A fetal wound healing program after intrauterine bile duct injury may contribute to biliary atresia.

BACKGROUND & AIMS: Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause. Fetal tissues have increased levels of hyaluronic acid (HA), which plays an integral role in fetal wound healing. The objective of this study was to determine whether a program of fetal wound healing is part of the response to fetal EHBD injury. METHODS: Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. RESULTS: A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. CONCLUSION: The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with prolonged high levels of HA typical of fetal wound healing. The expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD. IMPACT AND IMPLICATIONS: Biliary atresia is a pediatric cholangiopathy associated with high morbidity and mortality rates; although multiple etiologies have been proposed, the fetal response to bile duct damage is largely unknown. This study explores the fetal pathogenesis after extrahepatic bile duct damage, thereby opening a completely new avenue to study therapeutic targets in the context of biliary atresia.

Synthesis, Characterization, and Digital Light Processing of a Hydrolytically Degradable Hyaluronic Acid Hydrogel.

Numerous chemical modifications of hyaluronic acid (HA) have been explored for the formation of degradable hydrogels that are suitable for a variety of biomedical applications, including biofabrication and drug delivery. Thiol-ene step-growth chemistry is of particular interest due to its lower oxygen sensitivity and ability to precisely tune mechanical properties. Here, we utilize an aqueous esterification route via reaction with carbic anhydride to synthesize norbornene-modified HA (NorHACA) that is amenable to thiol-ene crosslinking to form hydrolytically unstable networks. NorHACA is first synthesized with varying degrees of modification (∼15-100%) by adjusting the ratio of reactive carbic anhydride to HA. Thereafter, NorHACA is reacted with dithiol crosslinker in the presence of visible light and photoinitiator to form hydrogels within tens of seconds. Unlike conventional NorHA, NorHACA hydrogels are highly susceptible to hydrolytic degradation through enhanced ester hydrolysis. Both the mechanical properties and the degradation timescales of NorHACA hydrogels are tuned via macromer concentration and/or the degree of modification. Moreover, the degradation behavior of NorHACA hydrogels is validated through a statistical-co-kinetic model of ester hydrolysis. The rapid degradation of NorHACA hydrogels can be adjusted by incorporating small amounts of slowly degrading NorHA macromer into the network. Further, NorHACA hydrogels are implemented as digital light processing (DLP) resins to fabricate hydrolytically degradable scaffolds with complex, macroporous structures that can incorporate cell-adhesive sites for cell attachment and proliferation after fabrication. Additionally, DLP bioprinting of NorHACA hydrogels to form cell-laden constructs with high viability is demonstrated, making them useful for applications in tissue engineering and regenerative medicine.

Simple production of cellulose nanofibril microcapsules and the rheology of their suspensions.

Microcapsules are commonly used in applications ranging from therapeutics to personal care products due to their ability to deliver encapsulated species through their porous shells. Here, we demonstrate a simple and scalable approach to fabricate microcapsules with porous shells by interfacial complexation of cellulose nanofibrils and oleylamine, and investigate the rheological properties of suspensions of the resulting microcapsules. The suspensions of neat capsules are viscous liquids whose viscosity increases with volume fraction according to a modified Kreiger-Dougherty relation with a maximum packing fraction of 0.74 and an intrinsic viscosity of 4.1. When polyacrylic acid (PAA) is added to the internal phase of the microcapsules, however, the suspensions become elastic and display yield stresses with power-law dependencies on capsule volume fraction and PAA concentration. The elasticity appears to originate from associative microcapsule interactions induced by PAA that is contained within and incorporated into the microcapsule shell. These results demonstrate that it is possible to tune the rheological properties of microcapsule suspensions by changing only the composition of the internal phase, thereby providing a novel method to tailor complex fluid rheology.

Additive manufacturing of highly entangled polymer networks.

Incorporation of polymer chain entanglements within a single network can synergistically improve stiffness and toughness, yet attaining such dense entanglements through vat photopolymerization additive manufacturing [e.g., digital light processing (DLP)] remains elusive. We report a facile strategy that combines light and dark polymerization to allow constituent polymer chains to densely entangle as they form within printed structures. This generalizable approach reaches high monomer conversion at room temperature without the need for additional stimuli, such as light or heat after printing, and enables additive manufacturing of highly entangled hydrogels and elastomers that exhibit fourfold- to sevenfold-higher extension energies in comparison to that of traditional DLP. We used this method to print high-resolution and multimaterial structures with features such as spatially programmed adhesion to wet tissues.

Multi-Material Volumetric Additive Manufacturing of Hydrogels using Gelatin as a Sacrificial Network and 3D Suspension Bath.

Volumetric additive manufacturing (VAM) is an emerging layerless method for the rapid processing of reactive resins into 3D structures, where printing is much faster (seconds) than other lithography and direct ink writing methods (minutes to hours). As a vial of resin rotates in the VAM process, patterned light exposure defines a 3D object and then resin that has not undergone gelation can be washed away. Despite the promise of VAM, there are challenges with the printing of soft hydrogel materials from non-viscous precursors, including multi-material constructs. To address this, sacrificial gelatin is used to modulate resin viscosity to support the cytocompatible VAM printing of macromers based on poly(ethylene glycol) (PEG), hyaluronic acid (HA), and polyacrylamide (PA). After printing, gelatin is removed by washing at an elevated temperature. To print multi-material constructs, the gelatin-containing resin is used as a shear-yielding suspension bath (including HA to further modulate bath properties) where ink can be extruded into the bath to define a multi-material resin that can then be processed with VAM into a defined object. Multi-material constructs of methacrylated HA (MeHA) and gelatin methacrylamide (GelMA) are printed (as proof-of-concept) with encapsulated mesenchymal stromal cells (MSCs), where the local hydrogel properties guide cell spreading behavior with culture.

Photochemical Control of Network Topology in PEG Hydrogels.

Hydrogels are often synthesized through photoinitiated step-, chain-, and mixed-mode polymerizations, generating diverse network topologies and resultant material properties that depend on the underlying network connectivity. While many photocrosslinking reactions are available, few afford controllable connectivity of the hydrogel network. Herein, a versatile photochemical strategy is introduced for tuning the structure of poly(ethylene glycol) (PEG) hydrogels using macromolecular monomers functionalized with maleimide and styrene moieties. Hydrogels are prepared along a gradient of topologies by varying the ratio of step-growth (maleimide dimerization) to chain-growth (maleimide-styrene alternating copolymerization) network-forming reactions. The initial PEG content and final network physical properties (e.g., modulus, swelling, diffusivity) are tailored in an independent manner, highlighting configurable gel mechanics and reactivity. These photochemical reactions allow high-fidelity photopatterning and 3D printing and are compatible with 2D and 3D cell culture. Ultimately, this photopolymer chemistry allows facile control over network connectivity to achieve adjustable material properties for broad applications.

Influence of Microgel and Interstitial Matrix Compositions on Granular Hydrogel Composite Properties.

Granular hydrogels are an emerging class of biomaterials formed by jamming hydrogel microparticles (i.e., microgels). These materials have many advantageous properties that can be tailored through microgel design and extent of packing. To enhance the range of properties, granular composites can be formed with a hydrogel interstitial matrix between the packed microgels, allowing for material flow and then stabilization after crosslinking. This approach allows for distinct compartments (i.e., microgels and interstitial space) with varied properties to engineer complex material behaviors. However, a thorough investigation of how the compositions and ratios of microgels and interstitial matrices influence material properties has not been performed. Herein, granular hydrogel composites are fabricated by combining fragmented hyaluronic acid (HA) microgels with interstitial matrices consisting of photocrosslinkable HA. Microgels of varying compressive moduli (10-70 kPa) are combined with interstitial matrices (0-30 vol.%) with compressive moduli varying from 2-120 kPa. Granular composite structure (confocal imaging), mechanics (local and bulk), flow behavior (rheology), and printability are thoroughly assessed. Lastly, variations in the interstitial matrix chemistry (covalent vs guest-host) and microgel degradability are investigated. Overall, this study describes the influence of granular composite composition on structure and mechanical properties of granular hydrogels towards informed designs for future applications.

Linguistic Analysis Identifies Emergent Biomaterial Fabrication Trends for Orthopaedic Applications.

The expanse of publications in tissue engineering (TE) and orthopedic TE (OTE) over the past 20 years presents an opportunity to probe emergent trends in the field to better guide future technologies that can make an impact on musculoskeletal therapies. Leveraging this trove of knowledge, a hierarchical systematic search method and trend analysis using connected network mapping of key terms is developed. Within discrete time intervals, an accelerated publication rate for anatomic orthopedic tissue engineering (AOTE) of osteochondral defects, tendons, menisci, and entheses is identified. Within these growing fields, the top-listed key terms are extracted and stratified into evident categories, such as biomaterials, delivery method, or 3D printing and biofabrication. It is then identified which categories decreased, remained constant, increased, or emerged over time, identifying the specific emergent categories currently driving innovation in orthopedic repair technologies. Together, these data demonstrate a significant convergence of material types and descriptors used across tissue types. From this convergence, design criteria to support future research of anatomic constructs that mimic both the form and function of native tissues are formulated. In summary, this review identifies large-scale trends and predicts new directions in orthopedics that will define future materials and technologies.

Simultaneous One-Pot Interpenetrating Network Formation to Expand 3D Processing Capabilities.

The incorporation of a secondary network into traditional single-network hydrogels can enhance mechanical properties, such as toughness and loading to failure. These features are important for many applications, including as biomedical materials; however, the processing of interpenetrating polymer network (IPN) hydrogels is often limited by their multistep fabrication procedures. Here, a one-pot scheme for the synthesis of biopolymer IPN hydrogels mediated by the simultaneous crosslinking of two independent networks with light, namely: i) free-radical crosslinking of methacrylate-modified hyaluronic acid (HA) to form the primary network and ii) thiol-ene crosslinking of norbornene-modified HA with thiolated guest-host assemblies of adamantane and ß-cyclodextrin to form the secondary network, is reported. The mechanical properties of the IPN hydrogels are tuned by changing the network composition, with high water content (≈94%) hydrogels exhibiting excellent work of fracture, tensile strength, and low hysteresis. As proof-of-concept, the IPN hydrogels are implemented as low-viscosity Digital Light Processing resins to fabricate complex structures that recover shape upon loading, as well as in microfluidic devices to form deformable microparticles. Further, the IPNs are cytocompatible with cell adhesion dependent on the inclusion of adhesive peptides. Overall, the enhanced processing of these IPN hydrogels will expand their utility across applications.

Enhancing Biopolymer Hydrogel Functionality through Interpenetrating Networks.

Traditional hydrogels are strong candidates for biomedical applications; however, they may suffer from drawbacks such as weak mechanics, static properties, and an inability to fully replicate aspects of the cellular microenvironment. These challenges can be addressed through the incorporation of second networks to form interpenetrating polymer network (IPN) hydrogels. The objective of this review is to establish clear trends on the enhanced functionality achieved by incorporating secondary networks into traditional, biopolymer-based hydrogels. These include mechanical reinforcement, 'smart' systems that respond to external stimuli, and the ability to tune cell-material interactions. Through attention to network structure and chemistry, IPN hydrogels may advance to meet challenging criteria for a wide range of biomedical fields.

Programmable and contractile materials through cell encapsulation in fibrous hydrogel assemblies.

The natural extracellular matrix (ECM) within tissues is physically contracted and remodeled by cells, allowing the collective shaping of functional tissue architectures. Synthetic materials that facilitate self-assembly similar to natural ECM are needed for cell culture, tissue engineering, and in vitro models of development and disease. To address this need, we develop fibrous hydrogel assemblies that are stabilized with photocrosslinking and display fiber density­dependent strain-responsive properties (strain stiffening and alignment). Encapsulated mesenchymal stromal cells locally contract low fiber density assemblies, resulting in macroscopic volumetric changes with increased cell densities and moduli. Because of properties such as shear-thinning and self-healing, assemblies can be processed into microtissues with aligned ECM deposition or through extrusion bioprinting and photopatterning to fabricate constructs with programmed shape changes due to cell contraction. These materials provide a synthetic approach to mimic features of natural ECM, which can now be processed for applications in biofabrication and tissue engineering.