Morphometric study of bioprinted hydrogel deformation during histological preparation

Histological preparation <i>via</i> paraffin embedding is the gold standard method for evaluating tissue structure and composition, whether it is originated from biopsy or engineered <i>in vitro</i>. Quite often, deformation and shrinkage occur during the histological preparation, which are difficult to predict and qualify. The present study investigates the morphometric changes in bioprinted hydrogels composed of alginate and gelatine, common tissue engineering materials, focusing on three morphologies: full slabs, porous slabs, and porous cubes. These structures underwent key histological steps, including fixation, processing (dehydration, clearing, and infiltration with melted paraffin), embedding, and slicing, to evaluate their shrinkage behavior. Shrinking factors were systematically measured, showing that processing had the most significant effect (34-40% shrinking), followed by fixation (20-28% shrinking). Porous structures exhibited greater shrinkage compared to full slabs due to their internal geometry. Additionally, anisotropic behavior was observed in porous cubes, with different shrinking factors in the XY plane (horizontal) and Z direction (vertical), leading to an overall volumetric shrinking factor of 81.3%. The results demonstrated the critical influence of hydrogel structure on deformation and emphasized the need for tailored histological protocols to maintain structural fidelity. While this study focused on hydrogels alone, future work will incorporate cellularized bioengineered tissues to evaluate the impact of cell-mediated remodeling and extracellular matrix deposition on histological outcomes. This research offers a framework for optimizing histological preparation in bioengineered tissues, enabling more accurate assessment of their structure and function for regenerative medicine applications.