In SituMonitoring of Tendon Structural Changes by Elastic Scattering Spectroscopy: Correlation with Changes in Collagen Fibril Diameter and Crimp

Abstract

The aim of this study was to monitor structural changes in loaded rabbit digital flexor tendons in situ and ex situ via elastic scattering spectroscopy (ESS). The optical setup consisted of a xenon white light source (lambda = 320-860 nm), connected to a fiber optic probe (with a source-detector separation of approximately 350 microm) and a spectrometer, controlled by a personal computer (PC). Cadaveric rabbit tendons were studied in situ under 3 tensional regimens: unloaded (no extrinsic tension applied), stretched, and 1-kg loaded and compared with excised tendons (i.e., no tension). Four times more light was detected in in situ unloaded tendons perpendicular to the tendon long axis than parallel to it. Backscatter anisotropy was expressed as the anisotropy factor (AF600nm: ratio of greatest to least backscatter intensity, measured with orthogonal probe positions). Differences in backscatter anisotropy between tendons from different digits were not significant. AF600nm had the smallest value (2.72 +/- 0.38) for the least aligned tendon preparations (excised tendons), and increased to 7.17 +/- 0.54 (1-kg loaded) as in situ loads were applied. Electron microscopy revealed that the distribution of collagen fibril diameters changed as loads were applied, with the diameter of larger fibrils decreasing approximately 33% for 1-kg loaded compared with excised tendons. Polarized light microscopy showed a characteristic crimp pattern in excised tendons, but this was hardly detectable in unloaded tendons and not detectable in tendons fixed in situ under a 1-kg load. We propose that the increase in optical anisotropy is a function of collagen fibril straightening and reducing fibril diameter as the tendon undergoes progressive loading. These findings are important for monitoring structure in vivo and in bioreactors for tissue engineers.

Mechanical response of individual collagen fibrils in loaded tendon as measured by atomic force microscopy

“…The observed decrease in mean fibril diameter corroborates an earlier study indicating smaller diameter fibrils in loaded tendons (Morgan et al, 2006).…”

“…For instance, we hypothesized that a multi-modal distribution of collagen fibril strains would be observed, and that this could be further related as a function of fibril diameter. Numerous authors (including ourselves) have conjectured that the bimodal distribution of large and small diameter fibrils may, respectively, enable load and creep resistance (Battaglia et al, 2003;Derwin and Soslowsky, 1999;Morgan et al, 2006;Rigozzi et al, 2010;Robinson et al, 2004b). This hypothesis was not born out by our data, which indicated that the collagen network stretches in a very uniform manner, both locally and across various anatomic tendon subregions, without obvious differences in mechanical strains between neighboring fibrils or between fibrils of different diameters.…”

“…Scanning electron microscopy studies of tendon sections allow excellent visualization of the collagen structures, but quantitative measures of collagen fibril dimensions (and eventually mechanical collagen strains) is limited by perspective distortion. Transmission electron microscopy (TEM) studies are better suited for morphometric analysis, but due to difficulties in aligning the sectional plane with the longitudinal axis of collagen fibrils, have been used almost exclusively for morphometric analysis of collagen fibril cross-section (Morgan et al, 2006;Rigozzi et al, 2010). X-ray spectroscopy has been successfully used to infer collagen fibril strain in loaded tendon fibers (Fratzl et al, 1998;Gupta et al, 2010;Puxkandl et al, 2002), but visualization of individual fibrils is not possible, precluding direct investigation of collagen network deformation.…”

Experimental and theoretical evaluation of rotating orthogonal polarization imaging

“…It has been demonstrated to be capable of providing coarse optical sectioning, which can be useful in characterizing superficial tissue. [1][2][3][4] In addition, many tissue types are known to have polarization properties that affect the polarization state of light such as linear dichroism in sickled red blood cells, 5 linear birefringence in collagen, 6 and chiral rotation in glucose. 7 Noninvasively characterizing the polarization properties of tissues has potential for in vivo clinical applications, such as monitoring the healing state of wounds and burns.…”

Engineering Functional Collagen Scaffolds: Cyclical Loading Increases Material Strength and Fibril Aggregation

“…[5] Despite the importance of fibril diameter to the material properties of tissues, our basic understanding of its control is poor. Control of fibril diameter is distinct from fibrillogenesis, which is a thermodynamically driven aggregation following neutralisation of a native collagen solution.…”