Towards the human bone organotypic model: From bioink to biocompatible 3-D printed model

In the scope of this project, we aim to establish a human 3-D organotypic model, using 3-D bioprinting technology for congruent representation of bone physiology in bone-related biomedical studies, e.g., in bone reparation mechanism. The goal is to obtain a reproducible 3-D in-vitro bone model with the engineered extracellular matrix (ECM) that includes bone cells, organized and mineralized collagen fibers.

Currently, state-of-the-art approaches in the evaluation of biocompatibility and osteoconductive/inductive effects of the new implants do not account for the lack of a native extracellular matrix (ECM) microenvironment. Ignoring the impact of ECM on the implant-bone interface leads to erroneous conclusions and distorts the real picture of the processes associated with bone regeneration, making the observations impracticable. Therefore, the development of an organotypic model, which reflects the correct physiology of human bone, can facilitate a valid approximation of the actual human in-vivo situation, contributing to the general understanding of physiological processes and hopefully, increasing the success rate in bone fracture treatments and prosthesis surgeries.

Expected results:
1. Bioink for the 3-DP application that: has structural integrity and geometrical fidelity up to 6 weeks in the culture; has tunable degradation kinetics and mechanical properties; can be used for printing cells with high viability with subsequent differentiation into the osteoblasts and collagen deposition; can form a viable tissue in the printed matrix; can be used for co-printing with different materials (different physicochemical properties) and create dynamically changing architecture.
2. 3-D organotypic model representing human bone physiology that: includes functional bone cells, the osteoblasts; has well-organized and mineralized collagen fibers at the micro- and nanoscale; has improved mechanical properties and is reproducible. Achieved results: Variable compositions of bioinks with alginate, gelatin, and GelMA were prepared to find the most promising compositions for 3D bioprinting. Bioinks were prepared by dissolving 1% alginate and varying concentrations of gelatin and GelMA from 0 to 10% and 2.5 to 10%. 3 of the 15 bioinks tested were determined to be print stable and will be used to print MSCs. An increase in degradation was observed with decreasing GelMA concentration. Printing parameters and conditions for bio-inks that provide structural stability and high printing accuracy: higher gelatin gelation temperature than GelMA improves the thermal sensitivity of hydrogel inks; inks containing more than 5% GelMA are more stable; printing temperature of about 10°C ensures the extrusion of stable bio-ink and high printing precision. The novelty of the results obtained lies in the production of new bioinks based on three materials such as alginate, gelatin, and GelMA. These materials allow the use of three methods of cross-linking hydrogels at once: ionic, thermal, and covalent, which made it possible to obtain inks with suitable parameters for bioprinting and are also mechanically stable for up to 3 weeks. The combination of covalent and ionic cross-linking mechanisms promotes better energy dissipation to create hydrogels of high strength and elasticity.