Understanding sub-cellular mechanochemical correlations of the neonatal extrahepatic bile duct extracellular matrix...

The neonatal extrahepatic bile duct (EHBD) is highly susceptible to injury and fibrotic remodeling, yet the sub-cellular mechanochemical basis of this vulnerability remains poorly understood. Atomic force microscopy coupled with infrared spectroscopy (AFM-IR) enables simultaneous mapping of stiffness and molecular composition at sub-micron resolution, providing new insight into extracellular matrix (ECM) heterogeneity. Here, we investigate how spatial variation in ECM structure and mechanics differs between neonatal and adult rat EHBDs to determine the mechanochemical features that may underlie neonatal susceptibility to injury.

AFM-IR spectral and nanomechanical mapping revealed that neonatal ECM is compositionally and mechanically heterogeneous, characterized by spatial gradients in stiffness and molecular composition across the duct wall. Spectral analysis revealed pronounced regional heterogeneity in neonatal ECM: lumican and hyaluronic acid (HA) were enriched in the outer submucosa near the duct wall, whereas non-fibromodulin (FMOD) matrix proteins predominated in the inner submucosa adjacent to the lumen. In contrast, adult EHBDs exhibited a more uniform and collagen-rich ECM with higher and more consistent stiffness. Principal component analysis (PCA) of AFM-IR spectra distinguished neonatal and adult ducts based on amide, carbonyl, and side-chain IR features, suggesting differences in ECM composition and organization. Nanomechanical mapping showed that neonatal ECM was significantly softer and more heterogeneous, with the lowest apparent Young's modulus near the duct edge. Random forest regression linked local stiffness to distinct chemical signatures, amide III features in neonatal inner submucosa and amide I, II, and carbonyl vibrations in the outer wall.

Neonatal EHBDs possess immature and spatially heterogeneous ECM organization that compromises mechanical integrity and may predispose the duct to cholangiopathies such as biliary atresia. This work establishes AFM-IR as a powerful mechanochemical tool for linking ECM composition to tissue vulnerability and remodeling in developing soft tissues.