Tissue engineering (TE) is an innovative approach to tackling many diseases and body parts that need to be replaced by developing artificial tissues and organs. Bioinks play an important role in the success of various TE applications. A bioink refers to a combination of a living cell, biomaterials, and bioactive molecules deposited in a layer-by-layer form to fabricate tissue-like structures. The research on bioink attempts to offer a 3D complex architecture and control cellular behavior that improve cell physical properties and viability. This research proposed a new multi-material bioink based on alginate (A), gelatin (G), and cholesteryl ester liquid crystals (CELC) biomaterials, namely (AGLC) bioinks. The development of AGLC was initiated with the optimization of different concentrations of A and G gels to obtain a printable formulation of AG gels. Subsequently, the influences of different concentrations of CELC with AG gels were investigated by using a microextrusion-based 3D bioprinting system to obtain a printed structure with high shape fidelity and minimum width. The AGLC bioinks were formulated using AG gel with 10% weight/volume (w/v) of A and 50% w/v G (AG10:50) and 1%, 5%, 10%, 20%, and 40% of CELC, respectively. The AGLC bioinks yield a high printability and resolution blend. The printed filament has a minimum width of 1.3 mm at a 1 mL/min extrusion rate when the A equals 10% w/v, G equals 50% w/v, and CELC equals 40% v/v (AGLC40). Polymerization of the AGLC bioinks with calcium (Ca²⁺) ions shows well-defined and more stable structures in the post-printing process. The physicochemical and viability properties of the AGLC bioinks were examined by FTIR, DSC, contact angle, FESEM, MTT assay, and cell interaction evaluation methods. The FTIR spectra of the AGLC bioinks exhibit a combination of characteristics vibrations of AG10:50 and CELC. The DSC analysis indicates the high thermal stability of the bioinks. Wettability analysis shows a reduction in the water absorption ability of the AGLC bioinks. FESEM analysis indicates that the surface morphologies of the bioinks exhibit varying microstructures. In vitro cytotoxicity by MTT assay shows the ability of the bioinks to support the biological activity of HeLa cells. The AGLC bioinks show average cell viability of 82.36% compared to the control (90%). Furthermore, cultured cells on the surface of AGLC bioinks showed that bioinks provide favorable interfaces for cell attachment.

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