Development of empirical hepatic biomechanical models through experimental characterization of porcine and bovine livers

INTRODUCTION Complex models of the liver are envisioned to support drug development with high-throughput experimental platforms. The chemo-mechanical niche, the set of biochemical and mechanical characteristics of a physiological or a pathological organ, greatly influences the fate and the behavior of cultured cells for the in vitro reproduction in vivo-like responses. The studies of the mechanical properties of liver are few, and empirical hepatic biomechanical models, dataset obtained through direct analyses of the organ, are widely dispersed. The differences between experimental techniques, conditions, or organ sources2 limits the possibility to compare them. Pioneering studies rely on indirect methods (i.e., magnetic resonance elastography) 3,4. Moreover, the intra-and inter-species variability and anisotropy of these tissues are not addressed. In this study we focus on the development of a methodology to experimentally evaluate the mechanical properties of the liver, in response to small shear deformations in oscillatory regime and to axial deformation applied in compression in quasi-static tests, to reproduce the physiological stresses on the organ by blood perfusion and surrounding organs. EXPERIMENTAL METHODS Porcine and bovine livers were kindly provided by a local butcher less than to 12 hours after the slaughter. Organs were sectioned along three spatial planes (Figure 1). From each slice, cylindrical specimens ( 25 mm) were cut. To prevent dehydration and coagulation, each specimen was injected from the top face, the bottom face, and from the side with 10 ml of isotonic (4% w/v) Na-citrate, in H2O, then immersed in the same solution, and finally stored at 4 °C until the measurements. Preliminary analyses were performed with a rheometer (MCR 502e, Anton-Paar, AT) mounting a 25 mm parallel plate geometry, and imposing different preload forces in the range 0.1 N – 5 N. The mechanical properties of the organs were then measured at small deformations in oscillatory regime in response to shear stresses and quasi-static compressions. Different livers were obtained and analyzed to evaluate the variability within the same species. Porcine and bovine livers were evaluated with the same test protocols, to compare interspecies variability. The effect of different preservation techniques was investigated on samples that were previously frozen at different temperatures up to -120°C. RESULTS AND DISCUSSION The study of relation between the gap among plates and the normal force indicates that preloads greater than 1 N progressively produced structural changes on specimens, as the response significantly varied with the higher preloads. The set preload was therefore considered for the following tests. Shear analyses highlighted that the liver tissues, both porcine and bovine, are characterized by a gel-like behavior, with a conservative modulus (G’) higher than the dissipative ones (G’’) at all the analyzed frequencies. Only sectioning the livers in the XZ plane (Fig. 1) resulted in reproducibility of G’, G’’, and loss factor (tan δ) data, with G’ and G’’ always ranging between 300-400 Pa and 70-100 Pa respectively, indicating the anisotropy of the tissues. Compressive modulus of organs did not display a significant variability between species and highlighted a similar anisotropy to the one observed with rheological analyses. The evaluated G’, G’’, and tan δ fall within the range of data from magnetic resonance elastography (MRE) 4. Up to now, the cryopreservation of samples seems appropriate. Thawed samples displayed mechanical properties that are aligned with the ones of fresh samples. CONCLUSION The developed procedure can be implemented to build an empirical model of the hepatic biomechanics as a reference when engineering a three-dimensional matrix to produce in vitro models of the liver. The protocol is proposed to characterize the hepatic biomechanics, even if, in principle, the same methodology can be applied to other types of biological soft tissue. The gel-like behavior of the hepatic tissues suggests the feasibility of producing hydrogels-based 3D models of the liver.