Micro-mechanical fingerprints of the rat bladder change in actinic cystitis and tumor presence

Abstract

Tissue mechanics determines tissue homeostasis, disease development and progression. Bladder strongly relies on its mechanical properties to perform its physiological function, but these are poorly unveiled under normal and pathological conditions. Here we characterize the mechanical fingerprints at the micro-scale level of the three tissue layers which compose the healthy bladder wall, and identify modifications associated with the onset and progression of pathological conditions (i.e., actinic cystitis and bladder cancer). We use two indentation-based instruments (an Atomic Force Microscope and a nanoindenter) and compare the micromechanical maps with a comprehensive histological analysis. We find that the healthy bladder wall is a mechanically inhomogeneous tissue, with a gradient of increasing stiffness from the urothelium to the lamina propria, which gradually decreases when reaching the muscle outer layer. Stiffening in fibrotic tissues correlate with increased deposition of dense extracellular matrix in the lamina propria. An increase in tissue compliance is observed before the onset and invasion of the tumor. By providing high resolution micromechanical investigation of each tissue layer of the bladder, we depict the intrinsic mechanical heterogeneity of the layers of a healthy bladder as compared with the mechanical properties alterations associated with either actinic cystitis or bladder tumor.

Native extracellular matrix probes to target patient- and tissue- specific cell-microenvironment interactions by force spectroscopy

“…While laminin seems to be consistent in all the matrix, fibronectin is more present in the detrusor compared to the other two proteins. We furthermore observed different mechanical properties of the ECM regions (Figure S5), in agreement with the results recently reported for the three tissue layers of the rat bladder (i.e., urothelium, lamina propria and muscle layer) 17 . These assessments confirm that the different parts of the bladder ECM reproduced by Probe SM and Probe D exhibit significant distinctions in their compositional and biophysical features.…”

“…In this work, 20µm x 20µm, 10µm thick squares (Figure 1A) were cut in different regions of decellularised rat bladder: submucosal, detrusor and adventitia 17,40,41 .…”

“…For the proof-of-principle presented here, we selected two main bladder regions for the creation of the ECM probes: the submucosal region, closer to the lumen of the bladder, rich in connective tissue to support the attachment of the urothelium, a type of stratified epithelium made of transitional epithelial cells, and the detrusor region, a central layer of the bladder made of muscle fibres that allow contraction 40,41 , as shown in Figure 5A-C. The ECM probes, named after their corresponding region of origin (Probe SM, submucosal, and Probe D, detrusor) are interesting because, albeit deriving from adjacent areas of the same bladder tissue, they are characterised by major differences in the composition and mechanical properties 17,40 .…”

“…Different techniques are used to study these cell-ECM interactions, among which Atomic Force Microscopy (AFM) stands out due to its ability to both sense and apply forces at the nanoscale, with sub-nanonewton sensitivity [13][14][15][16][17] . In the context of biophysical investigations, many AFM-based force spectroscopy (FS) configurations can be found 18 : a single-cell probe approaching a substrate coated with proteins 19-24 ; an ECM-mimicking probe approaching an adherent cell 11,[25][26][27][28][29] ; a single-cell probe approaching an adherent cell 15,30,31 .…”

Native extracellular matrix probes to target patient- and tissue-specific cell–microenvironment interactions by force spectroscopy

“…1A and 3A-E) were cut in different regions of decellularised rat bladder: submucosal, detrusor and adventitia. 18,42,43 2.2. Characterisation of ECM and ECM probes LMD provides many advantages including the possibility to precisely select the ROI, cut ECM pieces with predefined, reproducible geometry and dimensions, and collect the samples without damaging them.…”