Micro-mechanical properties of the tendon-to-bone attachment

Abstract

The tendon-to-bone attachment (enthesis) is a complex hierarchical tissue that connects stiff bone to compliant tendon. The attachment site at the micrometer scale exhibits gradients in mineral content and collagen orientation, which likely act to minimize stress concentrations. The physiological micromechanics of the attachment thus define resultant performance, but difficulties in sample preparation and mechanical testing at this scale have restricted understanding of structure-mechanical function. Here, microscale beams from entheses of wild type mice and mice with mineral defects were prepared using cryo-focused ion beam milling and pulled to failure using a modified atomic force microscopy system. Micromechanical behavior of tendon-to-bone structures, including elastic modulus, strength, resilience, and toughness, were obtained. Results demonstrated considerably higher mechanical performance at the micrometer length scale compared to the millimeter tissue length scale, describing enthesis material properties without the influence of higher order structural effects such as defects. Micromechanical investigation revealed a decrease in strength in entheses with mineral defects. To further examine structure-mechanical function relationships, local deformation behavior along the tendon-to-bone attachment was determined using local image correlation. A high compliance zone near the mineralized gradient of the attachment was clearly identified and highlighted the lack of correlation between mineral distribution and strain on the low-mineral end of the attachment. This compliant region is proposed to act as an energy absorbing component, limiting catastrophic failure within the tendon-to-bone attachment through higher local deformation. This understanding of tendon-to-bone micromechanics demonstrates the critical role of micrometer scale features in the mechanics of the tissue.

Statement of significance: The tendon-to-bone attachment (enthesis) is a complex hierarchical tissue with features at a numerous scales that dissipate stress concentrations between compliant tendon and stiff bone. At the micrometer scale, the enthesis exhibits gradients in collagen and mineral composition and organization. However, the physiological mechanics of the enthesis at this scale remained unknown due to difficulty in preparing and testing micrometer scale samples. This study is the first to measure the tensile mechanical properties of the enthesis at the micrometer scale. Results demonstrated considerably enhanced mechanical performance at the micrometer length scale compared to the millimeter tissue length scale and identified a high-compliance zone near the mineralized gradient of the attachment. This understanding of tendon-to-bone micromechanics demonstrates the critical role of micrometer scale features in the mechanics of the tissue.

Keywords: AFM; Enthesis; Gradient; Image correlation; Micromechanics; Tensile testing.

Fig. 1

Murine tendon-to-bone attachment sites were cut and milled into beams measuring approximately 60 μm long and 4.5 by 4.5 μm in diameter. (A) Dissected supraspinatus-to-humeral head complexes were fresh frozen and sectioned into 20–30 μm thick slices. (B) LCM was used to cut large beams, ~250 μm by 50 μm by 20–30 μm, in the fibrocartilaginous region of the attachment where there is a gradient in mineralization. (C–E) The LCM cut beams were further milled down to the final small beams via cryo-FIB.

Fig. 2

Micrometer scale beams of attachment tissue exhibiting a gradient in mineral content were attached to a modified AFM system. The mineralized end of the beam was attached to a motorized stage. The unmineralized end of the beam was attached to a blunted AFM tip with electron beam radiation-cured glue. The AFM tip was then retracted to apply a tensile load on the beam to failure.

Fig. 3

A beam bending model was developed to determine the effect of changes in composition on the beam modulus. The beam was modelled as a three phase composite in series. The modulus of the gradient region is defined as a linear extrapolation between the mineralized and unmineralized regions (E_G(x)). Values for the mineralized (E_M) and unmineralized (E_U) moduli were optimized to minimize error between the calculated and experimental beam moduli.

Fig. 4

The length and composition of the beams varied between beams. (A) Plot of calcium content as a function of position along the beams, with zero set as the center of the gradient region. The length of the beams ranged from 41 to 99 μm. (B) The length of the mineralized, unmineralized, and graded regions were determined from the plot in A and are shown normalized to beam length. Dark shades represent mineralized, medium shades represent graded, and light shades represent unmineralized. These variations in the amount of each phase between beams are expected to have mechanical consequences.

Fig. 5

(A) Stress vs. strain curves for the 11 micromechanical tests. There was no apparent difference between the WT and KO samples in terms of curve shape. Plots of elastic modulus (B), strength (C), toughness and resilience (D), and failure strain (E) are shown for the WT and KO beams. Strength was significantly decreased with in KO samples compared to WT samples. There was no significant change in modulus, toughness, resilience, or failure strain between the WT and KO samples.

Fig. 6

Comparison of the experimental and modelled moduli for each beam. The error was small for all beams, with an average of 14.6%. The errors were not significantly different for the WT and KO beams, suggesting that the mineral defect had little effect on the micromechanics of the tissue.

Fig. 7

Plots of local strain vs. position at multiple stresses (legend) for all of the samples for which local strains were measured. Dotted lines represent the calcium content as a function of position for each sample. The highest strain levels were not localized to the region closest to the tendon but instead within the beam near the gradient region. This indicates the presence of a region of high deformation within the enthesis.