Phase behaviour and structure formation of alginate-protein composite gels used in 3D bioprinting

Alginate-protein composite gels are widely used across various industries, with a key application in extrusion-based 3D bioprinting. Alginate offers favourable printability and rapid ionic gelation but lacks cell-adhesive properties. Gelatin addresses these limitations by providing RGD motifs essential for cell attachment and enhancing scaffold porosity through temperature-sensitive dissolution. However, its animal origin raises ethical and sustainability concerns, particularly in bioprinting applications targeting the reduction of animal testing or cultured meat production. This creates a need for animal-free alternatives that maintain the functional advantages of alginate-gelatin systems. However, successful substitution requires a comprehensive understanding of the molecular interactions and phase behaviour as well as the influence of different protein characteristics on the structure development. The overall aim of this thesis was to investigate the structure formation of alginate-gelatin composite gels and provide a scientific foundation for replacing gelatin with plant-based proteins – specifically, commercial pea protein – in the development of vegan bioinks. Therefore, the research was structured around three objectives: (1) understanding the molecular interactions in dilute alginate-gelatin systems, (2) characterisation of the structure formation in concentrated and gelled systems, and (3) evaluating the effect of pea protein on alginate gelation and its suitability as a gelatin substitute. At the first stage, dilute binary and ternary alginate-gelatin solutions were studied at neutral pH using zeta potential, rheology, viscometry, osmometry, and FTIR spectroscopy to assess molecular conformation, interaction parameters, and secondary structure changes under varying ionic strengths and solvent conditions. At the second stage, concentrated mixtures were analysed to investigate the impact of mixing ratio and gelatin state (liquid or gelled) on network formation. Alginate gelation was induced via internal cross-linking using Ca-EDTA/GDL, and structural properties were assessed through rheological analysis of the gel strength and gelation velocity as well as confocal laser scanning microscopy. At the third stage, alginate-pea protein composite gels were investigated using a novel two-component cartridge and static mixer system. This setup allowed for rapid mixing of the biopolymer mixture and calcium under shear, enabling rheological monitoring of the initial gelation behaviour, while closely resembling the bioink preparation. The effect of pea protein on the alginate gelation was analysed in solvents of varying ionic strength and in a cell culture medium. In dilute alginate-gelatin systems, weak attractive interactions and potential complex formation were indicated by negative cross-virial coefficients and the viscometric interaction parameter ε. These interactions were significantly influenced by the ionic strength and mixing ratio, but not by urea, suggesting that electrostatic forces rather than hydrogen bonds primarily govern the interaction. FTIR spectroscopy revealed no changes in the secondary structure of gelatin. In concentrated gelled systems, the temperature-dependent physical state of gelatin strongly influenced the alginate gelation. While liquid gelatin delayed the alginate gelation likely by steric hindrance and by buffering the pH drop facilitating the calcium release, gelled gelatin promoted faster alginate gelation due to inducing phase separation. The combination of rheological measurements and microstructural imaging revealed that the final network structure was governed by the competition between gelation rate and phase separation rate. For pea protein, the electrostatic repulsion between alginate and pea protein resulted in strong tendencies toward phase separation, proposing a challenge for homogenous gel formation. The rapid gelation using the novel mixing approach kinetically trapped the system before macroscopic phase separation occurred. Rheological characterisation showed that pea protein reduced the gel strength and delayed gelation, likely due to steric hindrance and potential calcium-binding capacity. Elevated ionic strength mitigated the delay of gelation to some extent, though this was less effective in cell culture medium. Overall, this thesis demonstrates that structurally stable and printable alginate-protein composite gels can be achieved without specific repulsive or attractive molecular interaction between components, provided that gelation occurs rapidly enough to prevent phase separation on a macroscopic level. The results highlight the critical role of formulation and process parameters – such as component concentrations, calcium availability, and mixing strategy – in tuning the properties of composite gels for bioink applications. These findings provide a framework for the development of functional, animal-free bioinks, offering a pathway toward more ethical and sustainable 3D bioprinting applications.

Author dc:creator

• Bäther, Sabrina

Advisor dc:contributor.advisor

• Wagemans, Anja Maria

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Identifier URI

https://doi.org/10.14279/depositonce-24819

OAI identifier oai:identifier

oai:depositonce.tu-berlin.de:11303/25991