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We have synthesized and characterized a collagen-hyaluronic acid hybrid network. The aim was to create a hydrogel mimicking the extracellular matrix of adipose tissue, primarily for use in in vitro studies of protein drug transport in the subcutaneous interstitial space. The network was created by covalently crosslinking methacryloyl-functionalized collagen type I and thiol-functionalized hyaluronic acid by means of thiol-Michael and thiol-ene photo-click reaction. The degree of modification corresponded to 74 % of the lysine and arginine groups on collagen, and 16 to 29 % of the carboxylate groups on hyaluronic acid, as determined with H-1 NMR. Circular dichroism measurements showed that the triple helix of modified collagen remained intact. Oscillatory shear rheology tests showed that the hydrated networks displayed viscoelastic properties characteristic of hydrogels. The storage modulus, measured at 1 Hz frequency in the linear viscoelastic range (<5%), varied in a controllable way between 1.5 and 4 kPa depending on the collagen concentration and collagen-to-hyaluronic acid ratio. The hydrogels had a lower collagen content (0.6--1.2 wt%) but similar hyaluronic acid content and shear modulus at low strain rates as the extracellular matrix in adipose tissue and were penetrable by albumin and lysozyme. The results show that the hydrogels are promising as model systems for investigations of drug transport.

Injectable hydrogels show great promise in developingnovel regenerativemedicine solutions and present advantages for minimally invasive applications.Hydrogels based on extracellular matrix components, such as collagen,have the benefits of cell adhesiveness, biocompatibility, and degradabilityby enzymes. However, to date, reported collagen hydrogels possesssevere shortcomings, such as nonbiocompatible cross-linking chemistry,significant swelling, limited range of mechanical properties, or gelationkinetics unsuitable for in vivo injection. To solvethese issues, we report the design and characterization of an injectablecollagen hydrogel based on covalently modified acetyl thiol collagencross-linked using thiol-maleimide click chemistry. The hydrogel isinjectable for up to 72 h after preparation, shows no noticeable swelling,is transparent, can be molded in situ, and retainsits shape in solution for at least one year. Notably, the hydrogelmechanical properties can be fine-tuned by simply adjusting the reactantstoichiometries, which to date was only reported for synthetic polymerhydrogels. The biocompatibility of the hydrogel is demonstrated in vitro using human corneal epithelial cells, which maintainviability and proliferation on the hydrogels for at least seven days.Furthermore, the developed hydrogel showed an adhesion strength onsoft tissues similar to fibrin glue. Additionally, the developed hydrogelcan be used as a sealant for repairing corneal perforations and canpotentially alleviate the off-label use of cyanoacrylate tissue adhesivefor repairing corneal perforations. Taken together, these characteristicsshow the potential of the thiol collagen hydrogel for future use asa prefabricated implant, injectable filler, or as sealant for cornealrepair and regeneration.

Hydrogels are used extensively as cell-culture scaffolds for both 2D and 3D cell cultures due to their biocompatibility and the ease in which their mechanical and biological properties can be tailored to mimic natural tissue. The challenge when working with hydrogel-based scaffolds is in their handling, as hydrogels that mimic e.g. brain tissue, are both fragile and brittle when prepared as thin (sub-mm) membranes. Here, we describe a method for facile handling of thin hydrogel cell culture scaffolds by molding them onto a polycaprolactone (PCL) mesh support attached to a commonly used Transwell set-up in which the original membrane has been removed. In addition to demonstrating the assembly of this set-up, we also show some applications for this type of biological membrane. A polyethylene glycol (PEG)-gelatin hydrogel supports cell adhesion, and the structures can be used for biological barrier models comprising either one or multiple hydrogel layers. Here, we demonstrate the formation of a tight layer of an epithelial cell model comprising MDCK cells cultured over 9 days by following the build-up of the transepithelial electrical resistances. Second, by integrating a pure PEG hydrogel into the PCL mesh, significant swelling is induced, which leads to the formation of a non-adherent biological scaffold with a large curvature that is useful for spheroid formation. In conclusion, we demonstrate the development of a handling platform for hydrogel cell culture scaffolds for easy integration with conventional measurement techniques and miniaturized organs-on-chip systems.

Visualizing cells, tissues, and their components specifically without interference with cellular functions, such as biochemical reactions, and cellular viability remains important for biomedical researchers worldwide. For an improved understanding of disease progression, tissue formation during development, and tissue regeneration, labeling extracellular matrix (ECM) components secreted by cells persists is required. Bioorthogonal chemistry approaches offer solutions to visualizing and labeling ECM constituents without interfering with other chemical or biological events. Although biorthogonal chemistry has been studied extensively for several applications, this review summarizes the recent advancements in using biorthogonal chemistry specifically for metabolic labeling and visualization of ECM proteins and glycosaminoglycans that are secreted by cells and living tissues. Challenges, limitations, and future directions surrounding biorthogonal chemistry involved in the labeling of ECM components are discussed. Finally, potential solutions for improvements to biorthogonal chemical approaches are suggested. This would provide theoretical guidance for labeling and visualization of de novo proteins and polysaccharides present in ECM that are cell-secreted for example during tissue remodeling or in vitro differentiation of stem cells.

Hyaluronic acid (HA), one of the main components of the extracellular matrix (ECM), is extensively used in the design of hydrogels and nanoparticles for different biomedical applications due to its critical role in vivo, degradability by endogenous enzymes, and absence of immunogenicity. HA-based hydrogels and nanoparticles have been developed by utilizing different crosslinking chemistries. The development of such crosslinking chemistries indicates that even subtle differences in the structure of reactive groups or the procedure of crosslinking may have a profound impact on the intended mechanical, physical and biological outcomes. There are widespread examples of modified HA polymers that can form either covalently or physically crosslinked biomaterials. More recently, studies have been focused on dynamic covalent crosslinked HA-based biomaterials since these types of crosslinking allow the preparation of dynamic structures with the ability to form in situ, be injectable, and have self-healing properties. In this review, HA-based hydrogels and nanomaterials that are crosslinked by dynamic-covalent coupling (DCC) chemistry have been critically assessed.

In severe malformations with a lack of native tissues, treatment options are limited. We aimed at expanding tissue in vivo using the body as a bioreactor and developing a sustainable single-staged procedure for autologous tissue reconstruction in malformation surgery. Autologous micro-epithelium from skin was integrated with plastically compressed collagen and a degradable knitted fabric mesh. Sixty-three scaffolds were implanted in nine rats for histological and mechanical analyses, up to 4 weeks after transplantation. Tissue integration, cell expansion, proliferation, inflammation, strength, and elasticity were evaluated over time in vivo and validated in vitro in a bladder wound healing model. After 5 days in vivo, we observed keratinocyte proliferation on top of the transplant, remodeling of the collagen, and neovascularization within the transplant. At 4 weeks, all transplants were fully integrated with the surrounding tissue. Tensile strength and elasticity were retained during the whole study period. In the in vitro models, a multilayered epithelium covered the defect after 4 weeks. Autologous micro-epithelial transplants allowed for cell expansion and reorganization in vivo without conventional pre-operative in vitro cell propagation. The method was easy to perform and did not require handling outside the operating theater.

The poor permeability of theranostic agents across the blood-brain barrier (BBB) significantly hampers the development of new treatment modalities for neurological diseases. A new biomimetic nanocarrier is discovered using heparin (HP) that effectively passes the BBB and targets glioblastoma. Specifically, HP-coated gold nanoparticles (HP-AuNPs) are designed that are labeled with three different imaging modalities namely, fluorescein (FITC-HP-AuNP), radioisotope (68)Gallium (Ga-68-HP-AuNPs), and MRI active gadolinium (Gd-HP-AuNPs). The systemic infusion of FITC-HP-AuNPs in three different mouse strains (C57BL/6JRj, FVB, and NMRI-nude) displays excellent penetration and reveals uniform distribution of fluorescent particles in the brain parenchyma (69-86%) with some accumulation in neurons (8-18%) and microglia (4-10%). Tail-vein administration of radiolabeled Ga-68-HP-AuNPs in healthy rats also show Ga-68-HP-AuNP inside the brain parenchyma and in areas containing cerebrospinal fluid, such as the lateral ventricles, the cerebellum, and brain stem. Finally, tail-vein administration of Gd-HP-AuNPs (that displays approximate to threefold higher relaxivity than that of commercial Gd-DTPA) in an orthotopic glioblastoma (U87MG xenograft) model in nude mice demonstrates enrichment of T1-contrast at the intracranial tumor with a gradual increase in the contrast in the tumor region between 1 and 3 h. It is believed, the finding offers the untapped potential of HP-derived-NPs to deliver cargo molecules for treating neurological disorders.

Click chemistry is not a single specific reaction, but describes ways of generating products which emulate examples in nature. Click reactions occur in one pot, are not disturbed by water, generate minimal and inoffensive byproducts, and are characterized by a high thermodynamic driving force, driving the reaction quickly and irreversibly towards a high yield of a single reaction product. As a result, over the past 15 years it has become a very useful bio-orthogonal method for the preparation of chemical cross-linked biopolymer-based hydrogel, in the presence of e.g. growth factors and live cells, or in-vivo. Biopolymers are renewable and non-toxic, providing a myriad of potential backbone toolboxes for hydrogel design. The goal of this review is to summarize recent advances in the development of click chemistry-based biopolymeric hydrogels, and their applications in regenerative medicine. In particular, various click chemistry approaches, including copper-catalyzed azide-alkyne cycloaddition reactions, copper-free click reactions (e.g. the Diels-Alder reactions, the strain-promoted azide-alkyne cycloaddition reactions, the radical mediated thiol-ene reactions, and the oxime-forming reactions), and pseudo-click reactions (e.g. the thiol-Michael addition reactions and the Schiff base reactions) are highlighted in the first section. In addition, numerous biopolymers, including proteins (e.g. collagen, gelatin, silk, and mucin), polysaccharides (e.g. hyaluronic acid, alginate, dextran, and chitosan) and polynucleotides (e.g. deoxyribonucleic acid), are discussed. Finally, we discuss biopolymeric hydrogels, cross-linked by click chemistry, intended for the regeneration of skin, bone, spinal cord, cartilage, and cornea. This article provides new insights for readers in terms of the design of regenerative medicine, and the use of biopolymeric hydrogels based on click chemistry reactions.

Self-assembled structures primarily arise through enzyme-regulated phenomena in nature under persistent conditions. Enzymatic reactions are one of the main biological processes constructing supramolecular hydrogel networks required for biomedical applications. Such enzymatic processes provide a unique opportunity to integrate hydrogel formation. In most cases, the structure and substrates of hydrogels are adjusted by enzyme catalysis due to enzymes' chemo-, regio- and stereo-selectivity. Such hydrogels processed using various enzyme schemes showed remarkable characteristics as dynamic frames for cells, bioactive molecules, and drugs in tissue engineering, drug delivery, and regenerative medicine. The enzyme-mediated crosslinking hydrogels mimic the extracellular matrices by displaying unique physicochemical properties and functionalities such as water-retention capacity, biodegradability, biocompatibility, biostability, bioactivity, optoelectronic properties, self-healing ability, and shape memory ability. In recent years, many enzymatic systems investigated polymer crosslinking. Herein, we review efficient strategies for enzymatic hydrogelation, including hydrogel synthesis and chemistry, and demonstrate their applicability in biomedical systems. Furthermore, the advantages, challenges, and prospects of enzymatic-crosslinkable hydrogels are discussed. The results of biocompatible hydrogel products show that these crosslinking mechanisms can fulfill requirements for a variety of biomedical applications, including tissue engineering, wound healing, and drug delivery.