Optimizing Alginate-Based Hydrogels with Phytotherapeutics, Human-Origin Biomolecules, and Inorganic Fillers for Biofabrication

Tissue engineering (TE) involves the use of a temporary structure (scaffolds), mimicking the native tissue, that supports tissue regeneration in cases where the body's natural healing mechanisms are insufficient, by enhancing inherent repair processes. The essential components of a TE strategy consist of scaffolds made by suitable biomaterials, cells, and signaling molecules, which must be carefully chosen to meet the specific requirements of each application. Advances in biomaterial development for tissue repair are progressing towards more multifunctional and advanced materials, particularly through 3D bioprinting approaches. These novel materials aim to better monitor, influence, and manage tissue healing compared to traditional materials. Hydrogels, composed of a polymeric water-swollen structure, have the capability to mimic the natural extracellular matrix (ECM) of human tissue and can also be engineered to possess antibacterial, antioxidant, and bioactive agents for additional functionality. However, creating hydrogels that are both cytocompatible and suitable for 3D bioprinting into complex mechanically competent structures resembling natural tissue remains a significant challenge in TE. This doctoral dissertation presents the development of novel hydrogels that possess antibacterial, antioxidant, cell-compatible, bioactive, and 3D bioprinting capabilities. The developed biomaterials are based on a matrix composed of oxidized alginate (ADA) and gelatin (GEL), named ADA-GEL, supplemented with the phytotherapeutic agent ferulic acid (FA), human platelet lysate (HPL) biomolecules, and/or inorganic fillers, such as mesoporous bioactive glass nanoparticles (MBGNs), to enhance the hydrogel properties. FA is a plant-derived compound known for its antioxidant and antibacterial properties which is capable of crosslinking with proteins like GEL. HPL is a commonly used substitute for fetal bovine serum (FBS) in cell culture and contains a diverse array of growth factors that enhance angiogenesis, cell adhesion, and cell growth, thereby improving the cell-adhesive properties of the ADA-GEL hydrogels. MBGNs are silicate nanoparticles capable of releasing biologically active ions when implanted in the body due to their high surface reactivity in contact with aqueous environment. MBGNs were incorporated into the ADA-GEL hydrogels to improve their bioactive properties to promote the formation of a hydroxyapatite (HA) layer on the surface considering the intended application of the materials in bone TE. Moreover, the addition of MBGNs facilitates osteogenic differentiation and accelerates the formation of new blood vessels, ultimately improving bone tissue regeneration. The resulting 3D printed scaffolds and films were crosslinked using a combination of enzymatic crosslinking of GEL with microbial transglutaminase (mTG), ionic crosslinking of alginate, and crosslinking of FA with GEL, resulting in improved 3D construct stability. The impact on the mechanical characteristics, degradation behavior, and release properties of the hydrogel were investigated. The first part of this thesis introduces the addition of FA into ADA-GEL hydrogels to create an antibacterial and antioxidant master bioink, and to determine the most suitable FA concentration for future investigation. The second part focuses on the incorporation of HPL into ADA-GEL-(FA) bioinks to enrich the hydrogel with cell-adhesive and cell-attractive biomolecules. In the third part of this project, the ink is directed towards bone TE by adding MBGNs and extensively investigating their 3D (bio)printability and bioactivity. The research successfully demonstrated the creation of a multifunctional bioink with antibacterial properties against S. epidermidis, S. aureus, and E. coli bacteria. Furthermore, bioactive properties were confirmed through Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive X-ray analysis (EDX), which revealed the formation of a HA layer on the surface of ADA-GEL-based samples due to the presence of MBGNs. 3D (bio)printability investigations included several tests, such as grid structure test, filament fusion test, filament collapse test, and complex shape 3D printing, all of which showed improved 3D printing characteristics with 0.1% (w/v) MBGN content and the presence of additional FA-GEL crosslinking, as confirmed by an analysis of the crosslinking degree. Moreover, the created bioinks exhibited an adjustable and controllable degradation behavior and suitable (time dependent) mechanical characteristics. Furthermore, it was demonstrated that the addition of the chosen additives did not hinder the FA release ability of the hydrogel. Most importantly, cytocompatibility tests were performed, including direct testing and 3D bioprinting with the created multifunctional bioinks, to demonstrate the beneficial addition of additives that promoted cell attachment and exhibited high biocompatibility with pre-osteoblastic MC3T3-E1 cells, guiding the final application of the bioink towards bone TE. Summarizing, in this thesis the 3D bioprinting approach was utilized to successfully create hierarchically structured ADA-GEL based constructs exhibiting suitable properties and biological activity for TE. The scaffold fabrication method developed in this study enables the development of intricate 3D constructs that mimic the layered natural ECM. The composite hydrogels developed and investigated in this thesis show promise for further studies in TE and BTE, with a focus on utilizing their bioactive and cell adhesive properties. The 3D printed ADA-GEL-based constructs have the potential to offer enhanced capability to heal bone defects by increasing bone cell proliferation, attachment, and differentiation. Collectively, these results indicate that enhancing ADA-GEL hydrogels by organic and inorganic additives leads to composite biomaterials, which can be used as promising bioinks for bone TE and regenerative medicine.