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

Abstract

We applied Raman spectroscopy to monitor the distribution of minerals and the degree of mineralization across the tendon-bone insertion site in the shoulders of five rats. We acquired Raman spectra from 100 to 4000 Δcm -1 on individual 1 μm points across the 120 μm 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 Δcm -1 P-O stretch for apatite (normalized to either the 2940 Δcm -1 C-H stretch or the 1003 Δcm -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-tocollagen ratio at the insertion site increases linearly (R 2 = 0.8 for five samples) over the distance of 120 μm from tendon to bone, rather than abruptly, as previously inferred from histological observations. In addition, narrowing of the 960 Δcm -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 stress concentrations 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.

Raman Assessment of Bone Quality

“…The mineral-to-matrix ratios across the insertion site increased linearly (R 2 = 0.8 for five specimens) over a distance of 120 lm from tendon to bone rather than abruptly as previously inferred from histologic observations [8]. Moreover, narrowing of the 960 cm À1 peak bandwidth indicated that the crystalline ordering within the apatite increases concomitantly with the degree of mineralization [79]. This finding of mineral gradation may explain why uninjured tendon-to-bone connection of the rotator cuff can sustain very high loads.…”

“…Injuries to the rotator cuff tendons of the shoulder are particularly problematic, because they heal poorly even after surgical intervention. In this preliminary study, the insertion sites of five rats were monitored using the relative intensity of the phosphate band at 960 cm À1 to either the 2490 cm À1 or 1003 cm À1 collagen band [79]. The mineral-to-matrix ratios across the insertion site increased linearly (R 2 = 0.8 for five specimens) over a distance of 120 lm from tendon to bone rather than abruptly as previously inferred from histologic observations [8].…”

“…1), most of the bands of a bone Raman spectrum can be assigned to mineral phosphate, carbonate, or matrix collagen. However, absolute band intensities are seldom used in Raman spectroscopy because they are affected by Raman scattering efficiency and other optical effects such as grain size, refractive index, and surface roughness of the specimen [79]. For these reasons, many studies report the relative peak intensities or peak areas (ie, integrated peak intensities) of select pairs of bands from the Raman spectrum.…”

“…The unique ability of Raman spectroscopy to detect spatial changes in mineral composition lends itself to the study of mineral distributions across tendon-to-bone insertion sites of the rotator cuff [79]. Injuries to the rotator cuff tendons of the shoulder are particularly problematic, because they heal poorly even after surgical intervention.…”

Allometry of the Tendon Enthesis: Mechanisms of Load Transfer Between Tendon and Bone

“…The proper scaling of these findings from small animals, however, to the attachments of larger animals such as humans is unknown. When designing scaffolds for human tendonto-bone repair, for example, does the $20 lm long mineral gradient found in the rat tendon-to-bone attachment [9] translate to $120 lm in humans, whose mass is $250 times that of rats? Allometry provides a way to study the relationship between the shapes and sizes of stress-mitigating structures at the interface and increasing animal mass and applied loads [10].…”