On-chip fabrication and in-flow 3D-printing of microgel constructs: from chip to scaffold materials in one integral process.

Bioprinting has evolved into a thriving technology for the fabrication of cell-laden scaffolds. Bioinks are the most critical component for bioprinting. Recently, microgels have been introduced as a very promising bioink, enabling cell protection and the control of the cellular microenvironment. However, the fabrication of the bioinks involves the microfluidic production of the microgels, with a subsequent multistep process to obtain the bioink, which so far has limited its application potential. Here we introduce a direct coupling of microfluidics and 3D-printing for the continuous microfluidic production of microgels with direct in-flow printing into stable scaffolds. The 3D-channel design of the microfluidic chip provides access to different hydrodynamic microdroplet formation regimes to cover a broad range of droplet and microgel diameters. After exiting a microtubing the produced microgels are hydrodynamically jammed into thin microgel filaments for direct 3D-printing into two- and three-dimensional scaffolds. The methodology enables the continuous on-chip encapsulation of cells into monodisperse microdroplets with subsequent in-flow cross-linking to produce cell-laden microgels. The method is demonstrated for different cross-linking methods and cell lines. This advancement will enable a direct coupling of microfluidics and 3D-bioprinting for scaffold fabrication.

DBD-FISH Using Specific Chromosomal Region Probes for the Study of Cervical Carcinoma Progression.

Genomic instability is an important biomarker in the progression of cervical carcinoma. DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) is a sensitive method that detects strand breaks, alkali-labile sites, and incomplete DNA excision repair in cells of the cervical epithelium. This technique integrates the microgel immersion of cells from a vaginal lesion scraping and the DNA unwinding treatment with the capacity of FISH integrated into digital image analysis. Cells captured within an agarose matrix are lysed and submerged in an alkaline unwinding solution that generates single-stranded DNA motifs at the ends of internal DNA strand breaks. After neutralization, the microgel is dehydrated and the cells are incubated with DNA-labeled probes. The quantity of a hybridized probe at a target sequence corresponds to the measure of the single-stranded DNA produced during the unwinding step, which is equivalent to the degree of local DNA breakage. DNA damage does not show uniformly throughout the entire DNA of a cell; rather, it is confined to specific chromosomal sites. In this chapter, an overview of the technique is supplied, focusing on its ability for assessing the association between DNA damage in specific sequences and in the progressive stages of cervical carcinoma.

ROS-responsive drug-releasing injectable microgels for ameliorating myocardial infarction.

Despite of the recent advances in regulatory T cell (Treg) therapy, a limited number of available cells and specificity at the desired tissue site have severely compromised their efficacy. Herein, an injectable drug-releasing (MTK-TK-drug) microgel system in response to in situ stimulation by reactive oxygen species (ROS) was constructed with a coaxial capillary microfluidic system and UV curing. The spherical microgels with a size of 150 µm were obtained. The MTK-TK-drug microgels efficiently converted the pro-inflammatory Th17 cells into anti-inflammatory regulatory T cells (Treg) cells in vitro, and the ROS-scavenging materials synergistically enhanced the effect by modulating the inflammation microenvironment. Thus, the microgels significantly reduced cardiomyocyte apoptosis and decreased the inflammatory response in the early stages of post-myocardial infarction (MI) in vivo, thereby reducing fibrosis, promoting vascularization, and preserving cardiac function. Overall, our results indicate that the MTK-TK-drug microgels can attenuate the inflammatory response and improve MI therapeutic effects in vivo.

Plant mucus-derived microgels: Blood-triggered gelation and strong hemostatic adhesion.

Arrest of bleeding usually applies clotting agents to trigger coagulation procedures or adhesives to interrupt blood flow through sealing the vessel; however, the efficiency is compromised. Here, we propose a concept of integration of hemostasis and adhesion via yam mucus's microgels. The mucus microgels exhibit attractive attributes of hydrogel with uniform size and shape. Their shear-thinning, self-healing and strong adhesion make them feasible as injectable bioadhesion. Exceptionally, the blood can trigger the microgels' gelation with the outcome of super extensibility, which leads to the microgels a strong hemostatic agent. We also found a tight gel adhesive layer formed upon microgels' contacting the blood on the tissue, where there is the coagulation factor XIII triggered to form a dense three-dimensional fibrin meshwork. The generated structures show that the microgels look like hard balls in the dispersed phase into the blood-produced fibrin mesh of a soft net phase. Both phases work together for a super-extension gel. We demonstrated the microgels' fast adhesion and hemostasis in the livers and hearts of rabbits and mini pigs. The microgels also promoted wound healing with good biocompatibility and biodegradability.

Survival-Associated Cellular Response Maintained in Pancreatic Ductal Adenocarcinoma (PDAC) Switched Between Soft and Stiff 3D Microgel Culture.

Pancreatic ductal adenocarcinoma (PDAC) accounts for about 90% of all pancreatic cancer cases. Five-year survival rates have remained below 12% since the 1970s, in part due to the difficulty in detection prior to metastasis (migration and invasion into neighboring organs and glands). Mechanical memory is a concept that has emerged over the past decade that may provide a path toward understanding how invading PDAC cells "remember" the mechanical properties of their diseased ("stiff", elastic modulus, E ≈ 10 kPa) microenvironment even while invading a healthy ("soft", E ≈ 1 kPa) microenvironment. Here, we investigated the role of mechanical priming by culturing a dilute suspension of PDAC (FG) cells within a 3D, rheologically tunable microgel platform from hydrogels with tunable mechanical properties. We conducted a suite of acute (short-term) priming studies where we cultured PDAC cells in either a soft (E ≈ 1 kPa) or stiff (E ≈ 10 kPa) environment for 6 h, then removed and placed them into a new soft or stiff 3D environment for another 18 h. Following these steps, we conducted RNA-seq analyses to quantify gene expression. Initial priming in the 3D culture showed persistent gene expression for the duration of the study, regardless of the subsequent environments (stiff or soft). Stiff 3D culture was associated with the downregulation of tumor suppressors (LATS1, BCAR3, CDKN2C), as well as the upregulation of cancer-associated genes (RAC3). Immunofluorescence staining (BCAR3, RAC3) further supported the persistence of this cellular response, with BCAR3 upregulated in soft culture and RAC3 upregulated in stiff-primed culture. Stiff-primed genes were stratified against patient data found in The Cancer Genome Atlas (TCGA). Upregulated genes in stiff-primed 3D culture were associated with decreased survival in patient data, suggesting a link between patient survival and mechanical priming.

In vitro development and optimization of cell-laden injectable bioprinted gelatin methacryloyl (GelMA) microgels mineralized on the nanoscale.

Bone defects may occur in different sizes and shapes due to trauma, infections, and cancer resection. Autografts are still considered the primary treatment choice for bone regeneration. However, they are hard to source and often create donor-site morbidity. Injectable microgels have attracted much attention in tissue engineering and regenerative medicine due to their ability to replace inert implants with a minimally invasive delivery. Here, we developed novel cell-laden bioprinted gelatin methacrylate (GelMA) injectable microgels, with controllable shapes and sizes that can be controllably mineralized on the nanoscale, while stimulating the response of cells embedded within the matrix. The injectable microgels were mineralized using a calcium and phosphate-rich medium that resulted in nanoscale crystalline hydroxyapatite deposition and increased stiffness within the crosslinked matrix of bioprinted GelMA microparticles. Next, we studied the effect of mineralization in osteocytes, a key bone homeostasis regulator. Viability stains showed that osteocytes were maintained at 98 % viability after mineralization with elevated expression of sclerostin in mineralized compared to non-mineralized microgels, showing that mineralization can effectively enhances osteocyte maturation. Based on our findings, bioprinted mineralized GelMA microgels appear to be an efficient material to approximate the bone microarchitecture and composition with desirable control of sample injectability and polymerization. These bone-like bioprinted mineralized biomaterials are exciting platforms for potential minimally invasive translational methods in bone regenerative therapies.

Production of letrozole-loaded alginate oxide-gelatin microgels using microfluidic systems for drug delivery applications.

Microfluidic systems are capable of producing microgels with a monodisperse size distribution and a spherical shape due to their laminar flow and superior flow. A significant challenge in producing these drug-carrying microgels is simultaneous drug loading into microgels. Various factors such as the type of polymer, the type of drug, the volume ratio of the drug to the polymer, and the geometry of the microfluidic system used to generate microgels can effectively address these challenges. The overall goal of this study was to produce mono-disperse drug-carrying microgels capable of controlled drug release. To achieve this goal, this study used a stream-focused microfluidic chip containing a coating current to prevent chip clogging. Alginate oxide was synthesized with a 30 % oxidation percentage. Alginate oxide, gelatin, and compositions of them with volume ratios of 50-50, 70-30, and 30-70, by determining their appropriate weight percentage, were used for the controlled release of letrozole. The properties of the produced microgels were measured through various tests such as drug release test, loading percentage, SEM, FTIR, swelling ratio, and dimensional stability. It was found that microgels made of a combination of alginate oxide-gelatin with volume ratios of 70-30 had a good swelling ratio and structural stability. The drug loading percentages for alginate, alginate oxide, and alginate oxide-gelatin with volume ratios of 50-50 and 30-70, respectively, were 56 %, 68 %, and 66 %, 61 % and the alginate oxide-gelatin with a volume ratio of 70-30 compared to other samples had over 70 % drug loading percentages. Furthermore, samples of alginate, alginate oxide, and alginate oxide-gelatin with volume ratios of 50-50 and 30-70 had 94 %, 63 %, 56 %, and 68 % drug release in 13 days, respectively. However, alginate oxide-gelatin with a volume ratio of 70-30 had a release rate of about 50 % in 13 days, which is a more controlled release for letrozole compared to the volume ratios of 50-50 and 30-70. Examining the drug release profile, it was concluded that drug release follows the Higuchi model and therefore follows Fick's first law of diffusion. It can be concluded that the combination of alginate oxide-gelatin produces more suitable microgels than alginate and alginate oxide for the controlled-release of letrozole. A comparison of microgels of alginate oxide and gelatin with volume ratios of 50-50 and 70-30 had better results for the cytotoxicity study compared to other samples.

Development and characterization of colloidal pNIPAM-methylcellulose microgels with potential application for drug delivery in dentoalveolar tissue engineering strategies.

Incorporation of growth factors, signaling molecules and drugs can be vital for the success of tissue engineering in complex structures such as the dentoalveolar region. This has led to the development of a variety of drug release systems. This study aimed to develop pNIPAM-methylcellulose microgels with different synthesis parameters based on a 23 full factorial design of experiments for this application. Microgel properties, including volume phase transition temperature (VPTT), hydrodynamic size, drug loading and release, and cytocompatibility were systematically evaluated. The results demonstrated successful copolymerization and development of the microgels, a hydrodynamic size ranging from ∼200 to ∼500 nm, and VPTT in the range of 34-39 °C. Furthermore, loading of genipin, capable of inducing odontoblastic differentiation, and its sustained release over a week was shown in all formulations. Together, this can serve as a solid basis for the development of tunable drug-delivering pNIPAM-methylcellulose microgels for specific tissue engineering applications.

Combinatorial Microgels for 3D ECM Screening and Heterogeneous Microenvironmental Culture of Primary Human Hepatic Stellate Cells.

Nonalcoholic fatty liver disease affects 30% of the United States population and its progression can lead to nonalcoholic steatohepatitis (NASH), and increased risks for cirrhosis and hepatocellular carcinoma. NASH is characterized by a highly heterogeneous liver microenvironment created by the fibrotic activity of hepatic stellate cells (HSCs). While HSCs have been widely studied in 2D, further advancements in physiologically relevant 3D culture platforms for the in vitro modeling of these heterogeneous environments are needed. In this study, the use of stiffness-variable, extracellular matrix (ECM) protein-conjugated polyethylene glycol microgels as 3D cell culture scaffolds to modulate HSC activation is demonstrated. These microgels as a high throughput ECM screening system to identify HSC matrix remodeling and metabolic activities in distinct heterogeneous microenvironmental conditions are further employed. The 6 kPa fibronectin microgels are shown to significantly increase HSC matrix remodeling and metabolic activities in single or multiple-component microenvironments. Overall, heterogeneous microenvironments consisting of multiple distinct ECM microgels promoted a decrease in HSC matrix remodeling and metabolic activities compared to homogeneous microenvironments. The study envisions this ECM screening platform being adapted to a broad number of cell types to aid the identification of ECM microenvironments that best recapitulate the desired phenotype, differentiation, or drug efficacy.

Biodegradable Dextrin-Based Microgels for Slow Release of Dual Fertilizers for Sustainable Agriculture.

In this research, we report dextrin-based biodegradable microgels (PDXE MGs) having phosphate-based cross-linking units for slow release of urea and a potential P source to improve fertilization. PDXE MGs (∼200 nm) are synthesized by cross-linking the lauroyl-functionalized dextrin chains with sodium tripolyphosphate. The developed PDXE MGs exhibit high loading (∼10%) and encapsulation efficiency (∼88%) for urea. It is observed that functionalization of PDXE MGs with lauroyl chains slows down the release of urea (90% in ∼24 days) as compared to nonfunctionalized microgels (PDX MGs) (99% in ∼17 days) in water. Further studies of the developed formulation display that Urea@PDXE MGs significantly boost maize seed germination and overall plant growth as compared to pure urea fertilizer. Moreover, analysis of maize leaves obtained from plants treated with Urea@PDXE MGs reveals 3.5 ± 0.3% nitrogen content and 90 ± 0.7 mg/g chlorophyll content. These values are significantly higher than 1.4 ± 0.6% nitrogen content and 48 ± 0.05 mg/g chlorophyll content obtained by using bare urea. Further, acid phosphatase activity in roots is reduced upon treatment with PDXE MGs and Urea@PDXE MGs, suggesting the availability of P upon degradation of PDXE MGs by the amylase enzyme in soil. These experimental results present the developed microgel-based biodegradable formulation with a slow release feature as a potential candidate to move toward sustainable agriculture practices.

Selenium-silk microgels as antifungal and antibacterial agents.

Antimicrobial resistance is a leading threat to global health. Alternative therapeutics to combat the rise in drug-resistant strains of bacteria and fungi are thus needed, but the development of new classes of small molecule therapeutics has remained challenging. Here, we explore an orthogonal approach and address this issue by synthesising micro-scale, protein colloidal particles that possess potent antimicrobial properties. We describe an approach for forming silk-based microgels that contain selenium nanoparticles embedded within the protein scaffold. We demonstrate that these materials have both antibacterial and antifungal properties while, crucially, also remaining highly biocompatible with mammalian cell lines. By combing the nanoparticles with silk, the protein microgel is able to fulfill two critical functions; it protects the mammalian cells from the cytotoxic effects of the bare nanoparticles, while simultaneously serving as a carrier for microbial eradication. Furthermore, since the antimicrobial activity originates from physical contact, bacteria and fungi are unlikely to develop resistance to our hybrid biomaterials, which remains a critical issue with current antibiotic and antifungal treatments. Therefore, taken together, these results provide the basis for innovative antimicrobial materials that can target drug-resistant microbial infections.

Supramolecular Peptoid Structure Strengthens Complexation with Polyacrylic Acid Microgels.

We have studied the complexation between cationic antimicrobials and polyanionic microgels to create self-defensive surfaces that responsively resist bacterial colonization. An essential property is the stable sequestration of the loaded (complexed) antimicrobial within the microgel under a physiological ionic strength. Here, we assess the complexation strength between poly(acrylic acid) [PAA] microgels and a series of cationic peptoids that display supramolecular structures ranging from an oligomeric monomer to a tetramer. We follow changes in loaded microgel diameter with increasing [Na+] as a measure of the counterion doping level. Consistent with prior findings on colistin/PAA complexation, we find that a monomeric peptoid is fully released at ionic strengths well below physiological conditions, despite its +5 charge. In contrast, progressively higher degrees of peptoid supramolecular structure display progressively greater resistance to salting out, which we attribute to the greater entropic stability associated with the complexation of multimeric peptoid bundles.

Ionically annealed zwitterionic microgels for bioprinting of cartilaginous constructs.

Foreign body response (FBR) is a pervasive problem for biomaterials used in tissue engineering. Zwitterionic hydrogels have emerged as an effective solution to this problem, due to their ultra-low fouling properties, which enable them to effectively inhibit FBRin vivo. However, no versatile zwitterionic bioink that allows for high resolution extrusion bioprinting of tissue implants has thus far been reported. In this work, we introduce a simple, novel method for producing zwitterionic microgel bioink, using alginate methacrylate (AlgMA) as crosslinker and mechanical fragmentation as a microgel fabrication method. Photocrosslinked hydrogels made of zwitterionic carboxybetaine acrylamide (CBAA) and sulfobetaine methacrylate (SBMA) are mechanically fragmented through meshes with aperture diameters of 50 and 90µm to produce microgel bioink. The bioinks made with both microgel sizes showed excellent rheological properties and were used for high-resolution printing of objects with overhanging features without requiring a support structure or support bath. The AlgMA crosslinker has a dual role, allowing for both primary photocrosslinking of the bulk hydrogel as well as secondary ionic crosslinking of produced microgels, to quickly stabilize the printed construct in a calcium bath and to produce a microporous scaffold. Scaffolds showed ∼20% porosity, and they supported viability and chondrogenesis of encapsulated human primary chondrocytes. Finally, a meniscus model was bioprinted, to demonstrate the bioink's versatility at printing large, cell-laden constructs which are stable for furtherin vitroculture to promote cartilaginous tissue production. This easy and scalable strategy of producing zwitterionic microgel bioink for high resolution extrusion bioprinting allows for direct cell encapsulation in a microporous scaffold and has potential forin vivobiocompatibility due to the zwitterionic nature of the bioink.

Cation-crosslinkedκ-carrageenan sub-microgel medium for high-quality embedded bioprinting.

Three-dimensional (3D) bioprinting embedded within a microgel bath has emerged as a promising strategy for creating intricate biomimetic scaffolds. However, it remains a great challenge to construct tissue-scale structures with high resolution by using embedded 3D bioprinting due to the large particle size and polydispersity of the microgel medium, as well as its limited cytocompatibility. To address these issues, novel uniform sub-microgels of cell-friendly cationic-crosslinked kappa-carrageenan (κ-Car) are developed through an easy-to-operate mechanical grinding strategy. Theseκ-Car sub-microgels maintain a uniform submicron size of around 642 nm and display a rapid jamming-unjamming transition within 5 s, along with excellent shear-thinning and self-healing properties, which are critical for the high resolution and fidelity in the construction of tissue architecture via embedded 3D bioprinting. Utilizing this new sub-microgel medium, various intricate 3D tissue and organ structures, including the heart, lungs, trachea, branched vasculature, kidney, auricle, nose, and liver, are successfully fabricated with delicate fine structures and high shape fidelity. Moreover, the bone marrow mesenchymal stem cells encapsulated within the printed constructs exhibit remarkable viability exceeding 92.1% and robust growth. Thisκ-Car sub-microgel medium offers an innovative avenue for achieving high-quality embedded bioprinting, facilitating the fabrication of functional biological constructs with biomimetic structural organizations.

Recent developments in chitosan based microgels and their hybrids.

Chitosan based microgels have gained great attention because of their chemical stability, biocompatibility, easy functionalization and potential uses in numerous fields. Production, properties, characterization and applications of chitosan based microgels have been systematically reviewed in this article. Some of these systems exhibit responsive behavior towards external stimuli like pH, light, temperature, glucose, etc. in terms of swelling/deswelling in an aqueous medium depending upon the functionalities present in the network which makes them a potential candidate for various applications in the fields of biomedicine, agriculture, catalysis, sensing and nanotechnology. Current research development and critical overview in this field accompanying by future possibilities is presented. The discussion is concluded with recommended possible future works for further progress in this field.

Microgels for bioprinting: recent advancements and challenges.

Microgels have become a popular and powerful structural unit in the bioprinting field due to their advanced properties, ranging from the tiny size and well-connected hydrogel (nutrient) network to special rheological properties. Different microgels can be fabricated by a variety of fabrication methods including bulk crushing, auxiliary dripping, multiphase emulsion, and lithography technology. Traditionally, microgels can encapsulate specific cells and are used for in vitro disease models and in vivo organ regeneration. Furthermore, microgels can serve as a drug carrier to realize controlled release of drug molecules. Apart from being used as an independent application unit, recently, these microgels are widely applied as a specific bioink component in 3D bioprinting for in situ tissue repair or building special 3D structures. In this review, we introduce different methods used to generate microgels and the microgel-based bioink for bioprinting. Besides, the further tendency of microgel development in future is introduced and predicted to provide guidance for related researchers in exploring more effective ways to fabricate microgels and more potential bioprinting application cases as multifunctional bioink components.

Injectable Microgels with Hybrid Exosomes of Chondrocyte-Targeted FGF18 Gene-Editing and Self-Renewable Lubrication for Osteoarthritis Therapy.

Abnormal silencing of fibroblast growth factor (FGF) signaling significantly contributes to joint dysplasia and osteoarthritis (OA); However, the clinical translation of FGF18-based protein drugs is hindered by their short half-life, low delivery efficiency and the need for repeated articular injections. This study proposes a CRISPR/Cas9-based approach to effectively activate the FGF18 gene of OA chondrocytes at the genome level in vivo, using chondrocyte-affinity peptide (CAP) incorporated hybrid exosomes (CAP/FGF18-hyEXO) loaded with an FGF18-targeted gene-editing tool. Furthermore, CAP/FGF18-hyEXO are encapsulated in methacrylic anhydride-modified hyaluronic (HAMA) hydrogel microspheres via microfluidics and photopolymerization to create an injectable microgel system (CAP/FGF18-hyEXO@HMs) with self-renewable hydration layers to provide persistent lubrication in response to frictional wear. Together, the injectable CAP/FGF18-hyEXO@HMs, combined with in vivo FGF18 gene editing and continuous lubrication, have demonstrated their capacity to synergistically promote cartilage regeneration, decrease inflammation, and prevent ECM degradation both in vitro and in vivo, holding great potential for clinical translation.

Antioxidant Microgels Support Peroxide-Challenged Hepatic Cells.

Access to therapeutic strategies that counter cellular stress induced by reactive oxygen species (ROS) is an important, long-standing challenge. Here, the assembly of antioxidant artificial cells is based on alginate hydrogels equipped with non-native catalysts, namely platinum nanoparticles and an EUK compound. These artificial cells are able to preserve the viability and lower the intracellular ROS levels of challenged hepatic cells by removing peroxides from the extracellular environment. Conceptually, this strategy illustrates the potential use of artificial cells with a synthetic catalyst toward long-term support of hepatic cells and potentially other mammalian cells.

Injectable MSC Spheroid and Microgel Granular Composites for Engineering Tissue.

Many cell types require direct cell-cell interactions for differentiation and function; yet, this can be challenging to incorporate into 3-dimensional (3D) structures for the engineering of tissues. Here, a new approach is introduced that combines aggregates of cells (spheroids) with similarly-sized hydrogel particles (microgels) to form granular composites that are injectable, undergo interparticle crosslinking via light for initial stabilization, permit cell-cell contacts for cell signaling, and allow spheroid fusion and growth. One area where this is important is in cartilage tissue engineering, as cell-cell contacts are crucial to chondrogenesis and are missing in many tissue engineering approaches. To address this, granular composites are developed from adult porcine mesenchymal stromal cell (MSC) spheroids and hyaluronic acid microgels and simulations and experimental analyses are used to establish the importance of initial MSC spheroid to microgel volume ratios to balance mechanical support with tissue growth. Long-term chondrogenic cultures of granular composites produce engineered cartilage tissue with extensive matrix deposition and mechanical properties within the range of cartilage, as well as integration with native tissue. Altogether, a new strategy of injectable granular composites is developed that leverages the benefits of cell-cell interactions through spheroids with the mechanical stabilization afforded with engineered hydrogels.

Tilapia-Derived Granular Hydrogel as a 3D Scaffold Promoting Rapid Wound Healing.

The skin, a crucial organ that protects the body, is vulnerable to external damage. Traditional tissue regeneration methods, including bulk hydrogels, aim to facilitate wound healing by interacting with host cells and providing a conducive environment. However, the nanoscale porosity of conventional hydrogels limits cell penetration and tissue regeneration. To overcome this, hydrogels composed of microgels have emerged as promising alternatives. In this study, we propose a granular hydrogel using decellularized tilapia skin. The tilapia skin-based microgels are cost-effective, immune-friendly, and have a high collagen content. Microgels based on the decellularized extracellular matrix of tilapia were successfully fabricated by using microfluidics. Through the assembly of these microgels using adhesive hyaluronic acid-catechol, the resulting 3D granular hydrogel scaffold facilitated enhanced cell growth, accelerated cell differentiation, and successful healing of full-thickness wounds in a mouse model. This study reveals the potential of tilapia skin-based granular hydrogel assembly in wound healing, overcoming conventional hydrogel limits.