Mineral distributions at the developing tendon enthesis.

Tendon attaches to bone across a functionally graded interface, "the enthesis". A gradient of mineral content is believed to play an important role for dissipation of stress concentrations at mature fibrocartilaginous interfaces. Surgical repair of injured tendon to bone often fails, suggesting that the enthesis does not regenerate in a healing setting. Understanding the development and the micro/nano-meter structure of this unique interface may provide novel insights for the improvement of repair strategies. This study monitored the development of transitional tissue at the murine supraspinatus tendon enthesis, which begins postnatally and is completed by postnatal day 28. The micrometer-scale distribution of mineral across the developing enthesis was studied by X-ray micro-computed tomography and Raman microprobe spectroscopy. Analyzed regions were identified and further studied by histomorphometry. The nanometer-scale distribution of mineral and collagen fibrils at the developing interface was studied using transmission electron microscopy (TEM). A zone (∼20 µm) exhibiting a gradient in mineral relative to collagen was detected at the leading edge of the hard-soft tissue interface as early as postnatal day 7. Nanocharacterization by TEM suggested that this mineral gradient arose from intrinsic surface roughness on the scale of tens of nanometers at the mineralized front. Microcomputed tomography measurements indicated increases in bone mineral density with time. Raman spectroscopy measurements revealed that the mineral-to-collagen ratio on the mineralized side of the interface was constant throughout postnatal development. An increase in the carbonate concentration of the apatite mineral phase over time suggested possible matrix remodeling during postnatal development. Comparison of Raman-based observations of localized mineral content with histomorphological features indicated that development of the graded mineralized interface is linked to endochondral bone formation near the tendon insertion. These conserved and time-varying aspects of interface composition may have important implications for the growth and mechanical stability of the tendon-to-bone attachment throughout development

Raman microprobe analysis of developing supraspinatus tendon insertions.

<p>Top row (A–C) shows a P7 insertion, middle row (D–F) a P14 insertion, and bottom row (G–I) a P28 insertion. Left column, A, D, G: 20 µm-thick sections stained with toluidine blue and according to von Kossa's method (scale bar = 50 µm). Note that despite the sharp front of mineralization in these figures suggested by the von Kossa staining, a graded mineralization from is evident from the Raman scans. Middle column B, E, H: magnified view of square region of interest shown in images in the left column (scale bar = 10 µm). The relative mineral concentration determined by the ratio of the heights of the 960 Δcm⁻¹ to 1003 Δcm⁻¹ Raman peaks (corresponding to the ν1 P-O stretching band of hydroxylapatite and the aromatic ring stretching band of phenylalanine in collagen, respectively) is indicated by the color gradient of the overlaid points. SS: supraspinatus tendon and H: humeral head. Right column C, F, I: baseline-corrected Raman spectra corresponding to points along a traverse from tendon (no mineral - dark blue) to bone (high mineral - red). The mineral peak (960 Δcm⁻¹) is indicated with a hollow arrow and the collagen peak (1003 Δcm⁻¹) is indicated with a black-filled arrow.</p

Mineralized interface observed at the supraspinatus tendon-to-bone insertion 56 days after birth.

<p>The interface is not smooth and planar but rather exhibits significant “surface roughness” at the length scale of tens of nanometers. A) STEM- high angle annular dark field (HAADF) image. Brighter STEM-HAADF image intensity in regions of uniform thickness corresponds to higher mean atomic number (i.e., locations of mineral relative to collagen). B) STEM-electron energy loss spectroscopy (EELS) spectral image map of C (in atomic (at) % shown by green intensity) as well as summed Ca, O, and P (shown in at% shown by red intensity). Brighter color indicates higher concentrations. STEM-EELS mapped elemental composition (in at%) for C) carbon, D) calcium, E) oxygen, and F) phosphorous. While all tissues will contain calcium, oxygen, and phosphorus to varying amounts, the major sources of these signals are spatially well correlated to one another as well as correlated to the STEM-HAADF intensity and are indicative of apatie. The gray-scale ranges for the STEM-EELS maps are not normalized between 0 at% and 100 at% but rather have been independently adjusted to enhance visual clarity (consequently they are not directly comparable).</p