Mechanical characterization of the thymic niche for bio-mimetic approaches

Introduction The thymus is the primary lymphoid organ responsible for the development and selection of T-cells, which is supported by thymic extracellular matrix, organized in a 3D mesh structure, [1][2]. 3D scaffolds made of natural/synthetic polymers are currently of limited relevance due to the absence of micro-structural (e.g. 3D topography) and micro-mechanical cues (e..g. viscoelasticity) [3]. Indeed, to date, the mechanical properties of this organ have been poorly investigated, and its viscoelastic behavior is still unknown [4]. This work aims to tackle these issues, by performing a thorough viscoelastic characterization of the bovine thymus.

Methods Fresh bovine thymus was provided by a local slaughterhouse. Two cutting directions (longitudinal and transversal) were considered to collect samples of 20×20 mm base area and 10 mm height). All mechanical tests were carried out on a Mach-1 V500CSS (Biomomentum Inc., Montreal, Canada) mechanical tester equipped with a 17 N load cells. For indentation stress-relaxation (SR) mapping, a 5 mm tip was adopted, with a 10 mm/s indentation velocity and a 2.5 mm amplitude maintained for 60 s. A generalized maxwell model was used to derive the instantaneous (Einst) and equilibrium (Eeq) elastic modulus, as well as the characteristic relaxation time (t). Thymus samples were fixed overnight with 4% paraformaldehyde at 4°C, then rinsed in PBS and embedded in paraffin. Sections of 5 µm thickness were stained with hematoxylin and eosin (H&E) to visualize nuclear content and tissue microstructure.

Results Figure 1a reports the Einst, Eeq, and t distribution on the surface of a representative tissue sample. The parameters are heterogeneous throughout the entire area, with values ranging between 3.5 and 36.9 kPa (Einst), 0.8 and 10.1 kPa (Eeq), 7.9 and 17.7 s (t). Standard histology by H&E stains confirmed the distribution of cortical and medullary areas.

Discussion The indentation map outlined sample anisotropy, which is likely associated with the cortical and medullar regions of the thymus also reported in the microscopy image. Further studies to perform a comprehensive and quantitative viscoelastic characterization of the organ, considering local and bulk compressive, as well as shear properties are ongoing. Thymic tissue will be also further characterized in terms of its microstructure, using scanning electron microscopy and micro-computer tomography.