Bioprinting is gaining an ever-increasing research interest thanks to its promising new applications in the field of tissue engineering. The main issue regarding this biofabrication technique is the engineering of suitable bioinks, accommodating both the requirements for fabrication and for cell culture and signalling. Versatile platform bioinks, with easily tailorable rheological, mechanical and biological properties could be applied for various applications and represent a powerful tool to overcome issues related with the long process required to engineer and validate inks. A versatile platform based on tyramine modified alginate (AL-TY) with multiple available crosslinking routes was defined, with the possibility to introduce tissue-specific biological functionalizations with phenol moieties during crosslinking.
Alginate was functionalized with tyramine through carbodiimide chemistry as already reported , obtaining a degree of substitution of 36 ± 2 % (characterized by 1H.NMR). Various crosslinking methods were tested and AL-TY was found suitable for: ionic gelation (Ca2+), enzymatic gelation (Horseradish peroxidase), photo-crosslinking (riboflavin (Rb) as photosensitizer and sodium persulfate as an electron acceptor). A novel crosslinking photo-enzymatic crosslinking route was investigated, as well, by activating the enzyme with light and Rb. The crosslinking kinetics of the various methods alone and combined were analysed through rheology and photo-rheology. The effect of crosslinking parameters (i.e. concentration of various reagents, light exposure time, light source power, etc.) was assessed to identify the best way to optimize the crosslinking kinetics and the mechanical properties of the constructs. Given the bioinert nature of alginate, tripeptide Arg-Gly-Asp (RGD) was introduced to favour cell adhesion. Cyclo(RGDyk) was selected, as containing a phenol group, it can be introduced during crosslinking . The RGD concentration effect was evaluated on encapsulated human bone marrow-derived mesenchymal stem cells (hMSC). As a proof of concept, deferoxamine (DFO) was grafted onto AL-TY to validate the possibility of introducing additional biological cues in the bioink. DFO has been reported to behave as a hypoxia simulator, promoting the expression of genes associated with angiogenesis . The platform was finally validated for bioprinting, by fabricating constructs under different conditions and measuring their mechanical properties, swelling and degradation time.
RESULTS AND CONCLUSIONS
All tested crosslinking routes proved to be suitable to produce hydrogels. However, photocrosslinking alone produced hydrogels with extremely fast degradation times, due to damage of the polymeric chains caused by excessive radical production. The extensive rheological characterization of the crosslinking methods allowed to verify the effect of several process parameters on the reaction kinetics and yield, and to build preliminary guidelines to tailor the properties of the gels and effectively create a versatile platform system in terms of mechanical properties and crosslinking routes and kinetics. Similarly, the addition of cyclo(RGDyk) and the effect of its concentration on cell adhesion was tested by preliminary cell encapsulation experiments. In conclusion, the material is versatile and can be easily tailored in terms of rheological properties, final mechanical properties, crosslinking kinetics and required biological properties. Additionally, the material is suitable for bioprinting and is showing promising results in building a “universal” bioink.