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We applied Raman spectroscopy to monitor the distribution of mineral and the degree of mineralization across the tendon-bone insertion site in the shoulders of five rats. We acquired Raman spectra from 100 to 4,000 Deltacm(-1) on individual 1 microm points across the 120 microm wide transition zone of each tissue sample and identified all the peaks detected in pure tendon and in pure bone, as well as in the transition zone. The intensity of the 960 Deltacm(-1) P-O stretch for apatite (normalized to either the 2,940 Deltacm(-1) C-H stretch or the 1,003 Deltacm(-1) C-C stretch for collagen) was used as an indicator of the abundance of mineral. We relate the observed histological morphology in the tissue thin section with the observed Raman peaks for both the organic component (mostly collagen) and the inorganic component (a carbonated form of the mineral apatite) and discuss spectroscopic issues related to peak deconvolution and quantification of overlapping Raman peaks. We show that the mineral-to-collagen ratio at the insertion site increases linearly (R(2) = 0.8 for five samples) over the distance of 120 microm from tendon to bone, rather than abruptly, as previously inferred from histological observations. In addition, narrowing of the 960 Deltacm(-1) band across the traverse indicates that the crystalline ordering within the apatite increases concomitantly with the degree of mineralization. This finding of mineral gradation has important clinical implications and may explain why the uninjured tendon-to-bone connection of the rotator cuff can sustain very high loads without failure. Our finding is also consistent with recent mechanical models and calculations developed to better understand the materials properties of this unusually strong interface.

applied spectroscopy

The Tendon-to-Bone Transition of the Rotator Cuff: A Preliminary Raman Spectroscopic Study Documenting the Gradual Mineralization across the Insertion in Rat Tissue Samples