Cell Adhesion Sites Research Articles

Quercetin-enriched animal-free scaffolds for promoted cell proliferation and differentiation in cultured meat production

To solve the challenges posed by increasing meat consumption, it is imperative to develop polysaccharide composite edible scaffolds for cell-cultured meat production using novel food production technologies. This study developed some non-animal-origin polysaccharide composite scaffolds for cell-cultured meat production, based on chitin‑sodium alginate and crosslinked with quercetin. The results indicated that the chitin‑sodium alginate-quercetin (CH-SA-QR) scaffolds exhibited a porous and loose structure, and excellent mechanical support capability and cell adhesion sites. Meanwhile, QR1 and QR1.5 scaffolds were screened by immunofluorescence staining, RT-qPCR, and SEM imaging results to facilitate the proliferation and differentiation of cells and enhance the generation of myotube fusion and extracellular matrix protein. Importantly, the texture of the fried cultured meat was similar to traditional beef or even more tender, while the color appearance of the cultured meat was different from beef, but more similar to fried meat cutlets. These results suggest that cultured meat produced based on CH-SA-QR scaffolds could provide consumers with more choices.

Methods to achieve tissue-mimetic physicochemical properties in hydrogels for regenerative medicine and tissue engineering.

Hydrogels are water-swollen polymeric matrices with properties that are remarkably similar in function to the extracellular matrix. For example, the polymer matrix provides structural support and adhesion sites for cells in much of the same way as the fibers of the extracellular matrix. In addition, depending on the polymer used, bioactive sites on the polymer may provide signals to initiate certain cell behavior. However, despite their potential as biomaterials for tissue engineering and regenerative medicine applications, fabricating hydrogels that truly mimic the physicochemical properties of the extracellular matrix to physiologically-relevant values is a challenge. Recent efforts in the field have sought to improve the physicochemical properties of hydrogels using advanced materials science and engineering methods. In this review, we highlight some of the most promising methods, including crosslinking strategies and manufacturing approaches such as 3D bioprinting and granular hydrogels. We also provide a brief perspective on the future outlook of this field and how these methods may lead to the clinical translation of hydrogel biomaterials for tissue engineering and regenerative medicine applications.

The effect of mesoporous bioglass on hemostatic, antibacterial and biocompatible properties of composite sponge

AbstractHemostatic materials used in penetrating injuries or incompressible wounds must possess exceptional efficacy in preventing bleeding. In this study, mesoporous bioglass (MBG) was synthesized using a two‐step acid‐catalyzed self‐assembly method, and a novel hemostatic sponge (MBG/CH/GEL) was prepared by combining chitosan (CH), gelatin (GEL), and MBG using a freeze‐drying method. The characteristics and hemostatic effects of the MBG/CH/GEL composite hemostatic sponge were analyzed and evaluated. Research has shown that the high specific surface area of MBG (730 m2/g) provides more blood cell adhesion sites during hemostasis, resulting in a low hemolysis rate, favorable swelling rate, and porosity of the hemostatic sponge. Additionally, MBG can release Si4+ and Ca2+ ions during hemostasis, giving the composite hemostatic sponge excellent cell compatibility and promoting cell growth. Compared with commercially available gelatin hemostatic sponges, it cannot only quickly stop bleeding but also has a greater compressive strength (212.07 kPa) and adhesion strength (11.54 ± 0.16 kPa), making it suitable for use in hemostasis of incompressible wounds. Furthermore, the composite hemostatic sponge exhibited significant antibacterial effects against Staphylococcus aureus and Escherichia coli. These results indicate that the MBG/CH/GEL composite hemostatic sponge, which is a hemostatic material, has promising applications.

Multifunctional Hydrogel Based on Silk Fibroin Promotes Tissue Repair and Regeneration

AbstractThe creation of functional hydrogels with robust load‐bearing capacity adaptable to complex tissue regeneration remains challenging. Silk fibroin (SF) is a natural biomaterial with excellent mechanical strength and cell adhesion capacity, possessing tremendous potential to solve the aforementioned dilemma. The excellent biodegradability and biocompatibility and the molecular structure with multiple modifiable moieties provide opportunities for the injectability and multifunctionality of hydrogels. Furthermore, the incorporation of other polymers or active ingredients can improve the basic properties, confer biological activities and pharmacological effects, and provide the prerequisites for hydrogels to fulfill specific requirements. Therefore, SF‐based hydrogels are widely applied in tissue repair and regeneration, especially in bone, skin, nerve, liver, myocardium, and cornea, which have effectively addressed the challenges of scaffolds' lack of cell adhesion sites, excessive degradation, as well as the lack of efficient, long‐lasting antimicrobial properties. This review outlines the properties of SF, discusses the types of materials commonly used in the preparation of hydrogels and their characteristics, and describes the current construction methods of SF hydrogels. Simultaneously, recent advancements in applying SF‐based multifunctional hydrogels within biological tissues are explored, with a focus on their role in tissue repair, highlighting the repair mechanism of SF based on bone and skin.

Tunable gelatin methacrylate polyethylene glycol diacrylate hydrogels for cell mechanosensing applications

Three-dimensional (3D) tissue-engineered scaffolds mimic the physiological environment of cells by providing essential structural support, biochemical cues, and the mechanical strength needed for cell adhesion, proliferation, migration, and differentiation. Hydrogels like polyethylene glycol diacrylate (PEGDA) are commonly used biomaterials for cell culture due to their affordability, tunable stiffness, and ability to efficiently transport nutrients and gases. However, PEGDA lacks cell adhesion sites essential for cell proliferation and migration and has limited degradability. Methacrylated gelatin (GelMA) produced from denatured bovine collagen, crosslinks under ultraviolet light (UV) resulting in a degradable hydrogel with cell adhesion sites. Here, we synthesized GelMA with variable degree of methacrylation and crosslinked it with PEGDA to produce cell scaffolds with independently tunable mechanical and biochemical properties by varying the ratios of the two polymers. We determined polymer ratios that resulted in scaffolds with different mechanical properties but the same gelatin concentrations (providing cell adhesion and degradation sites) as well as different gelatin concentrations but the same mechanical properties. With the developed scaffold library, we further used a design of experiments approach to probe the parameter space and perform detailed analysis on chemical composition-scaffold properties as well as scaffold properties-cell behavior correlations. Our findings showed that hydrogel properties such as modulus, swelling, pore size, and permeability, strongly depended on total polymer concentration and not on the GelMA fraction. GelMA significantly influenced cell spreading, while addition of any amount of PEGDA delayed cell spreading significantly. We suggest that such analysis will broaden the utility of the GelMA/PEGDA hydrogels, presenting a versatile platform for mechanosensing research in 3D environments.

Focal adhesion kinase signaling - tumor vulnerabilities and clinical opportunities.

Focal adhesion kinase (FAK; encoded by PTK2) was discovered over 30 years ago as a cytoplasmic protein tyrosine kinase that is localized to cell adhesion sites, where it is activated by integrin receptor binding to extracellular matrix proteins. FAK is ubiquitously expressed and functions as a signaling scaffold for a variety of proteins at adhesions and in the cell cytoplasm, and with transcription factors in the nucleus. FAK expression and intrinsic activity are essential for mouse development, with molecular connections to cell motility, cell survival and gene expression. Notably, elevated FAK tyrosine phosphorylation is common in tumors, including pancreatic and ovarian cancers, where it is associated with decreased survival. Small molecule and orally available FAK inhibitors show on-target inhibition in tumor and stromal cells with effects on chemotherapy resistance, stromal fibrosis and tumor microenvironment immune function. Herein, we discuss recent insights regarding mechanisms of FAK activation and signaling, its roles as a cytoplasmic and nuclear scaffold, and the tumor-intrinsic and -extrinsic effects of FAK inhibitors. We also discuss results from ongoing and advanced clinical trials targeting FAK in low- and high-grade serous ovarian cancers, where FAK acts as a master regulator of drug resistance. Although FAK is not known to be mutationally activated, preventing FAK activity has revealed multiple tumor vulnerabilities that support expanding clinical combinatorial targeting possibilities.

Supragel-mediated efficient generation of pancreatic progenitor clusters and functional glucose-responsive islet-like clusters

Although several synthetic hydrogels with defined stiffness have been developed to facilitate the proliferation and maintenance of human pluripotent stem cells (hPSCs), the influence of biochemical cues in lineage-specific differentiation and functional cluster formation has been rarely reported. Here, we present the application of Supragel, a supramolecular hydrogel formed by synthesized biotinylated peptides, for islet-like cluster differentiation. We observed that Supragel, with a peptide concentration of 5 mg/mL promoted spontaneous hPSCs formation into uniform clusters, which is mainly attributable to a supporting stiffness of ∼1.5 kPa as provided by the Supragel matrix. Supragel was also found to interact with the hPSCs and facilitate endodermal and subsequent insulin-secreting cell differentiation, partially through its components: the sequences of RGD and YIGSR that interacts with cell membrane molecules of integrin receptor. Compared to Matrigel and suspension culturing conditions, more efficient differentiation of the hPSCs was also observed at the stages 3 and 4, as well as the final stage toward generation of insulin-secreting cells. This could be explained by 1) suitable average size of the hPSCs clusters cultured on Supragel; 2) appropriate level of cell adhesive sites provided by Supragel during differentiation. It is worth noting that the Supragel culture system was more tolerance in terms of the initial seeding densities and less demanding, since a standard static cell culture condition was sufficient for the entire differentiation process. Our observations demonstrate a positive role of Supragel for hPSCs differentiation into islet-like cells, with additional potential in facilitating germ layer differentiation.

Albumin Nanoparticle-Based Drug Delivery Systems.

Nanoparticle-based systems are extensively investigated for drug delivery. Among others, with superior biocompatibility and enhanced targeting capacity, albumin appears to be a promising carrier for drug delivery. Albumin nanoparticles are highly favored in many disease therapies, as they have the proper chemical groups for modification, cell-binding sites for cell adhesion, and affinity to protein drugs for nanocomplex generation. Herein, this review summarizes the recent fabrication techniques, modification strategies, and application of albumin nanoparticles. We first discuss various albumin nanoparticle fabrication methods, from both pros and cons. Then, we provide a comprehensive introduction to the modification section, including organic albumin nanoparticles, metal albumin nanoparticles, inorganic albumin nanoparticles, and albumin nanoparticle-based hybrids. We finally bring further perspectives on albumin nanoparticles used for various critical diseases.

Integrin mechanosensing relies on a pivot-clip mechanism to reinforce cell adhesion

Cells intricately sense mechanical forces from their surroundings, driving biophysical and biochemical activities. This mechanosensing phenomenon occurs at the cell-matrix interface, where mechanical forces resulting from cellular motion, such as migration or matrix stretching, are exchanged through surface receptors, primarily integrins, and their corresponding matrix ligands. A pivotal player in this interaction is the α5β1 integrin and fibronectin (FN) bond, known for its role in establishing cell adhesion sites for migration. However, upregulation of the α5β1-FN bond is associated with uncontrolled cell metastasis. This bond operates through catch bond dynamics, wherein the bond lifetime paradoxically increases with greater force. The mechanism sustaining the characteristic catch bond dynamics of α5β1-FN remains unclear. Leveraging molecular dynamics simulations, our approach unveils a pivot-clip mechanism. Two key binding sites on FN, namely the synergy site and the RGD (Arg-Gly-Asp) motif, act as active points for structural changes in α5β1 integrin. Conformational adaptations at these sites are induced by a series of hydrogen bond formations and breaks at the synergy site. We disrupt these adaptations through a double mutation on FN, known to reduce cell adhesion. A whole-cell finite-element model is employed to elucidate how the synergy site may promote dynamic α5β1-FN binding, resisting cell contraction. In summary, our study integrates molecular- and cellular-level modeling to propose that FN’s synergy site reinforces cell adhesion through enhanced binding dynamics and a mechanosensitive pivot-clip mechanism. This work sheds light on the interplay between mechanical forces and cell-matrix interactions, contributing to our understanding of cellular behaviors in physiological and pathological contexts.

Cartilage-like protein-polysaccharide hybrid hydrogel for enhancing chondrogenic differentiation of bone marrow mesenchymal stem cells

The regeneration of articular cartilage posed a formidable challenge due to the restricted treatment efficacy of existing therapies. Scaffold-based tissue engineering emerges as a promising avenue for cartilage reconstitution. However, most scaffolds exhibit inadequate mechanical characteristics, poor biocompatibility, or absent cell adhesion sites. In this study, cartilage-like protein-polysaccharide hybrid hydrogel based on DOPA-modified hyaluronic acid, bovine type I collagen (Col I), and recombinant humanized type II collagen (rhCol II), denoted as HDCR. HDCR hydrogels possessed the advantage of injectability and in situ crosslinking through pH adjustment. Moreover, HDCR hydrogels exhibited a manipulable degradation rate and favorable biocompatibility. Notably, HDCR hydrogels significantly induced chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells in vitro, as demonstrated by the upregulation of crucial chondrogenic genes (type II collagen, aggrecan) and the abundant accumulation of glycosaminoglycan. This approach presented a strategy to manufacture injectable, biodegradable scaffolds based on cartilage-like protein-polysaccharide polymers, offering a minimally invasive solution for cartilage repair.Graphical abstract

Construction of 3D tumor in vitro models with an immune microenvironment exhibiting similar tumor properties and biomimetic physiological functionality.

Tumors pose a serious threat to people's lives and health, and the complex tumor microenvironment is the biggest obstacle to their treatment. In contrast to the basic protein matrices typically employed in 2D or 3D cell culture systems, decellularized extracellular matrix (dECM) can create complex microenvironments. In this study, a combination of physicochemical methods was established to obtain liver decellularized extracellular matrix scaffolds (dLECMs) to provide mechanical support and cell adhesion sites. By co-culturing tumor cells, tumor-associated stromal cells and immune cells, an in vitro 3D tumor model with a biomimetic immune microenvironment was constructed. By utilizing microenvironment data obtained from human liver tumor tissues and refining the double seeding modeling process, 3D in vitro liver tumor-like tissues with a tumor immune microenvironment (TIME) were obtained and designated as reconstructed human liver cancer (RHLC). These tissues demonstrated similar tumor characteristics and exhibited satisfactory physiological functionality. The results of metabolic characterisation and mouse tumorigenicity testing verified that the constructed RHLC significantly increased in vitro drug resistance while also closely mimicking in vivo tissue metabolism. This opens up new possibilities for creating effective in vitro models for screening chemotherapy drugs.

Magnetic Polymer Microcarriers for Cell Engineering Applications with Enzyme‐Free Cell Harvesting and Rapid Recovery in Large‐Scale Cell Culture

AbstractStimulus‐responsive large‐scale cell culture microcarriers are promising biological materials for large‐scale cell production. Herein, multifunctional thermal and magnetic microcarriers (TMMs) made of poly(N‐isopropylacrylamide) (PNIPAM), polylysines (PLLs), gelatin methacryloyl (GelMA), poly(ethylene glycol) diacrylate (PEGDA), N,N‐methylenebis(acrylamide) (MBA), Fe3O4 and SiO2 are prepared to achieve large‐scale enzyme‐free cell harvesting and also can be used as cells stereogrowth scaffold to mimic cells suspension state to develop novel application, such as recombinant proteins produced. In TMMs, NIPAM and other materials are mixed and then underwent in situ radical polymerization by UV irradition with 2‐hydroxy‐2‐methylpropiophenone as initiator, in which GelMA provides cell adhesion sites and PLLs enable a positive microenvironment with SiO2 as filler. As enzyme‐free cell microcarriers, cell harvesting from TMMs by PNIPAM are achieved at 25 °C through vortex oscillation within 15 min and TMMs can be recycled at least for four times by magnetic recovery. Moreover, TMMs can be used as stereoculture scaffold to let cells in suspension as adherent state to improve gene transfection efficiency. Combined with magnetothermal effect, TMMs can further improve gene transfer to obtain industrialization of proteins expression. Therefore, TMMs are potential for the development of cell manufacturing and the expansion of cell stereogrowth application for recombinant proteins production.

Programmable scaffolds with aligned porous structures for cell cultured meat

Porous scaffolds for cell cultured meat are currently limited in the food-grade material requirements, the cell adhesion, proliferation, and differentiation capacities, and the ignored appearance design. We proposed programmable scaffolds specially tailored for cell cultured meat. The scaffold with aligned porous structures was fabricated with the ice-templated directional freeze-drying of the food-grade collagen hydrogel. Due to the abundant tripeptide presence and well-aligned porous structures, the scaffold could not only provide sites for cell adhesion and proliferation, but also promote the oriented growth and differentiation of cells. The up-regulation of myogenic related genes, synthesis of myogenic related proteins and formation of matured myotubes furtherly proved the differentiation of cells on aligned scaffold. These characteristics would facilitate the traditional meat characteristics simulation of cell cultured meat in term of texture and microstructure. Meanwhile, patterned scaffolds were achievable as well with the help of mold-assisted ice templating, which would improve the people’s interest, recognition, and acceptance of the tailored cell cultured meat. These characteristics indicate great application prospects of the proposed programmable scaffolds in cell cultured meat.

Investigating the Interplay Between Matrix Compliance and Passaging History on Chondrogenic Differentiation of Mesenchymal Stem Cells Encapsulated Within Alginate-Gelatin Hybrid Hydrogels

Mesenchymal stem cells (MSCs) are used widely in tissue engineering and regenerative medicine because of their ease of isolation and their pluripotency. The low survival and retention rate of MSCs at the target site upon implantation can be addressed via encapsulation within hydrogels capable of directing their fate. In this study, the interplay between matrix mechanics and the passage number of MSCs on their chondrogenic differentiation was assessed. Human bone marrow-derived MSCs between passages 4 and 6 were encapsulated within alginate-gelatin hybrid gels. The stiffness of the gels was varied by varying alginate concentrations while maintaining the concentration of gelatin and consequently, the cell adhesion sites, constant. The study revealed that within 4.8 kPa gels, GAG deposition was higher by P4 MSCs compared to P6 MSCs. However, an opposite trend was observed with collagen type 2 deposition. Further, we observed enhanced chondrogenic differentiation upon encapsulation of MSCs within 6.7 kPa hydrogel irrespective of passaging history. However, the effect of matrix compliance was more prominent in the case of higher passaged MSCs suggesting that matrix stiffness can help rescue the reduced differentiation capability of these cells.

Extracellular Matrix-Mimetic Immunomodulatory Hydrogel for Accelerating Wound Healing.

Macrophages play a crucial role in the complete processes of tissue repair and regeneration, and the activation of M2 polarization is an effective approach to provide a pro-regenerative immune microenvironment. Natural extracellular matrix (ECM) has the capability to modulate macrophage activities via its molecular, physical, and mechanical properties. Inspired by this, an ECM-mimetic hydrogel strategy to modulate macrophages via its dynamic structural characteristics and bioactive cell adhesion sites is proposed. The LZM-SC/SS hydrogel is in situ formed through the amidation reaction between lysozyme (LZM), 4-arm-PEG-SC, and 4-arm-PEG-SS, where LZM provides DGR tripeptide for cell adhesion, 4-arm-PEG-SS provides succinyl ester for dynamic hydrolysis, and 4-arm-PEG-SC balances the stability and dynamics of the network. In vitro and subcutaneous tests indicate the dynamic structural evolution and cell adhesion capacity promotes macrophage movement and M2 polarization synergistically. Comprehensive bioinformatic analysis further confirms the immunomodulatory ability, and reveals a significant correlation between M2 polarization and cell adhesion. A full-thickness wound model is employed to validate the induced M2 polarization, vessel development, and accelerated healing by LZM-SC/SS. This study represents a pioneering exploration of macrophage modulation by biomaterials' structures and components rather than drug or cytokines and provides new strategies to promote tissue repair and regeneration.

Interplay of heparan sulfate chains with the core proteins of syndecans 2 and 4

AbstractWe have previously shown that the extracellular domains of the four syndecans are intrinsically disordered, and adopt a wide range of conformations. We report here the building of coarse‐grained models of the extracellular domains of human syndecans 2 and 4 using small‐angle X‐ray scattering restraints. One, two or three heparan sulfate (HS) hexadecasaccharides, (IdoA[2S]GlcNS[6S])8, were attached to three serine residues of the core proteins, resulting in eight variants for each syndecan that were used for all‐atom molecular dynamics (MD) simulations (0.5–1 µs). Syndecan‐4 had a larger conformational diversity than syndecan‐2, and remained extended during MD simulations in absence of HS whereas syndecan‐2 adopted more compact conformations. Their core proteins thus appeared to be structurally distinct. The HS chains also behave differently, the middle chain being more flexible in syndecan‐4, and the third chain being able to interact with the core protein regions mediating cell adhesion. The cell adhesion sites on both core proteins were flexible, with or without HS chains, the NXIP motif of syndecan‐2 being located in a particularly flexible region. In conclusion, the HS chains induce moderate changes in the conformational dynamics of both syndecans, depending on the number of HS chains and their location on the core protein, and on the core protein itself.

The Design and Developments of Protein‐Polysaccharide Biomaterials for Corneal Tissue Engineering

AbstractCorneal blindness is explained by a large reduction in vision loss caused by acute and chronic corneal diseases and/or disorders. Due to the severe unavailability of donor corneas, immune rejection, and the side effects of keratoprosthesis (KPro), vision‐impaired patients are often unable to restore their vision. Corneal tissue engineering appears to be a promising method using natural polymers made from different layers of corneal cells such as epithelium, stroma, and endothelium or full tissue equivalents. Proteins and polysaccharides are able to mimic the chemical composition of the native extracellular matrix (ECM), which has been extensively studied for corneal tissue engineering. Additionally, protein‐based materials are highly favored in corneal regenerative therapy as they have the proper cell‐binding sites for cell adhesion, corneal innervation regeneration, and oxidative stress suppression, while polysaccharide‐based materials can also promote cell proliferation and adhesion as well as ocular permeability. Herein, a comprehensive review of various biomaterials based on natural proteins (fibrin, fibrinogen, silk fibroin, collagen, gelatin, and serum albumin) and polysaccharides (chitin, chitosan, hyaluronic acid, alginate, cellulose, and gellan gum) for corneal tissue engineering are provided. The protein‐polysaccharide biomaterials are mainly used in the form of hydrogels, films, scaffolds, sutures, eye drops, nanocarriers, and nanocapsules to treat ocular diseases that cause blindness, such as corneal wounds, corneal ulcers, Goldenhar syndrome, corneal endothelium and stroma defects, corneal neovascularization, etc. In this review, insight is provided into recent developments in the areas of biomaterial design, cross‐linking strategy, corneal cell selection, and frontiers that are expected to offer researchers support and creativity in the development of corneal regenerative medicine.

Construction and properties detection of 3D micro-structure scaffolds base on decellularized sheep kidney before and after crosslinking.

Decellularized extracellular matrix is one form of natural material in tissue engineering. The process of dECM retains the tissue microstructure, provides good cell adhesion sites, maintains most of biological signals that promotes the survival and differentiation ability of cells. In this study, sheep kidney was decellularized followed by histochemical staining, elemental analysis and scanning electron microscopy characterizations. The dECM scaffold was prepared with different sequences of freeze drying technology, crosslinking and the water absorption, porosity, mechanical strength with subsequent thermogravimetric analysis, Infrared spectroscopy and biocompatibility tests. Our results indicated that these decellularized treatments of sheep kidney can effectively remove DNA and retain uniform pore size distribution. After crosslinking the scaffold's water absorption decreased from 987.56 ± 40.21% to 934.39 ± 39.61%, the porosity decreased from 89.64 ± 3.2% to 85.09 ± 17.63%, and the compression modulus increased from 304.32 ± 25.43kPa to 459.53 ± 38.92kPa, with thermal process the percentage of weight loss decreased from 66.57% to 44.731%, in addition, the composition didn't change significantly, crosslinking could also promote the stability. In terms of biocompatibility, the number of viable cells increased significantly with the days. In conclusion, the crosslinked decellularized sheep kidney extracellular matrix scaffold reduced water absorption and porosity slightly, but has a significant increase in mechanical properties, and presented excellent biocompatibility which are beneficial to cell adhesion, growth and differentiation.

Label-free analysis of membrane protein binding kinetics and cell adhesions using evanescent scattering microscopy.

Measuring ligand interactions with membrane proteins in single live cells is critical for understanding many cellular processes and screening drugs. However, developing such a capability has been a difficult challenge. Here, we employ evanescent scattering microscopy (ESM) to show that ligand binding to membrane proteins can change the cell adhesion properties, which are intrinsic cell properties and independent of random cell micromotions and ligand mass, thus allowing the kinetics analyses of both proteins and small molecules binding to membrane proteins in both single fixed and live cells. In addition, utilizing the high spatiotemporal resolution of ESM, the positions of cell adhesion sites can be tracked in real-time to analyze the cell deformations and migrations, thus providing a potential approach for understanding the cell activity during the ligand binding process in detail. The presented method may pave the road for developing a versatile and easy-to-use label-free detection strategy for in situ analysis of molecular interaction dynamics in living biosystems with single-cell resolution.

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.