Conformal encapsulation of mammalian stem cells using modified hyaluronic acid.

Micro- and nanoencapsulation of cells has been studied as a strategy to protect cells from environmental stress and promote survival during delivery. Hydrogels used in encapsulation can be modified to influence cell behaviors and direct assembly in their surroundings. Here, we report a system that conformally encapsulated stem cells using hyaluronic acid (HA). We successfully modified HA with lipid, thiol, and maleimide pendant groups to facilitate a hydrogel system in which HA was deposited onto cell plasma membranes and subsequently crosslinked through thiol-maleimide click chemistry. We demonstrated conformal encapsulation of both neural stem cells (NSCs) and mesenchymal stromal cells (MSCs), with viability of both cell types greater than 90% after encapsulation. Additional material could be added to the conformal hydrogel through alternating addition of thiol-modified and maleimide-modified HA in a layering process. After encapsulation, we tracked egress and viability of the cells over days and observed differential responses of cell types to conformal hydrogels both according to cell type and the amount of material deposited on the cell surfaces. Through the design of the conformal hydrogels, we showed that multicellular assembly could be created in suspension and that encapsulated cells could be immobilized on surfaces. In conjunction with photolithography, conformal hydrogels enabled rapid assembly of encapsulated cells on hydrogel substrates with resolution at the scale of 100 µm.

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, the first hydrogel biomaterial augmentation of USLS in a recently established rat model is described 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 six 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 can potentially reduce the high failure rate associated with USLS procedures.

User-defined, temporal presentation of bioactive molecules on hydrogel substrates using supramolecular coiled coil complexes.

The ability to spatiotemporally control the presentation of relevant biomolecules in synthetic culture systems has gained significant attention as researchers strive to recapitulate the endogenous extracellular matrix (ECM) in vitro. With the biochemical composition of the ECM constantly in flux, the development of platforms that allow for user-defined control of bioactivity is desired. Here, we reversibly conjugate bioactive molecules to hydrogel-based substrates through supramolecular coiled coil complexes that form between complementary peptides. Our system employs a thiolated peptide for tethering to hydrogel surfaces (T-peptide) through a spatially-controlled photomediated click reaction. The complementary association peptide (A-peptide), containing the bioactive domain, forms a heterodimeric coiled coil complex with the T-peptide. Addition of a disruptor peptide (D-peptide) engineered specifically to target the A-peptide outcompetes the T-peptide for binding, and removes the A-peptide and the attached bioactive motif from the scaffold. We use this platform to demonstrate spatiotemporal control of biomolecule presentation within hydrogel systems in a repeatable process that can be extended to adhesive motifs for cell culture. NIH 3T3 fibroblasts seeded on hyaluronic acid hydrogels and polyethylene glycol-based fibrous substrates supramolecularly functionalized with an RGD motif demonstrated significant cell spreading over their nonfunctionalized counterparts. Upon displacement of the RGD motif, fibroblasts occupied less area and clustured on the substrates. Taken together, this platform enables facile user-defined incorporation and removal of biomolecules in a repeatable process for controlled presentation of bioactivity in engineered culture systems.

Guest-Host Supramolecular Assembly of Injectable Hydrogel Nanofibers 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 behavior, rapid self-healing, and cytocompatibility. HA was modified with methacrylates to permit fiber photo-cross-linking following electrospinning and either "guest" adamantane or "host" ß-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) or host hydrogel fibers (1.1 ± 0.1 kPa) 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 7 days of culture (>85%). Encapsulated hMSCs were more spread and elongated when cultured in viscoelastic guest-host hydrogels compared to nonfibrous elastic controls, with hMSCs also showing significantly decreased circularity in fibrous guest-host hydrogels compared to nonfibrous 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.

Electrospun hydrogels for dynamic culture systems: advantages, progress, and opportunities.

The extracellular matrix (ECM) is a water-swollen, tissue-specific material environment in which biophysiochemical signals are organized and influence cell behaviors. Electrospun nanofibrous substrates have been pursued as platforms for tissue engineering and cell studies that recapitulate features of the native ECM, in particular its fibrous nature. In recent years, progress in the design of electrospun hydrogel systems has demonstrated that molecular design also enables unique studies of cellular behaviors. In comparison to the use of hydrophobic polymeric materials, electrospinning hydrophilic materials that crosslink to form hydrogels offer the potential to achieve the water-swollen, nanofibrous characteristics of endogenous ECM. Although electrospun hydrogels require an additional crosslinking step to stabilize the fibers (allowing fibers to swell with water instead of dissolving) in comparison to their hydrophobic counterparts, researchers have made significant advances in leveraging hydrogel chemistries to incorporate biochemical and dynamic functionalities within the fibers. Consequently, dynamic biophysical and biochemical properties can be engineered into hydrophilic nanofibers that would be difficult to engineer in hydrophobic systems without strategic and sometimes intensive post-processing techniques. This Review describes common methodologies to control biophysical and biochemical properties of both electrospun hydrophobic and hydrogel nanofibers, with an emphasis on highlighting recent progress using hydrogel nanofibers with engineered dynamic complexities to develop culture systems for the study of biological function, dysfunction, development, and regeneration.

Selective and Improved Photoannealing of Microporous Annealed Particle (MAP) Scaffolds.

Microporous annealed particle (MAP) scaffolds consist of a slurry of hydrogel microspheres that undergo annealing to form a solid scaffold. MAP scaffolds have contained functional groups with dual abilities to participate in Michael-type addition (gelation) and radical polymerization (photoannealing). Functional groups with efficient Michael-type additions react with thiols and amines under physiological conditions, limiting usage for therapeutic delivery. We present a heterofunctional maleimide/methacrylamide 4-arm PEG macromer (MethMal) engineered for selective photopolymerization compatible with multiple polymer backbones. Rheology using two classes of photoinitiators demonstrates advantageous photopolymerization capabilities. Functional assays show benefits for therapeutic delivery and 3D printing without impacting cell viability.

3D Printing in Suspension Baths: Keeping the Promises of Bioprinting Afloat.

Extrusion-based 3D printers have been adopted in pursuit of engineering functional tissues through 3D bioprinting. However, we are still a long way from the promise of fabricating constructs approaching the complexity and function of native tissues. A major challenge is presented by the competing requirements of biomimicry and manufacturability. This opinion article discusses 3D printing in suspension baths as a novel strategy capable of disrupting the current bioprinting landscape. Suspension baths provide a semisolid medium to print into, voiding many of the inherent flaws of printing onto a flat surface in air. We review the state-of-the-art of this approach and extrapolate toward future possibilities that this technology might bring, including the fabrication of vascularized tissue constructs.

Jammed Microgel Inks for 3D Printing Applications.

3D printing involves the development of inks that exhibit the requisite properties for both printing and the intended application. In bioprinting, these inks are often hydrogels with controlled rheological properties that can be stabilized after deposition. Here, an alternate approach is developed where the ink is composed exclusively of jammed microgels, which are designed to incorporate a range of properties through microgel design (e.g., composition, size) and through the mixing of microgels. The jammed microgel inks are shear-thinning to permit flow and rapidly recover upon deposition, including on surfaces or when deposited in 3D within hydrogel supports, and can be further stabilized with secondary cross-linking. This platform allows the use of microgels engineered from various materials (e.g., thiol-ene cross-linked hyaluronic acid (HA), photo-cross-linked poly(ethylene glycol), thermo-sensitive agarose) and that incorporate cells, where the jamming process and printing do not decrease cell viability. The versatility of this particle-based approach opens up numerous potential biomedical applications through the printing of a more diverse set of inks.

Three-dimensional extrusion bioprinting of single- and double-network hydrogels containing dynamic covalent crosslinks.

The fabrication of three-dimensional (3D) scaffolds is indispensable to tissue engineering and 3D printing is emerging as an important approach towards this. Hydrogels are often used as inks in extrusion-based 3D printing, including with encapsulated cells; however, numerous challenging requirements exist, including appropriate viscosity, the ability to stabilize after extrusion, and cytocompatibility. Here, we present a shear-thinning and self-healing hydrogel crosslinked through dynamic covalent chemistry for 3D bioprinting. Specifically, hyaluronic acid was modified with either hydrazide or aldehyde groups and mixed to form hydrogels containing a dynamic hydrazone bond. Due to their shear-thinning and self-healing properties, the hydrogels could be extruded for 3D printing of structures with high shape fidelity, stability to relaxation, and cytocompatibility with encapsulated fibroblasts (>80% viability). Forces for extrusion and filament sizes were dependent on parameters such as material concentration and needle gauge. To increase scaffold functionality, a second photocrosslinkable interpenetrating network was included that was used for orthogonal photostiffening and photopatterning through a thiol-ene reaction. Photostiffening increased the scaffold's modulus (∼300%) while significantly decreasing erosion (∼70%), whereas photopatterning allowed for spatial modification of scaffolds with dyes. Overall, this work introduces a simple approach to both fabricate and modify 3D printed scaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 865-875, 2018.

Ruthenium-Crosslinked Hydrogels with Rapid, Visible-Light Degradation.

Incorporation of photoresponsive molecules within soft materials can provide spatiotemporal control over bulk properties and address challenges in targeted delivery and mechanical variability. However, the kinetics of in situ photochemical reactions are often slow and typically employ ultraviolet wavelengths. Here, we present a novel photoactive crosslinker Ru(bipyridine)2 (3-pyridinaldehyde)2 (RuAldehyde), which was reacted with hydrazide-functionalized hyaluronic acid to form hydrogels capable of encapsulating protein cargo. Visible light irradiation (400-500 nm) initiated rapid ligand exchange on the ruthenium center, which degraded the hydrogel within seconds to minutes, depending on gel thickness. An exemplar enzyme cargo, TEM1 ß-lactamase, was loaded into and photoreleased from the Ru-hydrogel. To expand their applications, Ru-hydrogels were also processed into microgels using a microfluidic platform.

A Generalizable Strategy for the 3D Bioprinting of Hydrogels from Nonviscous Photo-crosslinkable Inks.

An in situ crosslinking strategy is used for 3D bioprinting of nonviscous photo-crosslinkable hydrogels. This method can be generalized to various photo-crosslinkable formulations, maintaining high embedded cell viability and tunable cell behavior. Heterogeneous and hollow filaments can be printed using this strategy, allowing fabrication of complex engineered cell-laden constructs.

Evolution of hierarchical porous structures in supramolecular guest-host hydrogels.

Macromolecular interactions are used to form supramolecular assemblies, including through the interaction of guest-host chemical pairs. Microstructural heterogeneity has been observed within such physical hydrogels; yet, systematic investigation of the microstructure and its determining inputs are lacking. Herein, we investigated the hierarchical self-assembly of hyaluronic acid (HA) modified by the guest-host pair adamantane (Ad-HA, guest) and ß-cyclodextrin (CD-HA, host), as well as with methacrylate groups to both tether fluorescent agents and to covalently stabilize the material structure. We observed microporous materials in the hydrated state, which temporally arose from initially homogenous hydrogels composed of the two polymers. Independent fluorescent labeling of Ad-HA and CD-HA demonstrated spatiotemporal co-localization, indicative of guest-host polymer condensation on the microscale. The hydrogel void fractions and pore diameters were independently tuned through incubation time (0-7 days), polymer concentration (1.25-10 wt%), and polymer modification (25-50% Ad-HA modification). Void fractions as great as 93.3 ± 2.4% were achieved and pore diameters ranged from 2.1 ± 0.5 to 1025.4 ± 209.4 µm. The segregation of discrete solid and solute phases was measured with both atomic force microscopy and diffusive microparticle tracking analysis, where the solute phase contained only dilute polymer. The study represents a systematic investigation of hierarchical self-assembly in binary associating hydrogels, and provides insights on mechanisms that control microstructure within supramolecular hydrogels.

3D printing of photocurable poly(glycerol sebacate) elastomers.

Three-dimensional (3D) printed scaffolds have great potential in biomedicine; however, it is important that we are able to design such scaffolds with a range of diverse properties towards specific applications. Here, we report the extrusion-based 3D printing of biodegradable and photocurable acrylated polyglycerol sebacate (Acr-PGS) to fabricate scaffolds with elastic properties. Two Acr-PGS macromers were synthesized with varied molecular weights and viscosity, which were then blended to obtain photocurable macromer inks with a range of viscosities. The quality of extruded and photocured scaffolds was dependent on the initial ink viscosity, with flow of printed material resulting in a loss of structural resolution or sample breaking observed with too low or too high viscosity inks, respectively. However, scaffolds with high print resolution and up to ten layers were fabricated with an optimal ink viscosity. The mechanical properties of printed scaffolds were dependent on printing density, where the scaffolds with lower printing density possessed lower moduli and failure properties than higher density scaffolds. The 3D printed scaffolds supported the culture of 3T3 fibroblasts and both spreading and proliferation were observed, indicating that 3D printed Acr-PGS scaffolds are cytocompatible. These results demonstrate that Acr-PGS is a promising material for the fabrication of elastomeric scaffolds for biomedical applications.

Injectable and Cytocompatible Tough Double-Network Hydrogels through Tandem Supramolecular and Covalent Crosslinking.

Double-network theory is extended to include guest-host interactions, enabling injectability and cytcompatibility of tough hydrogels. Noncovalent interactions are used as a sacrificial network to toughen covalently crosslinked hydrogels formed from hyaluronic acid. Shear thinning of supramolecular bonds allows hydrogel injection and rapid self-healing, while gentle reaction conditions permit cell encapsulation with high viability.

Near-infrared light triggered release of molecules from supramolecular hydrogel-nanorod composites.

AIM: To develop a stimulus-responsive material platform capable of releasing entrapped molecules in response to near infrared (NIR) light. MATERIALS & METHODS: Gold nanorods were mixed with hyaluronic acid derivatives modified with ß-cyclodextrin or adamantane to create a NIR-responsive hydrogel-nanorod composite. Microfluidics were used to create responsive microgels and NIR-triggered release was evaluated. RESULTS & DISCUSSION: The hydrogel-nanorod composite material exhibited a rapid response to NIR-irradiation, allowing enhanced release of encapsulated payloads with material heating and network disruption. The release was dependent on the entrapped molecule size, the NIR exposure time and the light intensity. CONCLUSION: NIR irradiation of hydrogel-nanorods leads to plasmonic heating and triggered release of encapsulated molecules, a system that has potential for light-triggered release of therapeutics.

Recent advances in hyaluronic acid hydrogels for biomedical applications.

Hyaluronic acid (HA) is widely used in the design of engineered hydrogels, due to its biofunctionality, as well as numerous sites for modification with reactive groups. There are now widespread examples of modified HA macromers that form either covalent or physical hydrogels through crosslinking reactions such as with click chemistry or supramolecular assemblies of guest-host pairs. HA hydrogels range from relatively static matrices to those that exhibit spatiotemporally dynamic properties through external triggers like light. Such hydrogels are being explored for the culture of cells in vitro, as carriers for cells in vivo, or to deliver therapeutics, including in an environmentally responsive manner. The future will bring new examples of HA hydrogels due to the synthetic diversity of HA.

3D Printing of Shear-Thinning Hyaluronic Acid Hydrogels with Secondary Cross-Linking.

The development of printable biomaterial inks is critical to the application of 3D printing in biomedicine. To print high-resolution structures with fidelity to a computer-aided design, materials used in 3D printing must be capable of being deposited on a surface and maintaining a printed structure. A dual-cross-linking hyaluronic acid system was studied here as a printable hydrogel ink, which encompassed both shear-thinning and self-healing behaviors via guest-host bonding, as well as covalent cross-linking for stabilization using photopolymerization. When either guest-host assembly or covalent cross-linking was used alone, long-term stable structures were not formed, because of network relaxation after printing or dispersion of the ink filaments prior to stabilization, respectively. The dual-cross-linking hydrogel filaments formed structures with greater than 16 layers that were stable over a month with no loss in mechanical properties and the printed filament size ranged from 100 to 500 µm, depending on printing parameters (needle size, speed, and extrusion flux). Printed structures were further functionalized (i.e., RGD peptide) to support cell adhesion. This work highlights the importance of ink formulation and cross-linking on the printing of stable hydrogel structures.

Direct 3D Printing of Shear-Thinning Hydrogels into Self-Healing Hydrogels.

Supramolecular hydrogels are used in the 3D printing of high-resolution, multi-material structures. The non-covalent bonds allow the extrusion of the inks into support gels to directly write structures continuously in 3D space. This material system supports the patterning of multiple inks, cells, and void spaces.

One-Step Generation of Multifunctional Polyelectrolyte Microcapsules via Nanoscale Interfacial Complexation in Emulsion (NICE).

Polyelectrolyte microcapsules represent versatile stimuli-responsive structures that enable the encapsulation, protection, and release of active agents. Their conventional preparation methods, however, tend to be time-consuming, yield low encapsulation efficiency, and seldom allow for the dual incorporation of hydrophilic and hydrophobic materials, limiting their widespread utilization. In this work, we present a method to fabricate stimuli-responsive polyelectrolyte microcapsules in one step based on nanoscale interfacial complexation in emulsions (NICE) followed by spontaneous droplet hatching. NICE microcapsules can incorporate both hydrophilic and hydrophobic materials and also can be induced to trigger the release of encapsulated materials by changes in the solution pH or ionic strength. We also show that NICE microcapsules can be functionalized with nanomaterials to exhibit useful functionality, such as response to a magnetic field and disassembly in response to light. NICE represents a potentially transformative method to prepare multifunctional nanoengineered polyelectrolyte microcapsules for various applications such as drug delivery and cell mimicry.

Ordered, adherent layers of nanofibers enabled by supramolecular interactions.

Aligned nanofibrous substrates can be created by electrospinning, but methods for creating multilamellar structures of aligned fibers are limited. Here, apposed nanofibrous scaffolds with pendant ß-cyclodextrin (CD) were adhered together by adamantane (Ad) modified hyaluronic acid, exploiting the guest-host interactions of CD and Ad for macroscopic assembly. Stable user-defined multi-layered scaffolds were formed for cell culture or tissue engineering.