Improved specificity of MRI diagnosis of collagenous lesions in tendon

Comprehension of the molecular model of Collagen Type I hydration is essential for understanding the magnetic resonance imaging signal and contrast mechanisms observed in collagenous tissues. This dissertation is composed of four chapters.;Chapter one reviews biophysical studies of the tendon/collagen structure, with the purpose of understanding the molecular origin of the orientational variation in MRI signals from tendon, which is referred to as the "magic angle" effect. Briefly, the irreducible separation of charges on the collagen molecule main chain, held apart by steric restrictions of protein folding, results in electrostatic energy that is reduced by water molecules serving as dielectric molecules. These highly immobilized water molecules and the adjacent hydrogen bonded water network are confined to the transverse plane of the tendon. Orientation restriction causes residual dipole coupling, which is directly responsible for the frequency and phase shifts described by the magic angle effect. The hydration compartments of collagen are detailed in reference to multiple biophysical methods. Implications of the magic angle effect in clinical MRI are also reviewed.;In chapter two, a new model of protein melting is proposed to provide understanding of factors controlling protein folding. Differential scanning calorimetry studies of tendon (collagen) as a function of hydration show that thermal melting of proteins results from melting of single and double water bridges, absorbed on the collagen protein backbone to reduce electrostatic energy caused by irreducible separation of partial electric charges. A molecular model of protein hydration is proposed, which includes one single water bridge and one double water bridge (consisting of three water molecules) per collagen tripeptide unit. Detailed analysis of the melting thermodynamics shows that, of the total collagen enthalpy of melting DeltaH =70 J/g-protein, 69.9% originates from double water bridges, 24.0% from single water bridges and only 6.9% from collagen itself. The entropy of melting is even more dominated by the influence of water bridges. Of the total DeltaS = 0.206 J/g-protein°K, 63% originates from double water bridges, 21% from single water bridges, 11.9% from bridge associated dielectric water clusters, and only 3.6% from collagen itself.;Understanding the molecular model of collagen hydration allows interpretation of imaging experiments in the two following chapters, where disruption of collagen structure was achieved in-vitro using two different mechanisms, allowing detailed evaluation of contrast mechanisms of tendon MRI and the magic angle effect. Chapter three is a systematic evaluation of the magic angle effect on the appearance of tendinosis lesions and non-injured tendon using standard MRI and ultra-short time-to-echo (UTE) sequences. An in-vitro model of tendinosis was produced by injecting activated collagenase enzyme in bovine tendons. Controls were injected with heat denatured collagenase or buffer solutions. MRI characteristics of healthy tendon that are absent within tendon lesions are confirmed: (1) low signal in traditional MRI sequences, (2) magic angle effect, and (3) high signal on ultrashort-T2-component imaging. These criteria may help in establishing presence of a lesion in difficult clinical cases (i.e. due to magic angle effect). Quantitative analysis was performed on the appearance of tendon lesions (SNR and CNR) on different MRI sequences, with and without the magic angle orientation, as well as on the visualized lesion volume, transverse and longitudinal size.;Chapter four shows the feasibility of detecting glycation of collagen in tendon using MRI. In-vitro glycation of bovine collagen was achieved by incubation in phosphate-buffered-saline with 0.2 Mole/L glucose. Controls were incubated without glucose. Gradual increase of MRI signal and loss of the magic angle effect was seen on glycated samples but not in controls (Two-way ANOVA p<0.0001). Clinical studies are planned to test detection of advanced glycation end-products in diabetic patients.