Thermosensitive chitosan-based hydrogels for extrusion-based bioprinting and injectable scaffold for articular tissue engineering

Bioprinting is an advanced method that enables to engineer living 3D structures mimicking the tissue complexity found in -vivo. It allows to create 3D tissues to study drugs /biological mechanisms, also, in longer-term, implantable tissue to replace diseased/damaged body tissues /organs. Bioprinting is growing rapidly but faces several challenges. One of them is to find ideal bioinks which combine all the required properties. Hydrogels are generally used since cells require an aqueous environment. But it is very challenging to stack hydrogels into a 3D structure because hydrogels are weak by nature and cannot support the structure without collapsing. Among the potential candidates are thermosensitive chitosan hydrogels which form solutions at room temperature but rapidly gel at body temperature. However, their potential in bioprinting has not been yet studied. Moreover, comprehensive rheological methods to predict their printability are still missing. The general objective of this Ph.D. was to study and optimize the thermosensitive chitosan -based hydrogels for extrusion -based bioprinting and injectable scaffold for articular tissue engineering. The first objective was to develop a rheological approach to study printability of these time – and temperature-dependent hydrogels and assess their potential as bioinks . Chitosan-based physical hydrogels prepared by combining chitosan acidic solution with weak bases like beta -glycerophosphate and sodium- hydrogen-carbonate were studied. Gelation kinetics , shear-thinning viscosity as a function of shear rate corresponding to that applied during printing, and recovery tests were performed. The resolution and mechanical properties were characterized as a function of applied shear rate and results were correlated with rheological data. This work allowed us to determine the best chitosan hydrogel formulation for 3Dprinting and compare it with conventionally used bioink, alginate/gelatin. This methodology can also be useful for other temperature- and time-dependent materials. We demonstrated that printing structures with adequate fidelity and handability using chitosan -based hydrogels was feasible; however, a high concentration (3%w/v) was required, leading to high mortality rate of encapsulated cells. Decreasing chitosan concentration resulted in poor shape fidelity. The second objective was therefore to develop a method using Freeform reversible embedding of suspended hydrogel (FRESH) bioprinting to overcome these limitations. A warm support bath was designed to support chitosan- based bioprinted structures and enhance chitosan thermo -crosslinking during printing. This approach drastically increases the fidelity and mechanical properties of structures printed with low concentration chitosan (2%w/v) suitable for cell encapsulation. Lastly, we studied the impact of loading bioglass particles into such thermosensitive hydrogels for potential bone-mineralized tissue repair, which could promote bone ingrowth through osteoconductivity. The mechanical properties and in -vitro cytocompatibility are affected adversely by bioglass addition. A new strategy was implemented to encapsulate bioglass within chitosan-based microbeads, then incorporate these microbeads in the cell-laden chitosan-based hydrogel. This strategy improved mechanical properties and cell viability. This hybrid hydrogel could be used to form an injectable cell-loaded scaffold. The bioactive microbeads were freezable, increasing their potential for clinical applications. We demonstrated the potential of the thermosensitive chitosan-based hydrogels for bioprinting, especially with the FRESH approach. This opens interesting avenues toward tissue engineering. However, much works still remain to be done before bioprinting complex gradient tissues.