Hyaluronan improves photoreceptor differentiation and maturation in human retinal organoids.

Human stem cell-derived organoids enable both disease modeling and serve as a source of cells for transplantation. Human retinal organoids are particularly important as a source of human photoreceptors; however, the long differentiation period required and lack of vascularization in the organoid often results in a necrotic core and death of inner retinal cells before photoreceptors are fully mature. Manipulating the in vitro environment of differentiating retinal organoids through the incorporation of extracellular matrix components could influence retinal development. We investigated the addition of hyaluronan (HA), a component of the interphotoreceptor matrix, as an additive to promote long-term organoid survival and enhance retinal maturation. HA treatment had a significant reduction in the proportion of proliferating (Ki67+) cells and increase in the proportion of photoreceptors (CRX+), suggesting that HA accelerated photoreceptor commitment in vitro. HA significantly upregulated genes specific to photoreceptor maturation and outer segment development. Interestingly, prolonged HA-treatment significantly decreased the length of the brush border layer compared to those in control retinal organoids, where the photoreceptor outer segments reside; however, HA-treated organoids also had more mature outer segments with organized discs structures, as revealed by transmission electron microscopy. The brush border layer length was inversely proportional to the molar mass and viscosity of the hyaluronan added. This is the first study to investigate the role of exogenous HA, viscosity, and polymer molar mass on photoreceptor maturation, emphasizing the importance of material properties on organoid culture. STATEMENT OF SIGNIFICANCE: Retinal organoids are a powerful tool to study retinal development in vitro, though like many other organoid systems, can be highly variable. In this work, Shoichet and colleagues investigated the use of hyaluronan (HA), a native component of the interphotoreceptor matrix, to improve photoreceptor maturation in developing human retinal organoids. HA promoted human photoreceptor differentiation leading to mature outer segments with disc formation and more uniform and healthy retinal organoids. These findings highlight the importance of adding components native to the developing retina to generate more physiologically relevant photoreceptors for cell therapy and in vitro models to drive drug discovery and uncover novel disease mechanisms.

Transplanted human photoreceptors transfer cytoplasmic material but not to the recipient mouse retina.

BACKGROUND: The discovery of material transfer between transplanted and host mouse photoreceptors has expanded the possibilities for utilizing transplanted photoreceptors as potential vehicles for delivering therapeutic cargo. However, previous research has not directly explored the capacity for human photoreceptors to engage in material transfer, as human photoreceptor transplantation has primarily been investigated in rodent models of late-stage retinal disease, which lack host photoreceptors. METHODS: In this study, we transplanted human stem-cell derived photoreceptors purified from human retinal organoids at different ontological ages (weeks 10, 14, or 20) into mouse models with intact photoreceptors and assessed transfer of human proteins and organelles to mouse photoreceptors. RESULTS: Unexpectedly, regardless of donor age or mouse recipient background, human photoreceptors did not transfer material in the mouse retina, though a rare subset of donor cells (< 5%) integrated into the mouse photoreceptor cell layer. To investigate the possibility that a species barrier impeded transfer, we used a flow cytometric assay to examine material transfer in vitro. Interestingly, dissociated human photoreceptors transferred fluorescent protein with each other in vitro, yet no transfer was detected in co-cultures of human and mouse photoreceptors, suggesting that material transfer is species specific. CONCLUSIONS: While xenograft models are not a tractable system to study material transfer of human photoreceptors, these findings demonstrate that human retinal organoid-derived photoreceptors are competent donors for material transfer and thus may be useful to treat retinal degenerative disease.

Engineering Biomaterials to Model Immune-Tumor Interactions In Vitro.

Engineered biomaterial scaffolds are becoming more prominent in research laboratories to study drug efficacy for oncological applications in vitro, but do they have a place in pharmaceutical drug screening pipelines? The low efficacy of cancer drugs in phase II/III clinical trials suggests that there are critical mechanisms not properly accounted for in the pre-clinical evaluation of drug candidates. Immune cells associated with the tumor may account for some of these failures given recent successes with cancer immunotherapies; however, there are few representative platforms to study immune cells in the context of cancer as traditional 2D culture is typically monocultures and humanized animal models have a weakened immune composition. Biomaterials that replicate tumor microenvironmental cues may provide a more relevant model with greater in vitro complexity. In this review, the authors explore the pertinent microenvironmental cues that drive tumor progression in the context of the immune system, discuss how these cues can be incorporated into hydrogel design to culture immune cells, and describe progress toward precision oncological drug screening with engineered tissues.

Long-Acting Ocular Injectables: Are We Looking In The Right Direction?

The complex anatomy and physiological barriers of the eye make delivering ocular therapeutics challenging. Generally, effective drug delivery to the eye is hindered by rapid clearance and limited drug bioavailability. Biomaterial-based approaches have emerged to enhance drug delivery to ocular tissues and overcome existing limitations. In this review, some of the most promising long-acting injectables (LAIs) in ocular drug delivery are explored, focusing on novel design strategies to improve therapeutic outcomes. LAIs are designed to enable sustained therapeutic effects, thereby extending local drug residence time and facilitating controlled and targeted drug delivery. Moreover, LAIs can be engineered to enhance drug targeting and penetration across ocular physiological barriers.

Engineered In Vitro Tumor Model Recapitulates Molecular Signatures of Invasion in Glioblastoma.

Glioblastoma stem cells (GSCs) play an important role in the invasive nature of glioblastoma (GBM); yet, the mechanisms driving this behavior are poorly understood. To recapitulate tumor invasion in vitro, we developed a GBM tumor-mimetic hydrogel using extracellular matrix components upregulated in patients. We show that our hydrogel facilitates the infiltration of a subset of patient-derived GSCs, differentiating samples based on phenotypic invasion. Invasive GSCs are enriched for injury-responsive pathways while noninvasive GSCs are enriched for developmental pathways, reflecting established GSC stratifications. Using small molecule inhibitors, we demonstrate that the suppression of matrix metalloprotease and rho-associated protein kinase processes results in a significant reduction of cell invasion into the hydrogel, reflecting mesenchymal- and amoeboid-dependent mechanisms. Similar reduction in cell invasion was observed by siRNA knockdown of ITGB1 and FAK focal adhesion pathways. We elucidate the transcriptomic profile of cells invading in the hydrogel by performing bulk RNA sequencing of cells cultured in the hydrogel and compare these to cells cultured in conventional tissue culture polystyrene (TCP). In our 3D hydrogel cultures, invasion-related molecular signatures along with proliferation and injury response pathways are upregulated while development processes are downregulated compared to culture on 2D TCP. With this validated in vitro model, we establish a valuable tool to find therapeutic intervention strategies against cellular invasion in glioblastoma.

Soluble CX3CL1-expressing retinal pigment epithelium cells protect rod photoreceptors in a mouse model of retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa (RP) is an inherited retinal disease that results in photoreceptor degeneration, leading to severe vision loss or blindness. Due to its genetic heterogeneity, developing a new gene therapy to correct every genetic mutation contributing to its progression is infeasible. Photoreceptor transplantation can be harnessed to restore vision; however, this approach is limited by poor cell survival and synaptic integration into the neural retina. Thus, we developed a combined cell and gene therapy that is expected to protect photoreceptors in most, if not all, cases of RP. METHODS: Human embryonic stem cells (hESCs) modified with our FailSafe™ system were genetically engineered to overexpress sCX3CL1, an inhibitor of microglia activation that has been shown to preserve photoreceptor survival and function in mouse models of RP, independent of the genetic cause. These cells were differentiated into human retinal pigment epithelium (hRPE) cells and used as therapeutic cells due to their longevity and safety, both of which have been demonstrated in preclinical and clinical studies. Transgenic hRPE were delivered into the subretinal space of immunodeficient mice and the rd10 mouse model of RP to evaluate donor cell survival and retention of transgene expression. The outer nuclear layer was quantified to assess photoreceptor protection. RESULTS: Transgenic FailSafe™ hRPE (FS-hRPE) cells can survive for at least four months in the retina of immunodeficient mice and retain transgene expression. However, these cells do not persist beyond two weeks post-injection in the retina of immunocompetent rd10 recipients, despite Cyclosporine A treatment. Nevertheless, sCX3CL1-expressing FailSafe™ hRPE cells prevented photoreceptor degeneration in a local acting manner during the duration of their presence in the subretinal space. CONCLUSIONS: Transgenic hESCs differentiate into hRPE cells and retain sCX3CL1 transgene expression both in vitro and in vivo. Moreover, hRPE cells delivered to the subretinal space of rd10 mice prevented photoreceptor degeneration in a local-acting manner, suggesting that this approach could have applications for preserving photoreceptors in specific subregions of the retina, such as the macula. Overall, our study not only reveals the potential of a combined cell and gene therapy for the treatment of RP, but also the possibility of using hRPE cells to deliver therapeutic biologics in situ to treat diseases over long-term.

Tissue-Engineered Disease Modeling of Lymphangioleiomyomatosis Exposes a Therapeutic Vulnerability to HDAC Inhibition.

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring hyperactive mTORC1 signaling. Here, tissue engineering tools are employed to model LAM and identify new therapeutic candidates. Biomimetic hydrogel culture of LAM cells is found to recapitulate the molecular and phenotypic characteristics of human disease more faithfully than culture on plastic. A 3D drug screen is conducted, identifying histone deacetylase (HDAC) inhibitors as anti-invasive agents that are also selectively cytotoxic toward TSC2-/- cells. The anti-invasive effects of HDAC inhibitors are independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Genotype-selective cytotoxicity is seen exclusively in hydrogel culture due to potentiated differential mTORC1 signaling, a feature that is abrogated in cell culture on plastic. Importantly, HDAC inhibitors block invasion and selectively eradicate LAM cells in vivo in zebrafish xenografts. These findings demonstrate that tissue-engineered disease modeling exposes a physiologically relevant therapeutic vulnerability that would be otherwise missed by conventional culture on plastic. This work substantiates HDAC inhibitors as possible therapeutic candidates for the treatment of patients with LAM and requires further study.

Hydrogel assisted photoreceptor delivery inhibits material transfer.

Cell therapy holds tremendous promise for vision restoration; yet donor cell survival and integration continue to limit efficacy of these strategies. Transplanted photoreceptors, which mediate light sensitivity in the retina, transfer cytoplasmic components to host photoreceptors instead of integrating into the tissue. Donor cell material transfer could, therefore, function as a protein augmentation strategy to restore photoreceptor function. Biomaterials, such as hyaluronan-based hydrogels, can support donor cell survival but have not been evaluated for effects on material transfer. With increased survival, we hypothesized that we would achieve greater material transfer; however, the opposite occurred. Photoreceptors delivered to the subretinal space in mice in a hyaluronan and methylcellulose (HAMC) hydrogel showed reduced material transfer. We examined mitochondria transfer in vitro and cytosolic protein transfer in vivo and demonstrate that HAMC significantly reduced transfer in both contexts, which we ascribe to reduced cell-cell contact. Nanotube-like donor cell protrusions were significantly reduced in the hydrogel-transplanted photoreceptors compared to the saline control group, which suggests that HAMC limits the contact required to the host retina for transfer. Thus, HAMC can be used to manipulate the behaviour of transplanted donor cells in cell therapy strategies.

Targeting tumour-associated macrophages in hodgkin lymphoma using engineered extracellular matrix-mimicking cryogels.

Tumour-associated macrophages are linked with poor prognosis and resistance to therapy in Hodgkin lymphoma; however, there are no suitable preclinical models to identify macrophage-targeting therapeutics. We used primary human tumours to guide the development of a mimetic cryogel, wherein Hodgkin (but not Non-Hodgkin) lymphoma cells promoted primary human macrophage invasion. In an invasion inhibitor screen, we identified five drug hits that significantly reduced tumour-associated macrophage invasion: marimastat, batimastat, AS1517499, ruxolitinib, and PD-169316. Importantly, ruxolitinib has demonstrated recent success in Hodgkin lymphoma clinical trials. Both ruxolitinib and PD-169316 (a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor) decreased the percent of M2-like macrophages; however, only PD-169316 enhanced the percentage of M1-like macrophages. We validated p38 MAPK as an anti-invasion drug target with five additional drugs using a high-content imaging platform. With our biomimetic cryogel, we modeled macrophage invasion in Hodgkin lymphoma and then used it for target discovery and drug screening, ultimately identifying potential future therapeutics.

Synthetic Ionizable Colloidal Drug Aggregates Enable Endosomal Disruption.

Colloidal drug aggregates enable the design of drug-rich nanoparticles; however, the efficacy of stabilized colloidal drug aggregates is limited by entrapment in the endo-lysosomal pathway. Although ionizable drugs are used to elicit lysosomal escape, this approach is hindered by toxicity associated with phospholipidosis. It is hypothesized that tuning the pKa of the drug would enable endosomal disruption while avoiding phospholipidosis and minimizing toxicity. To test this idea, 12 analogs of the nonionizable colloidal drug fulvestrant are synthesized with ionizable groups to enable pH-dependent endosomal disruption while maintaining bioactivity. Lipid-stabilized fulvestrant analog colloids are endocytosed by cancer cells, and the pKa of these ionizable colloids influenced the mechanism of endosomal and lysosomal disruption. Four fulvestrant analogs-those with pKa values between 5.1 and 5.7-disrupted endo-lysosomes without measurable phospholipidosis. Thus, by manipulating the pKa of colloid-forming drugs, a tunable and generalizable strategy for endosomal disruption is established.

Affinity-controlled release of rod-derived cone viability factor enhances cone photoreceptor survival.

Retinitis pigmentosa (RP) is a group of genetic diseases that results in rod photoreceptor cell degeneration, which subsequently leads to cone photoreceptor cell death, impaired vision and eventual blindness. Rod-derived cone viability factor (RdCVF) is a protein which has two isoforms: a short form (RdCVF) and a long form (RdCVFL) which act on cone photoreceptors in the retina. RdCVFL protects photoreceptors by reducing hyperoxia in the retina; however, sustained delivery of RdCVFL remains challenging. We developed an affinity-controlled release strategy for RdCVFL. An injectable physical blend of hyaluronan and methylcellulose (HAMC) was covalently modified with a peptide binding partner of the Src homology 3 (SH3) domain. This domain was expressed as a fusion protein with RdCVFL, thereby enabling its controlled release from HAMC-binding peptide. Sustained release of RdCVFL was demonstrated for the first time as RdCVFL-SH3 from HAMC-binding peptide for 7 d in vitro. To assess bioactivity, chick retinal dissociates were harvested and treated with the affinity-released recombinant protein from the HAMC-binding peptide vehicle. After 6 d in culture, cone cell viability was greater when cultured with released RdCVFL-SH3 relative to controls. We utilized computational fluid dynamics to model release of RdCVFL-SH3 from our delivery vehicle in the vitreous of the human eye. We demonstrate that our delivery vehicle can prolong the bioavailability of RdCVFL-SH3 in the retina, potentially enhancing its therapeutic effects. Our affinity-based system constitutes a versatile delivery platform for ultimate intraocular injection in the treatment of retinal degenerative diseases. STATEMENT OF SIGNIFICANCE: Retinitis pigmentosa (RP) is the leading cause of inherited blindness in the world. Rod-derived cone viability factor (RdCVF), a novel protein paracrine factor, is effective in preclinical models of RP. To extend its therapeutic effects, we developed an affinity-controlled release strategy for the long form of RdCVF, RdCVFL. We expressed RdCVFL as a fusion protein with an Src homology 3 domain (SH3). We then utilized a hydrogel composed of hyaluronan and methylcellulose (HAMC) and modified it with SH3 binding peptides to investigate its release in vitro. Furthermore, we designed a mathematical model of the human eye to investigate delivery of the protein from the delivery vehicle. This work paves the way for future investigation of controlled release RdCVF.

Protein Release by Controlled Desorption from Transiently Cationic Nanoparticles.

Therapeutic release from hydrogels is traditionally controlled by encapsulation within nanoparticles; however, this strategy is limited for the release of proteins due to poor efficiency and denaturation. To overcome this problem, we designed an encapsulation-free release platform where negatively charged proteins are adsorbed to the exterior of transiently cationic nanoparticles, thus allowing the nanoparticles to be formulated separately from the proteins. Release is then governed by the change in nanoparticle surface charge from positive to neutral. To achieve this, we synthesized eight zwitterionic poly(lactide-block-carboxybetaine) copolymer derivatives and formulated them into nanoparticles with differing surface chemistry. The nanoparticles were colloidally stable and lost positive charge at rates dependent on the hydrolytic stability of their surface ester groups. The nanoparticles (NPs) were dispersed in a physically cross-linked hyaluronan-based hydrogel with one of three negatively charged proteins (transferrin, panitumumab, or granulocyte-macrophage colony-stimulating factor) to assess their ability to control release. For all three proteins, dispersing NPs within the gels resulted in significant attenuation of release, with the extent modulated by the hydrolytic stability of the surface groups. Release was rapid from fast-hydrolyzing ester groups, reduced with slow-hydrolyzing bulky ester groups, and very slow with nonhydrolyzing amide groups. When positively charged lysozyme was loaded into the nanocomposite gel, there was no significant attenuation of release compared to gel alone. These data demonstrate that electrostatic interactions between the protein and NP are the primary driver of protein release from the hydrogel. All released proteins retained bioactivity as determined with in vitro cell assays. This release strategy shows tremendous versatility and provides a promising new platform for controlled release of anionic protein therapeutics.

Tunable Surface Charge Enables the Electrostatic Adsorption-Controlled Release of Neuroprotective Peptides from a Hydrogel-Nanoparticle Drug Delivery System.

We exploit the electrostatic interactions between the positively charged neuroprotective peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), and negatively charged poly(lactic-co-glycolic acid) (PLGA) nanoparticles to control PACAP release from the surface of nanoparticles dispersed in a hyaluronan-methylcellulose (HAMC) hydrogel composite. PACAP is a promising therapeutic for the treatment of neurological disorders, yet it has been difficult to deliver in vivo. Herein, the PACAP release rate was tuned by manipulating peptide adsorption onto the surface of blank nanoparticles by modifying either nanoparticle loading in the hydrogel or nanoparticle surface charge. This peptide-nanoparticle interaction was controlled by the pH-responsive carboxylic acid end terminal groups of PLGA. We further validated this system with the controlled release of a novel stabilized PACAP analog: Ac-[Ala15, Ala20]PACAP-propylamide, which masks its recognition to peptidases in circulation. Both wild-type and stabilized PACAP released from the vehicle increased the production of neuroprotective Interleukin-6 from cultured primary astrocytes. Using computational fluid dynamics methods, PACAP release from the composite was predicted based on experimentally derived adsorption isotherms, which exhibited similar release profiles to experimental data. This versatile adsorption-based system was used to deliver PACAP locally to the brains of stroke-injured mice over a 10 day period in vivo, highlighting its effectiveness for the controlled release of PACAP to the central nervous system.

Affibody-mediated controlled release of fibroblast growth factor 2.

Protein therapeutics possess high target affinity and specificity, yet short residence times, which limit their broad utility. To overcome this challenge, we used affinity interactions to modulate protein release from a hydrogel delivery vehicle thereby prolonging therapeutic availability. Specifically, we designed an affibody-modified hyaluronan (HA)-based hydrogel as a delivery platform for fibroblast growth factor 2 (FGF2), a neuroprotective and neuroregenerative factor in the central nervous system (CNS). We identified a highly specific affibody binding partner with moderate affinity for FGF2 using yeast surface display and flow cytometry-based screening. Importantly, we demonstrated controlled release of bioactive FGF2 from the hydrogel by varying the ratio of affibody to protein and showed increased thermal stability of FGF2 in the presence of affibody. This versatile delivery platform will allow the distinct, simultaneous release of multiple proteins based on specific affinity interactions.

Viscoelastic Notch Signaling Hydrogel Induces Liver Bile Duct Organoid Growth and Morphogenesis.

Cholangiocyte organoids can be used to model liver biliary disease; however, both a defined matrix to emulate cholangiocyte self-assembly and the mechano-transduction pathways involved therein remain elusive. A series of defined viscoelastic hyaluronan hydrogels to culture primary cholangiocytes are designed and it is found that by mimicking the stress relaxation rate of liver tissue, cholangiocyte organoid growth can be induced and expression of Yes-associated protein (YAP) target genes could be significantly increased. Strikingly, inhibition of matrix metalloproteinases (MMPs) does not significantly affect organoid growth in 3D culture, suggesting that mechanical remodeling of the viscoelastic microenvironment-and not MMP-mediated degradation-is the key to cholangiocyte organoid growth. By immobilizing Jagged1 to the hyaluronan, stress relaxing hydrogel, self-assembled bile duct structures form in organoid culture, indicating the synergistic effects of Notch signaling and viscoelasticity. By uncovering critical roles of hydrogel viscoelasticity, YAP signaling, and Notch activation, cholangiocyte organogenesis is controlled, thereby paving the way for their use in disease modeling and/or transplantation.

Directed Evolution Enables Simultaneous Controlled Release of Multiple Therapeutic Proteins from Biopolymer-Based Hydrogels.

With the advent of increasingly complex combination strategies of biologics, independent control over their delivery is the key to their efficacy; however, current approaches are hindered by the limited independent tunability of their release rates. To overcome these limitations, directed evolution is used to engineer highly specific, low affinity affibody binding partners to multiple therapeutic proteins to independently control protein release rates. As a proof-of-concept, specific affibody binding partners for two proteins with broad therapeutic utility: insulin-like growth factor-1 (IGF-1) and pigment epithelium-derived factor (PEDF) are identified. Protein-affibody binding interactions specific to these target proteins with equilibrium dissociation constants (KD ) between 10-7 and 10-8 m are discovered. The affibodies are covalently bound to the backbone of crosslinked hydrogels using click chemistry, enabling sustained, independent, and simultaneous release of bioactive IGF-1 and PEDF over 7 days. The system is tested with C57BL/6J mice in vivo, and the affibody-controlled release of IGF-1 results in sustained activity when compared to bolus IGF-1 delivery. This work demonstrates a new, broadly applicable approach to tune the release of therapeutic proteins simultaneously and independently and thus the way for precise control over the delivery of multicomponent therapies is paved.

Temporally Controlled Photouncaged Epidermal Growth Factor Influences Cell Fate in Hydrogels.

Hydrogels are powerful materials that more accurately mimic the cellular microenvironment over static two-dimensional culture. Photochemical strategies enable dynamic complexity to be achieved within hydrogels to better mimic the extracellular matrix; however, many photochemical systems to pattern proteins within hydrogels are complicated by long reaction times to immobilize these proteins wherein the protein can lose activity. As proof-of-concept, we demonstrate an elegant method where photocaged proteins are immobilized in hydrogels and then directly photoactivated. Specifically, we immobilized streptavidin-ortho-nitrobenzyl-modified epidermal growth factor (EGF) to cross-linked hyaluronan hydrogels and cultured two EGF-responsive cancer cells of breast and lung therein. We used light to temporally uncage and control EGF activation, thereby inducing cell death in breast cancer cells and proliferation in lung cancer cells. These results show how temporal, photochemical, protein activation influences cellular response and lays the foundation for further advances in manipulating the in vitro environment to control cell fate.

Synthesis of an Enzyme-Mediated Reversible Cross-linked Hydrogel for Cell Culture.

Detachment of fragile cell types cultured on two-dimensional (2D) surfaces has been shown to be detrimental to their viability. For example, detachment of induced pluripotent stem cell (iPSC)-derived neurons grown in vitro in 2D typically results in loss of neuronal connections and/or cell death. Avoiding cell detachment altogether by changing the properties of the substrate on which the cells are grown is a compelling strategy to maintain cell viability. Here, we present the synthesis of a reversible cross-linked hydrogel that is sufficiently stable for cell culture and differentiation and is cleaved by an external stimulus, facilitating injection. Specifically, hyaluronan (HA) and methylcellulose (MC) were modified with ketone and aldehyde groups, respectively, and a TEV protease-degradable peptide was synthesized via solid-state synthesis and modified at both termini with oxyamine groups to cross-link HA-ketone and MC-aldehyde to produce oxime-cross-linked HA × MC. The HA × MC hydrogel demonstrated good stability, enzyme-sensitive degradation, and cytocompatibility with iPSC-derived neural progenitor cells, laying the framework for broad applicability.

Designing Hydrogels for 3D Cell Culture Using Dynamic Covalent Crosslinking.

Designing simple biomaterials to replicate the biochemical and mechanical properties of tissues is an ongoing challenge in tissue engineering. For several decades, new biomaterials have been engineered using cytocompatible chemical reactions and spontaneous ligations via click chemistries to generate scaffolds and water swollen polymer networks, known as hydrogels, with tunable properties. However, most of these materials are static in nature, providing only macroscopic tunability of the scaffold mechanics, and do not reflect the dynamic environment of natural extracellular microenvironment. For more complex applications such as organoids or co-culture systems, there remain opportunities to investigate cells that locally remodel and change the physicochemical properties within the matrices. In this review, advanced biomaterials where dynamic covalent chemistry is used to produce stable 3D cell culture models and high-resolution constructs for both in vitro and in vivo applications, are discussed. The implications of dynamic covalent chemistry on viscoelastic properties of in vitro models are summarized, case studies in 3D cell culture are critically analyzed, and opportunities to further improve the performance of biomaterials for 3D tissue engineering are discussed.

Stable oxime-crosslinked hyaluronan-based hydrogel as a biomimetic vitreous substitute.

Vitreous substitutes are clinically used to maintain retinal apposition and preserve retinal function; yet the most used substitutes are gases and oils which have disadvantages including strict face-down positioning post-surgery and the need for subsequent surgical removal, respectively. We have engineered a vitreous substitute comprised of a novel hyaluronan-oxime crosslinked hydrogel. Hyaluronan, which is naturally abundant in the vitreous of the eye, is chemically modified to crosslink with poly(ethylene glycol)-tetraoxyamine via oxime chemistry to produce a vitreous substitute that has similar physical properties to the native vitreous including refractive index, density and transparency. The oxime hydrogel is cytocompatible in vitro with photoreceptors from mouse retinal explants and biocompatible in rabbit eyes as determined by histology of the inner nuclear layer and photoreceptors in the outer nuclear layer. The ocular pressure in the rabbit eyes was consistent over 56 d, demonstrating limited to no swelling. Our vitreous substitute was stable in vivo over 28 d after which it began to degrade, with approximately 50% loss by day 56. We confirmed that the implanted hydrogel did not impact retina function using electroretinography over 90 days versus eyes injected with balanced saline solution. This new oxime hydrogel provides a significant improvement over the status quo as a vitreous substitute.