Hierarchical Detection and Assessment of Material Fatigue Damage of the Human Anterior Cruciate Ligament caused by Repetitive Sub-maximal Mechanical Loading

Abstract

Chapter 1 begins by describing the anatomy and function of the anterior cruciate ligament (ACL) followed by the significant health and economic burdens incurred by ACL injuries. Then the shortcomings of current diagnostic and treatment methods are reported, highlighting the need for higher sensitivity in diagnostic methods and the unresolved problem of re-injuries. The material fatigue hypothesis postulates that modulation of magnitude or frequency of loading patterns can alter the fatigue failure life of tissue. The structural and compositional heterogeneity of ACL demands high spatial resolution imaging techniques to capture changes in collagen structure at each hierarchical levels. Therefore, atomic force microscopy – infrared spectroscopy (AFM-IR) and second harmonic generation imaging (SHIM) are introduced as structural probes. In chapter 2, the fatigue damage signatures of ACL from a loaded cadaver are characterized at each hierarchical level of collagen organization. At the molecular level, the denaturation of collagen molecules were detected by the spectroscopic peak 1740 cm-1 with AFM-IR. This finding was validated by increased binding of collagen hybridizing peptide (CHP) – TAMRA dye in loaded cadavers’ ACL. Further structural degradation was seen as a form of fibril voids, and reduction in topographical thickness of collagen fibrils detected by the AFM. Lastly, a reduction in SHG signal intensity and reduction in fiber alignment suggest that the reduction in structural integrity from fatigue are at least in part responsible for leading to noncontact ACL failures. Similar damage signatures were found in patients who sustained an ACL injury. In chapter 3, collagen’s intrinsic autofluorescence (AF) and second harmonic generation (SHG) signals are used as optical probes to detect accumulation of fatigue damage. A decrease in SHG signal, increase in AF signal intensity with increased anisotropy of collagen fiber orientations in loaded cadavers were found to be consistent with fatigue damage signatures from chapter 2. The feasibility of using a confocal laser endomicroscope (CLE) to detect progression of fatigue damage is explored. Evaluation of damage progression by AF signal detection utilizing CLE showed that a larger signal change may indicate larger plastic deformation to the tissue. Additionally, BMI category and posterior lateral tibial slope (PTS) may influence the AF signal due to influence on fiber structure from load distribution. In chapter 4, a detailed spectroscopic analysis of the 1740 cm-1 IR peak was carried out to investigate the conformation change in collagen as a result of fatigue. Examination of IR frequencies in the Amide I, Amide III region, and wavenumber associated with tyrosine residue increased along with the increase in 1740 cm-1 peak intensity. The conformation changes describe disruption in the hydrogen bonding network within the helix to increased water mediated hydrogen bonds from the environment, and a disruption in the hydrophobic interactions at the C-telopeptides attributed to tyrosine. In chapter 5, the results of hierarchical fatigue damage signatures (Ch.2), collagen autofluorescence detection with CLE during progression of fatigue damage (Ch.3) and spectroscopic analysis of 1740 cm-1 (Ch.4) are summarized. Future works include three projects expanding on characterization of the fatigue damage signatures: i) AFM-IR detection of local stiffness and composition changes in the ACL as a result of material fatigue ii) O-PTIR (optical photothermal infrared spectroscopy) detection of changes in proteoglycans levels in in vivo fatigue tested mouse ACL and iii) Autofluorescence detection by CLE as a sign of fatigue damage progression.