Quantifying the Effect of Intrinsic Aging on Skin Microstructure and Mechanical Function Using Multiphoton Microscopy

Over the next couple of decades, the number of elderly individuals is expected to double, bringing increased healthcare spending due to seemingly minor injuries to skin. In skin, the 3D dermal collagen fiber network, which is the primary load-bearing structure, undergoes changes in organization and composition due to intrinsic aging. However, the relationships between altered microstructure and mechanical function is not well understood. Quantitative imaging techniques have been used in the past to link skin structure to mechanical function, but previous analysis has been limited to 2D assessments. Multiphoton microscopy (MPM) is a non-destructive imaging technique with intrinsic depth-sectioning capabilities, making it well-suited to quantify the 3D organization of the collagen fiber network via second harmonic generation (SHG). The goal of this dissertation was to combine multiphoton microscopy with mechanical testing of mouse skin to better understand the relationship between microstructure and mechanical function. Using young and aged mouse skin, the micro-scale fiber kinematics and tissue kinematics were measured and related to mechanical function. The skin underwent significant realignment in the direction of loading and volume loss at increasing magnitudes of strain during tensile loading. The collagen fibers within the dermis of young mouse skin were found to act like a highly connected 3D network, rather than rotating independently during mechanical loading. Aged mouse skin had an increased amount of non-enzymatic crosslink autofluorescence present in the dermal collagen, which likely resulted in less organization within the collagen fiber network and a reduced ability to recruit collagen fibers into a stress-bearing role. Finally, a notched tissue model was used to observe the tensile failure mechanics of young and aged mouse skin. The fiber kinematics of young tissue indicated an ability of fibers to realign and create a stress shield at the tip of the notch. Although young and aged skin displayed similar average levels of fiber realignment throughout the skin during tensile loading, aged skin realigned significantly less at the tip of the notch. Additionally, the strain threshold for failure of the epidermis and the dermis were measured based on the occurrence of visible rupture and indicated that epidermal failure occurs at strains far lower than dermal failure and complete tissue rupture. This work provides valuable insights on how the structure of skin is related to its biomechanical function, and aids in understanding how skin biomechanical properties change with age