Detection of the Tendon Properties in Posterior Tibial Tendinopathy in Three-Dimensional Space Using High Resolution Multiphoton and Second Harmonic Generation Imaging

Abstract

Category: Hindfoot Introduction/Purpose: Posterior tibial tendon dysfunction is the most common cause of acquired flat foot deformity in adults, and results in significant morbidity. The exact etiology of this condition is still unknown. Posterior tibial tendon is predominately made up of fibrillar collagens and we hypothesize that their structural properties such as the collagen fiber density, orientation properties and cross-linking density essentially control the biomechanical properties of posterior tibial tendons. In this study, our aim is to visualize and quantitate fibrillar collagen distributions, their organization, cross-link densities and the fiber orientation (using Fourier analysis) in three distinct regions of pathologic posterior tibial tendon human samples namely proximal, middle and distal regions. Methods: After an institutional IRB approval we obtained diseased posterior tibial tendon total excision samples from 10 patients that had reconstructive procedures for posterior tibial tendinopathy at our institution and two fresh non-frozen healthy posterior tibial tendon samples from cadavers were used as the control. This research mainly utilized well established multiphoton microscopy and harmonic generation methodologies which compared proximal, middle and distal 1/3 of the tendon samples; multiphoton microscopy provides a powerful imaging method for evaluation of remodeling of fibrillar collagen structures deep within tissues. Ultra-short IR laser pulses served as an excitation source to produce multiphoton excitation fluorescence (MPEF) from endogenously fluorescent macromolecular systems and to induce highly specific second harmonic generation (SHG) signals from fibrillar collagens. Since MPEF and SHG involve different principles and contrast mechanisms, they were captured simultaneously to provide spectrally and spatially resolved, quantitative imaging of complex structures. Results: We systematically examined the nature of fibrillar collagen remodeling in relatively thick posterior tibial tendon tissues. Representative 3D forward SHG (FSHG), backward SHG (BSHG) and auto-fluorescence (MPEF) images originating from fibrillar collagen matrix from relatively thick (˜50 microns) posterior tibial tendon sections of three different regions namely proximal, middle and distal regions are shown in Figure 1. Computed Orientation Index values obtained from Fourier analysis show statistically significant differences particularly between proximal and middle regions (decreased orientation of collagen fibers in the middle region, p<0.0001). The cross-linking densities determined from the ratio of auto-fluorescence (MPEF) to BSHG again show differences particularly between proximal and middle regions (increased cross-linking in the middle region, p<0.01). Conclusion: The multiphoton and harmonic generation microscopy can be a powerful high resolution imaging method requiring minimal sample preparation that can provide structural information about spatially and spectrally resolved fibrillar collagens in three different posterior tibial tendon tendon regions. We found that middle third of the pathologic tendons show increased cross-linking and decreased orientation ratio of the collagen fibers which can provide insight into the regional based collagen remodeling and improved understanding of earliest stages of tendon destruction that could lead to improvements in the treatment of this condition.