Factor XIII Cross-Linked Adhesive Chitosan Hydrogels

Biomedical adhesives have been found to be an attractive alternative to suturing in several circumstances. However, to date most of the clinically approved formulations are based on synthetic and highly reactive toxic chemicals. In this work, they aimed to combine for the first time the bioactive properties of the cationic polysaccharide chitosan and its intrinsic electrostatic binding to negatively charged tissues with the biocompatible and clinically compliant enzymatic cross-linking scheme of fibrin glue. This synergistic activity led to the generation of a transglutaminase Factor XIII cross-linkable chitosan formulation with fast gelatin kinetics, tunable mechanical properties, antibacterial activity, and strong adhesion to cartilage.


In past decades tissue adhesives have increasingly gained attention as an alternative to suturing, in particular in those circumstances where suturing is impractical or ineffective. Tissue adhesives mainly include synthetic or naturally derived liquid formulations that undergo cross-linking upon delivery to the damaged site. Most biomedical adhesives are cyanoacrylate-, urethane-, formaldehyde-, and glutaraldehyde-based, which exhibit rapid cross-linking rates, as well as fast and strong adhesion to tissues.

However, the nonspecific reactions of chemical glues, which can cause irritation, and the concerns about toxicity and carcinogenicity of the degradation products (i.e., cyanoacetates and formaldehyde) represent major drawbacks.This has motivated the development of safe and biocompatible bioadhesion strategies. Enzymatic reactions emerged as a promising solution because of their fast kinetics, high substrate selectivity, physiological reaction conditions (pH, temperature, and aqueous environment), and no need for toxic chemicals. Fibrin glue is a commercially available adhesive based on enzymatic cross-linking and one of the most widely used in clinics. Its cross-linking strategy is based on the activity of transglutaminase Factor XIII (FXIII) and thrombin, which in the presence of fibrinogen replicates the last stage of the blood coagulation cascade. Despite the several advantages of this formulation, concentrated human fibrinogen is pulled from donated blood, which makes it relatively expensive and subject to the risk of transferring blood-borne diseases.

(A) Reaction scheme illustrating the consecutive grafting of arginine (orange), vinyl sulfone linker (gray), and transglutaminase substrate peptides TG-Q and TG-K. (B) Cross-linking strategy of the polycationic Arg-Chi-TG and illustration of in situ cross-linking in cartilage defect/damage with detail on hydrogel formation and cross-linking points. (C) Images showing that the formation of stable hydrogels (right) is due to the enzymatic activity. In the absence of thrombin and FXIII, Arg-Chi-TG does not form a gel (left).

In this context, polysaccharides have emerged as a highly attractive alternative because of their high abundance, safety, intrinsic bioactive effects, tunable mechanical and chemical properties, and ability to mimic the native extracellular matrix (ECM), thus promoting tissue regeneration.

Mechanical properties of Arg-Chi-TG hydrogels. (A) Average gelation curves (storage modulus, n = 3) monitored by oscillatory test at different polymer concentrations, showing fast gelation onset (2–3 min) with equilibrium reached after approximately 15 min. (B) Plateau storage modulus reached by hydrogels at different polymer concentrations, proving stiffness tunability. (C) Qualitative assessment of Arg-Chi-TG hydrogel cohesiveness. A hydrogel cube is cut in half with a scalpel, and the two resulting pieces are rejoined by simple juxtaposition showing cohesiveness and stability. (D) Shear-recovery test of 3% Arg-Chi-TG hydrogel showing recovery of hydrogel mechanical properties after high shear rate (light blue area, 500% strain, 1 Hz, 10 s). (E) Image showing Arg-Chi-TG hydrogel gluing together two bovine knee articular cartilage pieces. (F, G) Arg-Chi-TG hydrogel exhibits stronger adhesion to cartilage compared to fibrin glue and PBS during tensile test (n = 3).

Chitin, the most abundant natural biopolymer in the marine ecosystem and the second most abundant on Earth after cellulose, represents a valuable source of materials for various applications, including drug and gene delivery, tissue engineering, and modern biomimetics. Mainly found in fungi, diatoms, sponges, mollusks, and arthropods, chitin in its native form appears as a linear homopolymer of β(1,4)-linked N-acetyl-d-glucosamine. The tight parallel or antiparallel arrangement of the linear polysaccharide chains results in a crystalline form with poor water solubility.

(A) Schematic illustration of Arg-Chi-TG application on cartilage tissue defect and details on main adhesion mechanisms that take place in the material-to-tissue interface. (B) Infiltration and retention of fluorescently labeled Arg-Chi into cartilage explants over time (scale bar, 200 μm). (C) (i–iv) defect filling on osteochondral graft with Arg-Chi-TG 3% supplemented with TRITC-dextran (scale bar, 2 mm); (v) infiltration of fluorescently labeled Arg-Chi into cartilage defects (scale bar, 200 μm).

Its partially deacetylated derivative, chitosan, shows instead water solubility in acidic conditions (pH < 6) due to glucosamine amino group protonation, while retaining insolubility in aqueous solutions at neutral or basic pH. Despite this limited water solubility, chitosan has found use in many industries including food processing, cosmetics, and water treatment. To facilitate water solubility, several chitosan derivatives have been proposed in the most recent decades, for example, carboxymethyl-, sulfated-, quaternarized-,and arginine-chitosan.As with other kinds of polysaccharides, chitosan offers key advantages for biomedical applications, such as biocompatibility, biodegradability, and nontoxic degradation products. Although factors such as purity, source, degree of deacetylation, and molecular weight may influence its in vivo therapeutic effects, chitosan is considered nontoxic. In addition, mainly due to its polycationic nature, it offers a unique combination of biological properties such as antibacterial, antifungal, and antioxidant activity, as well as hemostatic potential and in vivo mucoadhesivity. These versatile properties have made chitosan an excellent candidate as a biomedical adhesive.

Biological properties of Arg-Chi-TG. (A) Chitosan derivatives Arg-Chi and Arg-Chi-TG showed clear antibacterial activity against Gram-positive S. aureus compared to PBS control. (B) MTS assay with articular chondrocytes showed improved proliferation, compared to medium only, when cultured in the presence of chitosan derivatives Arg-Chi and Arg-Chi-TG at different concentrations, as well as with medium preincubated in the presence of Arg-Chi-TG hydrogel (one-way ANOVA; n = 3; *, p < 0.05).

In this study they report for the first time the synthesis and characterization of a water-soluble transglutaminase (TG) cross-linkable chitosan to be used as an injectable adhesive. This formulation aims to combine the intrinsic bioactive and adhesive properties of chitosan with the clinically approved enzymatic cross-linking components of fibrin glue (FXIII and thrombin). First, to overcome a major disadvantage of chitosan that limits its biological applications, they have made it water-soluble at neutral pH by chemically grafting arginine through EDC/NHS activation of the amino acid α-carboxyl group. Conjugation of arginine allows one to generate a protonated water-soluble derivative, while imparting significant antibacterial activity.The degree of arginine substitution was determined by 1H NMR analysis to be ∼13–18%. EDC activation was exploited also in a second step, during which a short hetero-difunctional linker (VS-COOH), previously developed in their group, was grafted onto the arginine-chitosan (Arg-Chi) polymer, thereby providing a vinyl sulfone moiety to the polymer. In the final step, the vinyl sulfone reactive group was exploited to immobilize FXIII peptide substrates through click thiol-Michael addition.

The two peptides used in this study were previously reported by their group, with one containing a reactive glutamine (TG-Q) and the other a reactive lysine (TG-K). Arg-Chi derivatives with TG-Q and TG-K peptides (Arg-Chi-TG) were synthesized separately and then mixed in equal amounts. Their degree of substitution was calculated by 1H NMR to be ∼11–16%, based on the vinyl protons of the previous step, which are then found to be completely lost due to the formation of the thioether bond with the cysteine bearing peptides1.

  1. Berg, I. et al. (2021). Factor XIII Cross-Linked Adhesive Chitosan Hydrogels. ACS biomaterials science & engineering. doi:10.1021/acsbiomaterials.1c00298