Echinoderm connective tissue as a dynamic mechanically responsive biomaterial: insights from synchrotron small-angle X-ray scattering with in situ tensile testing

摘要

Introduction: Mutable collagenous tissue (MCT) of echinoderms is a remarkable example of a biological material which has the unique property of being able to change its stiffness and extensibility within an extremely short time scale'11 via neural mechanisms. The precise mechanisms are not well understood at the nanoscale, although they are believed to involve noncollagenous proteins such as tensilin and stiparin. Using synchrotron small-angle X-ray scattering to probe fibrillar structure during in situ tensile testing of MCT, we investigate the fibrillar mechanics of chemically-induced stiffened and softened sea cucumber dermis. Materials and Methods: Body-wall tissue from the black sea cucumber (Holothuria leucospilota) was dissected, retaining the collagenous part, for tensile testing combined with X-ray scattering. To induce the normal, stiff and soft state in the tissue, samples were incubated in artificial seawater (ASW), high potassium [K+] artificial seawater (KASW), and calcium-free artificial seawater (CaF-ASW). Samples were tested in a custom-built tensile tester installed in the ID02 beamline (European Synchrotron Radiation Facility, Grenoble, France). Simultaneous with tensile testing of the sea-cucumber tissue to failure, SAXS images were acquired (wavelength 0.995 A) with a FReLoN detector every 0.5-seconds. The fibril D-period was obtained by fitting the 5th order meridional peak in the radial SAXS intensity profile I(q). Fibril strain Ef was measured from percentage shifts in D-period from its unstressed value. The direction and degree of fibril orientation was obtained from the angular SAXS intensity profile of the 5th order meridional peak. Results and Discussion: Macroscopically, CaF-ASW treated samples had -4 times lower maximum stress compared to ASW while KASW had ～4 times higher. Concurrently, the maximum fibril strain developed in each state is highly different, with the fibrils in CaF-ASW having much lower extension (～0.16%) compared to the ASW (～0.60%), and the KASW much higher (～1.0%). When taken together with the larger absolute tissue strain in CaF-ASW, we see that the ε_F/ε_T is much smaller in the softened state. Furthermore, fibrils undergo a pronounced reorientation into the loading direction, characterized by a narrowing of the width of the I(X) profile and increase in peak intensity. The rate of reorientation is much slower for the CaF-ASW softened tissue. Conclusion: Insight into how the altered fibrillar mechanics observed can explain mutability can be obtained by considering MCT at the nanoscale to consist of collagen fibrils separated by a hydrated interfibrillar matrix of proteoglycans and noncollagenous proteins. Tensile strains in fibrils developed by shear-transfer in the interfibrillar matrix. The altered maximum fibril strains in KASW and CaF-ASW treated MCT suggest that the main mechanism for mechanical mutability in MCT is an alteration of the degree of crosslinking and shear stiffness in the interfibrillar matrix rather than a change in the properties of the collagen fibrils themselves. Our results will help in understanding the structure-function relations of MCT, and can serve as a source for designing new dynamic biomaterials, which would have potential applications in biomedical engineering.