Gelatin-Sodium Alginate Hydrogels Cross-Linked by Squaric Acid and Dialdehyde Starch as a Potential Bio-Ink.

Hydrogels as biomaterials possess appropriate physicochemical and mechanical properties that enable the formation of a three-dimensional, stable structure used in tissue engineering and 3D printing. The integrity of the hydrogel composition is due to the presence of covalent or noncovalent cross-linking bonds. Using various cross-linking methods and agents is crucial for adjusting the properties of the hydrogel to specific biomedical applications, e.g., for direct bioprinting. The research subject was mixtures of gel-forming polymers: sodium alginate and gelatin. The polymers were cross-linked ionically with the addition of CaCl2 solutions of various concentrations (10%, 5%, 2.5%, and 1%) and covalently using squaric acid (SQ) and dialdehyde starch (DAS). Initially, the polymer mixture's composition and the hydrogel cross-linking procedure were determined. The obtained materials were characterized by mechanical property tests, swelling degree, FTIR, SEM, thermal analysis, and biological research. It was found that the tensile strength of hydrogels cross-linked with 1% and 2.5% CaCl2 solutions was higher than after using a 10% solution (130 kPa and 80 kPa, respectively), and at the same time, the elongation at break increased (to 75%), and the stiffness decreased (Young Modulus is 169 kPa and 104 kPa, respectively). Moreover, lowering the concentration of the CaCl2 solution from 10% to 1% reduced the final material's toxicity. The hydrogels cross-linked with 1% CaCl2 showed lower degradation temperatures and higher weight losses than those cross-linked with 2.5% CaCl2 and therefore were less thermally stable. Additional cross-linking using SQ and DAS had only a minor effect on the strength of the hydrogels, but especially the use of 1% DAS increased the material's elasticity. All tested hydrogels possess a 3D porous structure, with pores of irregular shape and heterogenic size, and their swelling degree initially increased sharply to the value of approx. 1000% during the first 6 h, and finally, it stabilized at a level of 1200-1600% after 24 h. The viscosity of 6% gelatin and 2% alginate solutions with and without cross-linking agents was similar, and they were only slightly shear-thinning. It was concluded that a mixture containing 2% sodium alginate and 6% gelatin presented optimal properties after gel formation and lowering the concentration of the CaCl2 solution to 1% improved the hydrogel's biocompatibility and positively influenced the cross-linking efficiency. Moreover, chemical cross-linking by DAS or SQ additionally improved the final hydrogel's properties and the mixture's printability. In conclusion, among the tested systems, the cross-linking of 6% gelatin-2% alginate mixtures by 1% DAS addition and 1% CaCl2 solution is optimal for tissue engineering applications and potentially suitable for 3D printing.

The Impact of Gelatin and Fish Collagen on Alginate Hydrogel Properties: A Comparative Study.

Hydrogel materials based on sodium alginate find versatile applications in regenerative medicine and tissue engineering due to their unique properties, such as biocompatibility and biodegradability, and the possibility of the customization of their mechanical properties, such as in terms of the individual requirements of separate clinical applications. These materials, however, have numerous limitations in the area of biological activity. In order to eliminate their limitations, sodium alginate is popularly applied in combination with added gelatin, which represents a product of collagen hydrolysis. Despite numerous beneficial biological properties, matrix materials based on gelatin have poor mechanical properties and are characterized by their ability for rapid degradation in an aqueous environment, particularly at the physiological temperature of the body, which significantly limits the independent application opportunities of this type of composition in the range of scaffolding production dedicated for tissue engineering. Collagen hydrogels, unlike gelatin, are characterized by higher bioactivity, dictated by a greater number of ligands that allow for cell adhesion, as well as better stability under physiological conditions. Fish-derived collagen provides a material that may be efficiently extracted without the risk of mammalian prion infection and can be used in all patients without religious restrictions. Considering the numerous advantages of collagen indicating its superiority over gelatin, within the framework of this study, the compositions of hydrogel materials based on sodium alginate and fish collagen in different concentrations were developed. Prepared hydrogel materials were compared with the properties of a typical composition of alginate with the addition of gelatin. The rheological, mechanical, and physicochemical properties of the developed polymer compositions were evaluated. The first trials of 3D printing by extrusion technique using the analyzed polymer solutions were also conducted. The results obtained indicate that replacing gelatin with fish collagen at an analogous concentration leads to obtaining materials with a lower swelling degree, better mechanical properties, higher stability, limited release kinetics of calcium ions cross-linking the alginate matrix, a slowed process of protein release under physiological conditions, and the possibility of extrusion 3D printing. The conducted analysis highlights that the optimization of the applied concentrations of fish collagen additives to composition based on sodium alginate creates the possibility of designing materials with appropriate mechanical and rheological properties and degradation kinetics adjusted to the requirements of specific applications, leading to the prospective opportunity to produce materials capable of mimicking the properties of relevant soft tissues. Thanks to its excellent bioactivity and lower-than-gelatin viscosity of the polymer solution, fish collagen also provides a prospective solution for applications in the field of 3D bioprinting.

Solvent types used for the preparation of hydrogels determine their mechanical properties and influence cell viability through gelatine and calcium ions release.

Alginate-gelatin hydrogels are the most commonly used materials for 3D bioprinting. Their printability depends on their properties, and these derive from the way they are prepared and their very composition. Therefore, the aim of the study was to investigate the type of solvent (deionized water, phosphate buffer, and culture medium) and contents of gelatin in the composition of hydrogel (2% wt/vol alginate, 6% and 9% wt/vol of gelatin) on their biological, physicochemical, and mechanical properties, as well as printability and the ability of cells to proliferate in the printed structures. The results obtained revealed that all the manufactured hydrogel materials are biocompatible. The use of deionized water as a solvent results in the highest degree of cross-linking of hydrogels, thus obtaining a polymer with the highest rigidity. Moreover, an increase in gelatin content leads to an increase in the Young's modulus value, irrespectively of the solvent in which the hydrogels were prepared. Based on the chemical structure, it is more reasonable to use a culture medium for bioink preparation due to free NH and NH2 groups being present, which are ligands for cell attachment and their proliferation. For the selected material (2A9GM), the printability and high viability of the cells after printing were confirmed. In this case, the concentration of the cross-linking agent influences gelatin amount release and calcium ions release, and these two processes determine the change in the viability of the cells encapsulated in the bioink.

An Integration of a Peristaltic Pump-Based Extruder into a 3D Bioprinter Dedicated to Hydrogels.

The 3D printing technologies used for medical applications are mostly based on paste extruders. These are designed for high capacity, and thus often feature large material reservoirs and large diameter nozzles. A major challenge for most 3D printing platforms is a compromise between speed, accuracy, and/or volume/mass of moving elements. To address these issues, we integrated a peristaltic pump into a bioprinter. That allowed for combining the most important requirements: high precision, a large material reservoir, and safety of biological material. The system of a fully heated nozzle and a cooled print bed were developed to maintain the optimal hydrogel temperature and crosslinking speed. Our modifications of the bioprinter design improved the mechanical properties of the printouts and their accuracy while maintaining the maximal survival rate of cells and increasing the capacity of the bioink reservoir.

Sodium Alginate/Gelatine Hydrogels for Direct Bioprinting-The Effect of Composition Selection and Applied Solvents on the Bioink Properties.

Hydrogels tested and evaluated in this study were developed for the possibility of their use as the bioinks for 3D direct bioprinting. Procedures for preparation and sterilization of hydrogels and the speed of the bioprinting were developed. Sodium alginate gelatine hydrogels were characterized in terms of printability, mechanical, and biological properties (viability, proliferation ability, biocompatibility). A hydrogel with the best properties was selected to carry out direct bioprinting tests in order to determine the parameters of the bioink, adapted to print with use of the designed and constructed bioprinter and provide the best conditions for cell growth. The obtained results showed the ability to control mechanical properties, biological response, and degradation rate of hydrogels through the use of various solvents. The use of a dedicated culture medium as a solvent for the preparation of a bioink, containing the predicted cell line, increases the proliferation of these cells. Modification of the percentage of individual components of the hydrogel gives the possibility of a controlled degradation process, which, in the case of printing of temporary medical devices, is a very important parameter for the hydrogels' usage possibility-both in terms of tissue engineering and printing of tissue elements replacement, implants, and organs.