Marine-based injectable platforms: development of cryogels for cartilage repair using fish collagen, chondroitin sulfate and hyaluronic acid

The application of marine-derived biomaterials is becoming increasingly popular as a biocompatible and eco-friendly alternative to the use of mammalian compounds in regenerative medicine. The current study investigates the prospect of creating injectable cryogels for cartilage regeneration based on three marine-sourced biopolymers — collagen, chondroitin sulfate (CS), and hyaluronic acid (HA) —. Collagen and CS were extracted from Prionace glauca skin and cartilage, respectively, while HA was produced by bacterial fermentation using marine peptones as protein substrate. After examining a rigorous physicochemical characterization of isolated compounds, several hydrogel formulations were prepared through mixing biopolymer solutions at low temperature, varying concentrations and ratios, with electrostatic intereactions being further complemented by covalent crosslinking approaches. Mechanical cohesiveness and injectability of resulting systems were evaluated in response to formulation variables. FTIR analyses confirmed the identity of the materials, revealing characteristic signals such as amide bands in collagen, sulfate vibrations in CS, and a carboxylate peak around 1610 cm⁻¹ in HA. Circular dichroism confirmed the preservation of the triple-helix structure in shark-derived collagen. Rheological assessment demonstrated shear-thinning behavior in both collagen and HA, with increased viscoelasticity at higher HA concentrations. The sulfation level of marine CS was evaluated using the DMMB assay, which showed significantly higher values (1.26 ± 0.32 µg/mL) compared to bovine-derived CS. Zeta potential analysis revealed a strong negative surface charge for HA (–45.1 mV), and a moderately negative value for CS (–20.2 mV), supporting their electrostatic stability. Additionally, marine-derived collagen showed a positive zeta potential of +19.4 mV, supporting the referred electrostatic interactions between the three components. The formulation method allowed for the adjustment of properties of cryogels such as cohesiveness and mechanical stability in PBS, with a direct effect on injectability. Blending marine-derived collagen, CS, and HA in predetermined ratios resulted in hydrogels of adjustable nature and potential application as injectable scaffolds for minimally invasive articular pathology treatment. Altogether, this research emphasizes the functional capabilities of marine biopolymers, aligned with circular bioeconomy approaches through valorization of fish by-products, and supports their use as sustainable alternatives in the design of next-generation regenerative materials.