Mechanical testing with in situ imaging offers new insight into normal physiological function and response to damaging loading.
Multiphoton microscopy (MPM) images collagen, elastin and other proteins with no exogenous staining permits image-based cell-level strain estimation. Ultra-small and wide-angle X-ray scattering (USAXS and WAXS) using the quasi-crystalline structure of proteins estimate in situ strain at molecular and fibrillar level. Collagen and elastin fibrils constraining swelling glycosaminoglycans is a constant structural motif for soft tissue so techniques and models developed for one tissue may be readily employed for multiple others.
Tendons resist millions of cycles at high load, however mechanobiological mechanisms of homeostasis and adaptation are poorly understood 5. MPM of fresh tendon samples showed the pericellular environment dominated by elastin motivating a finite element model that followed cell strain measurement well (Fig 1) 6 .Enzymatic removal of elastin confirmed the likely pericellular rather than macroscopic role of this protein 7. Mechanical overload that did not affect macroscopic stiffness still altered tendon stiffness at fibrillar (USAXS) and molecular (WAXS) levels, possibly localising damage to molecular cross links 8.
In peripheral nerves the microscopic mechanisms of damage from repetitive mechanical loading and trauma that can provoke functional deficit and pain are little understood. USAXS, WAXS and video extensometry showed similar collagen fibril recruitment and deformation mechanisms to tendons and highlighted the high relative stiffness of myelin sheaths protecting the axons from radial compression 9. In situ tensile loading with sodium ion channel staining and digital image correlation showed axons were also protected by uncrimping mechanisms in collagen fibrils and the axons themselves 10.
Bladder sustains large cyclic deformations and in situ uniaxial loading and MPM revealed widely differing mechanisms in the two tissue layers: low strain unfolding of the ruffled lamina propria then followed by rapid stiffening as two families of collagen fibrils in the detrusor engage at higher strains 11.
In both stiff and highly compliant tissues a mathematical analysis of collagen fibril recruitment probability density function (PDF) was supported by imaging enabling this PDF to be estimated from macroscopic stress-stretch characteristics 12.
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