Impact of Collagen Crosslinking on Dislocated Human Shoulder Capsules-Effect on Structural and Mechanical Properties

Abstract

Classical treatments of shoulder instability are associated with recurrence. To determine whether the modification of the capsule properties may be an alternative procedure, the effect of crosslinking treatment on the structure and mechanical properties of diseased human shoulder capsules was investigated. Joint capsules harvested from patients during shoulder surgery (n = 5) were treated or not with UV and/or riboflavin (0.1%, 1.0% and 2.5%). The structure and the mechanical properties of the capsules were determined by atomic force microscopy. The effect of treatments on cell death was investigated. Collagen fibrils were well-aligned and adjacent to each other with a D-periodicity of 66.9 ± 3.2 nm and a diameter of 71.8 ± 15.4 nm in control untreated capsules. No effect of treatments was observed on the organization of the collagen fibrils nor on their intrinsic characteristics, including D-periodicity or their mean diameter. The treatments also did not induce cell death. In contrast, UV + 2.5% riboflavin induced capsule stiffness, as revealed by the increased Young's modulus values (p < 0.0001 for each patient). Our results showed that the crosslinking procedure changed the biomechanics of diseased capsules, while keeping their structural organisation unchanged at the single fibril level. The UV/riboflavin crosslinking procedure may be a promising way to preserve the functions of collagen-based tissues and tune their elasticity for clinically relevant treatments.

Keywords: atomic force microscopy; shoulder instability; type I collagen fibrils.

Figure 1

Experimental design. (A) Tissue sampling. (B) Tissue treatment: Riboflavin treatment (R_H: 2.5%, R_M: 1.0%, R_L: 0.1%) with and without UV radiation, O.C.T. inclusion and subsequent cryosectioning. (C) Tissue characterizations: Fluorescent (C₁) for the detection of cell apoptosis and atomic force microscopy (C₂) for probing structural and mechanical properties.

Figure 2

Atomic force microscopy analysis of human capsule elasticity. (A) A schematic representation of the experimental set-up. (B) Representative force versus displacement curve (the approach part) showing two different behaviours: the probe pushing on a hard surface (dashed line, label B) or indenting in a soft surface (continuous line, label C) (C–F) Elastic modulus maps (2 µm × 2 µm) obtained for the joint capsule in the different conditions: control (C), UV treatment (D), 2.5% riboflavin treatment (R_H) (E) and 2.5% riboflavin + UV treatment (UV + R_H) (F). The different colours are representative of lower (blue/green) and higher (red/orange) values of the Young’s modulus. (G) Quantification of Young’s modulus obtained by AFM for the joint capsules in the four different conditions for one representative patient (box plots generated by pooling all of the data collected for at least three different elasticity maps per condition and for different regions of the tissue, *** p-value < 0.0001).

Figure 3

Increase tissue stiffness of human capsules by crosslinking treatment. Comparison of the quantification of Young’s modulus obtained by AFM for the joint capsules in the two different conditions (Control in black and 2.5% riboflavin + UV in red) and for five different patients (box plots generated by pooling all of the data collected for at least three different elasticity maps per condition and for different regions of the tissue, *** p-value < 0.0001).

Figure 4

Atomic force microscopy analysis of human capsule structure. Representative AFM (A,C) height and (B,D) deflection images (5 µm × 5 µm, contact mode) recorded on untreated joint capsule in (A,B) PBS buffer or in (C,D) air. (E–H) High-resolution images recorded in air of the joint capsule (E) prior to and after (F) 2.5% riboflavin (R_H), (G) UV or (H) 2.5% riboflavin + UV treatments (UV + R_H). (I) Scan line taken at the location indicated by the arrows and dashed line in the image (E). (J,K) Box plots generated by pooling all of the data as a function of the treatment showing the evolution of (J) D-period and (K) mean diameter of individual fibrils.

Figure 5

Absence of cell death induction by crosslinking treatment. Human articular capsules were treated or not (CTRL) by UV, 0.1% riboflavin (R_L), 2.5% riboflavin (R_H), UV + 0.1% riboflavin (UV + R_L) or UV + 2.5% riboflavin (UV + R_H) before TUNEL assay. No cell death was observed in the negative control, whereas many apoptotic cells (green) were present in the positive control. UV and riboflavin alone or in combination did not induce cell death. DAPI staining was used to check the location of the cell nuclei (blue).