Hydrogel Stiffness Research Articles

Gelatin maleimide microgels for hematopoietic progenitor cell encapsulation.

Hematopoietic stem cells (HSCs) are the apical cells of the hematopoietic system, giving rise to cells of the blood and lymph lineages. HSCs reside primarily within bone marrow niches that contain matrix and cell-derived signals that help inform stem cell fate. Aspects of the bone marrow microenvironment have been captured in vitro by encapsulating cells within hydrogel matrices that mimic native mechanical and biochemical properties. Hydrogel microparticles, or microgels, are increasingly being used to assemble granular biomaterials for cell culture and noninvasive delivery applications. Here, we report the optimization of a gelatin maleimide hydrogel system to create monodisperse gelatin microgels via a flow-focusing microfluidic process. We report characteristic hydrogel stiffness, stability, and swelling characteristics as well as encapsulation of murine hematopoietic stem and progenitor cells, and mesenchymal stem cells within microgels. Microgels support cell viability, confirming compatibility of the microfluidic encapsulation process with these sensitive bone marrow cell populations. Overall, this work presents a microgel-based gelatin maleimide hydrogel as a foundation for future development of a multicellular artificial bone marrow culture system.

Age-associated increased stiffness of the ovarian microenvironment impairs follicle development and oocyte quality and rapidly alters follicle gene expression.

In humans, aging triggers cellular and tissue deterioration, and the female reproductive system is the first to show signs of decline. Reproductive aging is associated with decreased ovarian reserve, decreased quality of the remaining oocytes, and decreased production of the ovarian hormones estrogen and progesterone. With aging, both mouse and human ovaries become pro-fibrotic and stiff. However, whether stiffness directly impairs ovarian function, folliculogenesis, and oocyte quality is unknown. To answer this question, we cultured mouse follicles in alginate gels that mimicked the stiffness of reproductively young and old ovaries. Follicles cultured in stiff hydrogels exhibited decreased survival and growth, decreased granulosa cell viability and estradiol synthesis, and decreased oocyte quality. We also observed a reduction in the number of granulosa cell-oocyte transzonal projections. RNA sequencing revealed early changes in the follicle transcriptome in response to stiffness. Follicles cultured in a stiff environment had lower expression of genes related to follicle development and greater expression of genes related to inflammation and extracellular matrix remodeling than follicles cultured in a soft environment. Altogether, our findings suggest that ovarian stiffness directly modulates folliculogenesis and contributes to the progressive decline in oocyte quantity and quality observed in women of advanced maternal age.

Crosslinker Architectures Impact Viscoelasticity in Dynamic Covalent Hydrogels.

Dynamic covalent crosslinked (DCC) hydrogels represent a significant advance in biomaterials for regenerative medicine and mechanobiology. These gels typically offer viscoelasticity and self-healing properties that more closely mimic in vivo tissue mechanics than traditional, predominantly elastic, covalent crosslinked hydrogels. Despite their promise, the effects of varying crosslinker architecture - side chain versus telechelic crosslinks - on the viscoelastic properties of DCC hydrogels have not been thoroughly investigated. This study introduces hydrazone-based alginate hydrogels and examines how side-chain and telechelic crosslinker architectures impact hydrogel viscoelasticity and stiffness. In hydrogels with side-chain crosslinking (SCX), higher polymer concentrations enhance stiffness and decelerates stress relaxation, while an off-stoichiometric hydrazine-to-aldehyde ratio leads to reduced stiffness and shorter relaxation time. In hydrogels with telechelic crosslinking, maximal stiffness and slowest stress relaxation occurs at intermediate crosslinker concentrations for both linear and star crosslinkers, with higher crosslinker valency further increasing stiffness and relaxation time. Our result suggested different ranges of stiffness and stress relaxation are accessible with the different crosslinker architectures, with SCX hydrogels leading to slower stress relaxation relative to the other architectures, and hydrogels with star crosslinking (SX) providing increased stiffness and slower stress relaxation relative to hydrogels with linear crosslinking (LX). The mechanical properties of SX hydrogels are more robust to changes induced by competing chemical reactions compared to LX hydrogels. Our research underscores the pivotal role of crosslinker architecture in defining hydrogel stiffness and viscoelasticity, providing crucial insights for the design of DCC hydrogels with tailored mechanical properties for specific biomedical applications.

Toward Bioactive Hydrogels: A Tunable Approach via Nucleic Acid-Collagen Complexation

Abstract Purpose Nucleic acid-collagen complexes (NACCs) are unique biomaterials formed by binding short, monodisperse single-stranded DNA (ssDNA) with type I collagen. These complexes spontaneously generate microfibers and nanoparticles of varying sizes, offering a versatile platform with potential applications in tissue engineering and regenerative medicine. However, the detailed mechanisms behind the nucleic acid-driven assembly of collagen fibers still need to be established. We aim to understand the relationship between microscopic structure and bulk material properties and demonstrate that NACCs can be engineered as mechanically tunable systems. Methods We present a study to test NACCs with varying molar ratios of collagen to random ssDNA oligonucleotides. Our methods encompass the assessment of molecular interactions through infrared spectroscopy and the characterization of gelation and rheological behavior. We also include phase contrast, confocal reflectance, and transmission electron microscopy to provide complementary information on the 3D structural organization of the hydrogels. Results We report that adding DNA oligonucleotides within collagen robustly reinforces and rearranges the hydrogel network and accelerates gelation by triggering rapid fiber formation and spontaneous self-assembly. The elasticity of NACC hydrogels can be tailored according to the collagen-to-DNA molar ratio, ssDNA length, and collagen species. Conclusion Our findings hold significant implications for the design of mechanically tunable DNA-based hydrogel systems. The ability to manipulate hydrogel stiffness by tailoring DNA content and collagen concentration offers new avenues for fine tuning material properties, enhancing the versatility of bioactive hydrogels in diverse biomedical applications. Lay Summary This work is an example of forming fibers and gels with tunable elasticity that stems from the complexation of short-length nucleic acids (on the order of size of aptamers) and collagen, which can be potentially extended to a variety of functionalized hydrogel designs and tailored biomedical applications. Incorporating DNA induces mechanical changes in NACCs. Graphical Abstract

In Vitro Investigation of Vocal Fold Cellular Response to Variations in Hydrogel Porosity and Elasticity.

Tissue regeneration is intricately influenced by the dynamic interplay between the physical attributes of tissue engineering scaffolds and the resulting biological responses. A tunable microporous hydrogel system was engineered using gelatin methacryloyl (GelMA) and polyethylene glycol diacrylate (PEGDA), with polyethylene glycol (PEG) serving as a porogen. Through systematic variation of PEGDA molecular weights, hydrogels with varying mechanical and architectural properties were obtained. The objective of the present study was to elucidate the impact of substrate mechanics and architecture on the immunological and reparative activities of vocal fold tissues. Mechanical characterization of the hydrogels was performed using tensile strength measurements and rheometry. Their morphological properties were investigated using scanning electron microscopy (SEM) and confocal microscopy. A series of biological assays were conducted. Cellular morphology, differentiation, and collagen synthesis of human vocal fold fibroblasts (hVFFs) were evaluated using immunostaining. Fibroblast proliferation was studied using the WST-1 assay, and cell migration was investigated via the Boyden chamber assay. Macrophage polarization and secretions were also examined using immunostaining and ELISA. The results revealed that increasing the molecular weight of PEGDA from 700 Da to 10,000 Da resulted in decreased hydrogel stiffness, from 62.6 to 8.8 kPa, and increased pore dimensions from approximately 64.9 to 137.4 μm. Biological evaluations revealed that hydrogels with a higher stiffness promoted fibroblast proliferation and spreading, albeit with an increased propensity for fibrosis, as indicated by a surge in myofibroblast differentiation and collagen synthesis. In contrast, hydrogels with greater molecular weights had a softer matrix with expanded pores, enhancing cellular migration and promoting an M2 macrophage phenotype conducive to tissue healing. The findings show that the hydrogels formulated with a PEGDA molecular weight of 6000 Da are best among the hydrogels considered for vocal fold repair. The microporous hydrogels could be tuned to serve in other tissue engineering applications.

Localized Ionic Reinforcement of Double Network Granular Hydrogels.

Nature produces soft materials with fascinating combinations of mechanical properties. For example, the mussel byssus embodies a combination of stiffness and toughness, a feature that is unmatched by synthetic hydrogels. Key to enabling these excellent mechanical properties are the well-defined structures of natural materials and their compositions controlled on lengths scales down to tens of nanometers. The composition of synthetic materials can be controlled on a micrometer length scale if processed into densely packed microgels. However, these microgels are typically soft. Microgels can be stiffened by enhancing interactions between particles, for example through the formation of covalent bonds between their surfaces or a second interpenetrating hydrogel network. Nonetheless, changes in the composition of these synthetic materials occur on a micrometer length scale. Here, 3D printable load-bearing granular hydrogels are introduced whose composition changes on the tens of nanometer length scale. The hydrogels are composed of jammed microgels encompassing tens of nm-sized ionically reinforced domains that increase the stiffness of double network granular hydrogels up to 18-fold. The printability of the ink and the local reinforcement of the resulting granular hydrogels are leveraged to 3D print a butterfly with composition and structural changes on a tens of nanometer length scale.

Synergistic Influence of Fibrous Pattern Orientation and Modulus on Cellular Mechanoresponse.

The fibrous extracellular matrix (ECM) is vital for tissue regeneration and impacts implanted device treatments. Previous research on fibrous biomaterials shows varying cellular reactions to surface orientation, often due to unclear interactions between surface topography and substrate elasticity. Our study addresses this gap by achieving the rapid creation of hydrogels with diverse fibrous topographies and varying substrate moduli through a surface printing strategy. Cells exhibit heightened traction force on nanopatterned soft hydrogels, particularly with randomly distributed patterns compared with regular soft hydrogels. Meanwhile, on stiff hydrogels featuring an aligned topography, optimal cellular mechanosensing is observed compared to random topography. Mechanistic investigations highlight that cellular force-sensing and adhesion are influenced by the interplay of pattern deformability and focal adhesion orientation, subsequently mediating stem cell differentiation. Our findings highlight the importance of combining substrate modulus and topography to guide cellular behavior in designing advanced tissue engineering biomaterials.

Phototuning of Hyaluronic-Acid-Based Hydrogel Properties to Control Network Formation in Human Vascular Endothelial Cells.

In vitro network formation by endothelial cells serves as a fundamental model for studies aimed at understanding angiogenesis. The morphogenesis of these cells to form a network is intricately regulated by the mechanical and biochemical properties of the extracellular matrix. Here the effects of modulating these properties in hydrogels derived from phenolated hyaluronic acid (HA-Ph) and phenolated gelatin (Gelatin-Ph) are presented. Visible-light irradiation in the presence of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate induces the crosslinking of these polymers, thereby forming a hydrogel and degrading HA-Ph. Human vascular endothelial cells form networks on the hydrogel prepared by visible-light irradiation for 45min (42W cm-2 at 450nm) but not on the hydrogels prepared by irradiation for 15, 30, or 60min. The irradiation time-dependent degradation of HA-Ph and the changes in the mechanical stiffness of the hydrogels, coupled with the expressions of RhoA and β-actin genes and CD44 receptors in the cells, reveal that the network formation is synergistically influenced by the hydrogel stiffness and HA-Ph degradation. These findings highlight the potential of tailoring HA-based hydrogel properties to modulate human vascular endothelial cell responses, which is critical for advancing their application in vascular tissue engineering.

Abstract 2147: Impact Of Donor Age On Microvasculature Self-assembly Of Induced Pluripotent Stem Cell Derived Endothelial Progenitors

Introduction: Current vascular tissue-engineering using induced pluripotent stem cells (iPSCs) predominantly relies on somatic cells obtained from newborn or fetal donors. However, iPSCs sourced from aged individuals exhibit epigenetic differences in angiogenic genes that may hinder their utility in iPSC derived vasculature. We hypothesized that, due to these differences, iPSCs from aged donors will exhibit decreased vasculogenic potential. Methods: We differentiated iPSCs into endothelial progenitors (iEPs) and encapsulated them in an interpenetrating polymer network hydrogel containing norbornene-functionalized hyaluronic acid and Collagen I at cell densities and hydrogel stiffness optimal for vasculogenesis. Seven days after encapsulation, the hydrogels were fixed, and the resulting microvasculature imaged. We then utilized a previously developed computational pipeline to quantify several vasculogenic properties. Results: We derived iEPs from four separate iPSC lines: 2 sourced from neonatal donors (ND) and 2 from mature donors (MD). Irrespective of donor age, differentiation yielded ~15% (n=4) iEPs of the total cell population. The two ND lines developed a similar plexus, whereas this structure was diminished in one adult line and absent in the second. Between iEPs matched for sex and somatic cell origin, there is a 50% decrease in vessel volume fraction and number of links between vessels, a 66% reduction in the percentage of vessels connected to the largest vessel, and a 43% reduction in branch points between vessels ( Fig. 1 ). Conclusion: Overall, these data suggest MD iEPs are less likely to form dense interconnected microvasculature as compared to ND iEPs. Interestingly, the average diameter did not change across all cell lines. This suggests that while MD iEPs exhibited cell clustering, they were less likely to branch out and interconnect with other clusters of iPSC-EPs to form interconnected networks

Disaggregation and fibrillation during sol-gel transition of alginate hydrogels

The rheological and morphological characteristics of Ca-crosslinked alginate hydrogels with two different M/G ratios, α-L-guluronate (G)-rich and β-D-mannuronate (M)-rich, each with one alginic acid concentration, were investigated. It was found that the stiffness and elasticity of alginate hydrogels are derived from the thickness and density of the fibril network structures. In aqueous alginate solution, ball-like aggregates of alginates are present. Time-resolved small-angle X-ray scattering and time-domain nuclear magnetic resonance measurements suggest that the disaggregation of alginate aggregates and loose fibrillation occur in the early stage of the sol-gel transition. After these induction stage, direct gelation is finally caused by the formation of the egg-box junction. G-rich alginate hydrogel has a higher stiffness and a thicker and denser fibril network structure than M-rich alginate hydrogel. The former also exhibits faster and more significant changes in physical properties during the sol-gel transition.

Peptide Sequence Variations Govern Hydrogel Stiffness: Insights from a Multi-Scale Structural Analysis of H-FQFQFK-NH2 Peptide Derivatives.

Throughout the past decades, amphipathic peptide-based hydrogels have proven to be promising materials for biomedical applications. Amphipathic peptides are known to adopt β-sheet configurations that self-assemble into fibers that then interact to form a hydrogel network. A fundamental understanding of how the peptide sequence alters the structural properties of the hydrogels would allow for a more rational design of novel peptides for a variety of biomedical applications in the future. Therefore, the current work investigates how changing the type of amino acid, the amphipathic pattern, and the peptide length affects the secondary structure, fiber characteristics, and stiffness of peptide-based hydrogels. Hereto, seven amphipathic peptides of different sequence and length, four of which have not been previously reported, based on and including the hexapeptide H-Phe-Gln-Phe-Gln-Phe-Lys-NH2, are synthesized and thoroughly characterized by circular dichroism (CD), Fourier Transform Infrared (FTIR) spectroscopy, Wide Angle X-ray Scattering (WAXS), Small Angle X-ray Scattering (SAXS), Transmission Electron Microscopy (TEM), and Thioflavin T (ThT) fibrillization assays. The results show that a high amount of regularly spaced β-sheets, a high amount of fibers, and fiber bundling contribute to the stiffness of the hydrogel. Furthermore, a study of the time-dependent fibril formation process reveals complex transient dynamics. The peptide strands structure through an intermediate helical state prior to β-sheet formation, which is found to be concentration- andtime-dependent.

Investigating the Effect of Processing and Material Parameters of Alginate Dialdehyde-Gelatin (ADA-GEL)-Based Hydrogels on Stiffness by XGB Machine Learning Model.

To address the limitations of alginate and gelatin as separate hydrogels, partially oxidized alginate, alginate dialdehyde (ADA), is usually combined with gelatin to prepare ADA-GEL hydrogels. These hydrogels offer tunable properties, controllable degradation, and suitable stiffness for 3D bioprinting and tissue engineering applications. Several processing variables affect the final properties of the hydrogel, including degree of oxidation, gelatin content and type of crosslinking agent. In addition, in 3D-printed structures, pore size and the possible addition of a filler to make a hydrogel composite also affect the final physical and biological properties. This study utilized datasets from 13 research papers, encompassing 33 unique combinations of ADA concentration, gelatin concentration, CaCl2 and microbial transglutaminase (mTG) concentrations (as crosslinkers), pore size, bioactive glass (BG) filler content, and one identified target property of the hydrogels, stiffness, utilizing the Extreme Boost (XGB) machine learning algorithm to create a predictive model for understanding the combined influence of these parameters on hydrogel stiffness. The stiffness of ADA-GEL hydrogels is notably affected by the ADA to GEL ratio, and higher gelatin content for different ADA gel concentrations weakens the scaffold, likely due to the presence of unbound gelatin. Pore size and the inclusion of a BG particulate filler also have a significant impact on stiffness; smaller pore sizes and higher BG content lead to increased stiffness. The optimization of ADA-GEL composition and the inclusion of BG fillers are key determinants to tailor the stiffness of these 3D printed hydrogels, as found by the analysis of the available data.

Mucus-coated, magnetically-propelled fecal surrogate to mimic fecal shear forces on colonic epithelium

The relationship between the mechanical forces associated with bowel movement and colonic mucosal physiology is understudied. This is partly due to the limited availability of physiologically relevant fecal models that can exert these mechanical stimuli in in vitro colon models in a simple-to-implement manner. In this report, we created a mucus-coated fecal surrogate that was magnetically propelled to produce a controllable sweeping mechanical stimulation on primary intestinal epithelial cell monolayers. The mucus layer was derived from purified porcine stomach mucins, which were first modified with reactive vinyl sulfone (VS) groups followed by reaction with a thiol crosslinker (PEG-4SH) via a Michael addition click reaction. Formation of mucus hydrogel network was achieved at the optimal mixing ratio at 2.5 % w/v mucin-VS and 0.5 % w/v PEG-4SH. The artificial mucus layer possessed similar properties as the native mucus in terms of its storage modulus (66 Pa) and barrier function (resistance to penetration by 1-μm microbeads). This soft, but mechanically resilient mucus layer was covalently linked to a stiff fecal hydrogel surrogate (based on agarose and magnetic particles, with a storage modulus of 4600 Pa). The covalent bonding between the mucus and agarose ensured its stability in the subsequent fecal sliding movement when tested at travel distances as long as 203 m. The mucus layer served as a lubricant and protected epithelial cells from the moving fecal surrogate over a 1 h time without cell damage. To demonstrate its utility, this mucus-coated fecal surrogate was used to mechanically stimulate a fully differentiated, in vitro primary colon epithelium, and the physiological stimulated response of mucin-2 (MUC2), interleukin-8 (IL-8) and serotonin (5HT) secretion was quantified. Compared with a static control, mechanical stimulation caused a significant increase in MUC2 secretion into luminal compartment (6.4 × ), a small but significant increase in IL-8 secretion (2.5 × and 3.5 × , at both luminal and basal compartments, respectively), and no detectable alteration in 5HT secretion. This mucus-coated fecal surrogate is expected to be useful in in vitro colon organ-on-chips and microphysiological systems to facilitate the investigation of feces-induced mechanical stimulation on intestinal physiology and pathology.

Peptoid-Cross-Linked Hydrogel Stiffness Modulates Human Mesenchymal Stromal Cell Immunoregulatory Potential in the Presence of Interferon-Gamma.

Human mesenchymal stromal cell (hMSC) manufacturing requires the production of large numbers of therapeutically potent cells. Licensing with soluble cytokines improves hMSC therapeutic potency by enhancing secretion of immunoactive factors but typically decreases proliferative ability. Soft hydrogels, however, have shown promise for boosting immunomodulatory potential, which may compensate for decreased proliferation. Here, hydrogels are cross-linked with peptoids of different secondary structures to generate substrates of various bulk stiffnesses but fixed network connectivity. Secretions of interleukin 6, monocyte chemoattractive protein-1, macrophage colony-stimulating factor, and vascular endothelial growth factor are shown to depend on hydrogel stiffness in the presence of interferon gamma (IFN-γ) supplementation, with soft substrates further improving secretion. The immunological function of these secreted cytokines is then investigated via coculture of hMSCs seeded on hydrogels with primary peripheral blood mononuclear cells (PBMCs) in the presence and absence of IFN-γ. Cocultures with hMSCs seeded on softer hydrogels show decreased PBMC proliferation with IFN-γ. To probe possible signaling pathways, immunofluorescent studies probe the nuclear factor kappa B pathway and demonstrate that IFN-γ supplementation and softer hydrogel mechanics lead to higher activation of this pathway. Overall, these studies may allow for production of more efficacious therapeutic hMSCs in the presence of IFN-γ.

Stiffness Mediated-Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels.

In obstructive airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), the extracellular matrix (ECM) protein amount and composition of the airway smooth muscle (ASM) is often remodelled, likely altering tissue stiffness. The underlying mechanism of how human ASM cell (hASMC) mechanosenses the aberrant microenvironment is not well understood. Physiological stiffnesses of the ASM were measured by uniaxial compression tester using porcine ASM layers under 0, 5 and 10% longitudinal stretch above in situ length. Linear stiffness gradient hydrogels (230 kPa range) were fabricated and functionalized with ECM proteins, collagen I (ColI), fibronectin (Fn) and laminin (Ln), to recapitulate the above-measured range of stiffnesses. Overall, hASMC mechanosensation exhibited a clear correlation with the underlying hydrogel stiffness. Cell size, nuclear size and contractile marker alpha-smooth muscle actin (αSMA) expression showed a strong correlation to substrate stiffness. Mechanosensation, assessed by Lamin-A intensity and nuc/cyto YAP, exhibited stiffness-mediated behaviour only on ColI and Fn-coated hydrogels. Inhibition studies using blebbistatin or Y27632 attenuated most mechanotransduction-derived cell morphological responses, αSMA and Lamin-A expression and nuc/cyto YAP (blebbistatin only). This study highlights the interplay and complexities between stiffness and ECM protein type on hASMC mechanosensation, relevant to airway remodelling in obstructive airway diseases.

Deep learning-assisted concentration gradient generation for the study of 3D cell cultures in hydrogel beads of varying stiffness.

The study of dose-response relationships underpins analytical biosciences. Droplet microfluidics platforms can automate the generation of microreactors encapsulating varying concentrations of an assay component, providing datasets across a large chemical space in a single experiment. A classical method consists in varying the flow rate of multiple solutions co-flowing into a single microchannel (producing different volume fractions) before encapsulating the contents into water-in-oil droplets. This process can be automated through controlling the pumping elements but lacks the ability to adapt to unpredictable experimental scenarios, often requiring constant human supervision. In this paper, we introduce an image-based, closed-loop control system for assessing and adjusting volume fractions, thereby generating unsupervised, uniform concentration gradients. We trained a shallow convolutional neural network to assess the position of the laminar flow interface between two co-flowing fluids and used this model to adjust flow rates in real-time. We apply the method to generate alginate microbeads in which HEK293FT cells could grow in three dimensions. The stiffnesses ranged from 50Pa to close to 1kPa in Young modulus and were encoded with a fluorescent marker. We trained deep learning models based on the YOLOv4 object detector to efficiently detect both microbeads and multicellular spheroids from high-content screening images. This allowed us to map relationships between hydrogel stiffness and multicellular spheroid growth.

A Biomimetic "Salting Out-Alignment-Locking" Tactic to Design Strong and Tough Hydrogel.

Recently, hydrogel-based soft materials have demonstrated huge potential in soft robotics, flexible electronics as well as artificial skins. Although various methods are developed to prepare tough and strong hydrogels, it is still challenging to simultaneously enhance the strength and toughness of hydrogels, especially for protein-based hydrogels. Herein, a biomimetic "salting out-alignment-locking" tactic (SALT) is introduced for enhancing mechanical properties through the synergy of alignment and the salting out effect. As a typical example, tensile strength and modulus of initially brittle gelatin hydrogels increase 940 folds to 10.12±0.50MPa and 2830 folds to 34.26±3.94MPa, respectively, and the toughness increases up to 1785 folds to 14.28±3.13MJ m-3. The obtained strength and toughness hold records for the previously reported gelatin-based hydrogel and are close to the tendons. It is further elucidated that the salting out effect engenders hydrophobic domains, while prestretching facilitates chain alignment, both synergistically contributing to the outstanding mechanical properties. It is noteworthy that the SALT demonstrates remarkable versatility across different salt types and polymer systems, thus opening up new avenues for engineering strong, tough, and stiff hydrogels.

Enzymatically gellable chitosan inks with enhanced printability by chitosan nanofibers for 3D printing of wound dressings

Chitosan-based hydrogels have received considerable attention as wound dressing materials. The utility of these materials can be further enhanced in combination with three-dimensional (3D) printing for the fabrication of dressings tailored to the specific needs of individual wounds. Here, we investigated the fabrication of wound dressings from hydrogel precursor inks containing phenolated chitosan (chitosan-Ph) and chitosan nanofibers (chitosan-NF) via 3D printing with horseradish peroxidase (HRP)-mediated hydrogelation. By adjusting the content of chitosan-Ph and chitosan-NFs, the ink viscosity, gelation time, and hydrogel stiffness could be controlled. The composite hydrogel exhibited antibacterial properties and cytocompatibility. In particular, hydrogels with 2% (w/w) chitosan-Ph and 1% (w/w) chitosan-NFs demonstrated superior design fidelity and promising wound healing properties compared to commercial dressings. These hydrogels promoted re-epithelialization, granulation tissue formation and extracellular matrix organization without exacerbating inflammation or foreign body reaction. These results underscore the efficacy of incorporating chitosan-NF into chitosan-Ph inks to produce patient-specific wound dressings by extrusion 3D printing, which represents a significant advancement in personalized wound care.

Abstract 5409: Phosphorylated focal adhesion kinase by mild reduction of cell surface proteins inhibits integrin α5β1-dependent patient- derived cancer cell migration and invasion

Abstract Although head and neck squamous cell carcinomas (HNSCCs), including tongue, vocal cord, and buccal cancers, are one of the most common cancers worldwide with a mortality rate of more than 40%, there have been no solid standard biomarkers for effectively diagnosing HNSCCs at early stages and no effective protocols available to support in vitro expansion of HNSCC cells. To establish a cell culture method to support in vitro self-renewal of HNSCC cells, patient-derived tumor was enzymatically digested, and the tongue cancer epithelial cells were seeded onto feeder cells with the Rho kinase inhibitor (Y-27632). Additionally, to test our hypothesis that free thiol groups in the cells, generated by a mild reduction of cell surface proteins using the reducing agent tris(2-carboxyethyl) phosphine hydrochloride (TCEP), would modulate cancer cell adhesion and migration, we assessed the expression levels of integrins, focal adhesion kinase (FAK), and phosphorylated FAK (pFAK) of tongue cancer epithelial cells cultured on both soft and rigid polyacrylamide (PAAm) hydrogel-based cell culture substrates. Patient-derived tongue cancer epithelial cells exhibited self-renewal ability and maintained cancerous phenotypes, with high expressions of P53, Ki67, E-cadherin, CD44, and AHDH1A1. Treatment with the reducing agent (TCEP) increased the degree of cell adhesion through the significantly upregulated integrin α5β1-mediated phosphorylation of FAK, resulting in inhibited single-cell and collective cell migration as well as invasion. Additionally, when these cells were cultured on stiff PAAm hydrogels mimicking the tumor microenvironment, the cells exhibited accelerated cell migration behaviors, compared to cells on soft hydrogels. These results highlight the synergetic contributions of feeder cells and Rho kinase inhibitor to support long-term self-renewal of tongue cancer patient-derived epithelial cells. Citation Format: Joo Hyun Kim, Laurensia D. Anggradita, Sung Sik Hur, Jae Hong Park, Sang-Heon Kim, Myung Jin Ban, Yongsung Hwang. Phosphorylated focal adhesion kinase by mild reduction of cell surface proteins inhibits integrin α5β1-dependent patient- derived cancer cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5409.

Decoupling stiffness and toughness of self-healing hydrogels for complex tissue regeneration via 3D bioprinting

Although polysaccharides are attractive biocompatible materials compared with synthetic polymers, most polysaccharide-based hydrogels exhibit suboptimal mechanical properties. In this study, we fabricated a hyaluronate (HA)-based self-healing hydrogel with enhanced toughness that can be used as a 3D bioink for complex tissue regeneration. We hypothesized that the incorporation of oxidation, aimed at minimizing the reduction in the molecular weight of HA, along with the introduction of a double network to the hydrogel, could facilitate the construction of 3D structures with decoupled stiffness and toughness. Various physicochemical properties of hydrogels comprising oxidized and hydrazide-modified HAs for potential use as bioinks were assessed. Bilayered constructs containing primary chondrocytes and osteoblasts were designed and fabricated using 3D printing to mimic the growth plate structure. Transplantation of the 3D-printed construct into a mouse model revealed the controlled differentiation stages of chondrocytes. This strategy for tailoring 3D scaffolds and addressing the limitations of polysaccharide-based hydrogels may hold promise for applications in the biomedical field, including the regeneration of complex tissues and organs.