)-degradable HA hydrogel create an injectable scaffold showing excellent biocompatibility and hemocompatibility. The hydrogel can be successfully delivered and localized to the suture sites of the USLS procedure, where it gradually degrades over 6 weeks. In situ mechanical testing 24 weeks post-operative in the multiparous USLS rat model shows the ultimate load (load at failure) to be 1.70 {+/-} 0.36 N for the intact uterosacral ligament (USL), 0.89 {+/-} 0.28 N for the USLS repair, and 1.37 {+/-} 0.31 N for the USLS + hydrogel (USLS+H) repair (n = 8). These results indicate that the hydrogel composite significantly improves load required for tissue failure compared to the standard USLS, even after the hydrogel degrades, and that this hydrogel-based approach could potentially reduce the high failure rate associated with USLS procedures.

bioengineering↗

bioRxiv2021

Guest-host supramolecular assembly of injectable hydrogel fibers for cell encapsulation

The fibrous architecture of the extracellular matrix (ECM) is recognized as an integral regulator of cell function. However, there is an unmet need to develop mechanically robust biomaterials mimicking nanofibrous tissue topography that are also injectable to enable minimally invasive delivery. In this study we have developed a fibrous hydrogel composed of supramolecularly-assembled hyaluronic acid (HA) nanofibers that exhibits mechanical integrity, shear-thinning, rapid self-healing, and cytocompatibility. HA was modified with methacrylates to permit fiber photocrosslinking following electrospinning and either guest adamantane or host {beta}-cyclodextrin groups to guide supramolecular fibrous hydrogel assembly. Analysis of fibrous hydrogel rheological properties showed that the mixed guest-host fibrous hydrogel was more mechanically robust (6.6 {+/-} 2.0 kPa, storage modulus (G')) than unmixed guest hydrogel fibers (1.0 {+/-} 0.1 kPa, G') or host hydrogel fibers (1.1 {+/-} 0.1 kPa, G') separately. The reversible nature of the guest-host supramolecular interactions also allowed for shear-thinning and self-healing behavior as demonstrated by cyclic deformation testing. Human mesenchymal stromal cells (hMSCs) encapsulated in fibrous hydrogels demonstrated satisfactory viability following injection and after seven days of culture (> 85%). Encapsulated hMSCs were more spread and elongated when cultured in viscoelastic guest-host hydrogels compared to non-fibrous elastic controls, with hMSCs also showing significantly decreased circularity in fibrous guest-host hydrogels compared to non-fibrous guest-host hydrogels. Together, these data highlight the potential of this injectable fibrous hydrogel platform for cell and tissue engineering applications requiring minimally invasive delivery.

bioengineering↗