Fig 1.
Bovine BCU explants and small angle X-ray scattering (SAXS) data analysis.
(A) Schematic of joint structure showing the collagen fibril orientation from parallel to the articulating surface in the superficial zone, to vertical in the deep zone and calcified plate. Dashed arrows indicate, schematically, the directions of shear (horizontal) and compressive (vertical) stresses. Abbreviations for region classification: SZ: superficial zone; TZ: transitional zone; DZ: deep zone: CP: calcified plate, TB: trabecular bone (B) Fibrillar microstructure in AC, CP, and TB zones. Type II collagen fibrils are found in AC and calcified cartilage (CC), with mineral particles as well in CC, whereas type I collagen fibrils are found in SCB and TB. (C) Bone-cartilage unit (BCU) cores of 5mm length and 2mm diameter extracted from bovine metacarpophalangeal joints and placed in cassette holder for SAXS scanning while kept hydrated. (D) 2D SAXS patterns from AC, CP, and TB. Low diffuse SAXS with clear peaks and oriented ellipse in AC, high diffuse SAXS with average peaks and oriented ellipse in CP and high diffuse SAXS with average peaks and no orientation for ellipse in TB. (E) Azimuthal and radial integration to get the I(q) and I(χ) profiles to infer nano structural parameters (D-period, fibrillar orientation and degree of alignment). (F) Fibril schematic: Collagen D-period, characterised by the repeating gap-overlap regions causes by the staggered fibril stacking and fibrillar orientation. (G) I(q) plot, showing peaks due to collagen D-period, particularly clear at the 3rd and 5th order after azimuthally integration (0–360°) around the diffraction pattern for the q-radial range 0.1–1.2nm-1. Right top plot is 3rd order collagen peak after background subtraction. (H) I(χ) profiles after corrected intensity with Gaussian model fitting to the peak. Gaussian fitting was used to infer parameters (fibril orientation and ρ).
Fig 2.
Variation of fibril pre-strain and ultrastructural SAXS measures across the BCU.
Colour plots of 2D SAXS scan on a bone-cartilage unit (BCU) (A) Region, with the acronyms following the region classification in Fig 1A, (B) Fibrillar D-period (nm), (C) Total SAXS intensity (a.u.) and (D) Total diffraction intensity from the background-corrected meridional collagen peak intensity (a.u.). The plots show the variation in these nano structural parameters across the BCU.
Fig 3.
Fibril orientation and alignment across the BCU.
Colour map (for the sample in Fig 2) for collagen fibril orientation (degrees) and ρ (a.u.) parameter, where the length of the white line and the colour scale corresponds to ρ, and the direction of the white line indicates the fibrillar orientation across BCU. There is a clear increase in the degree of fibrillar alignment at the bone-cartilage interface, with the fibrillar orientation perpendicular to articulating surface. However, there is random orientation with lower degree of alignment in TB.
Fig 4.
Significant difference in fibrillar pre-strain, alignment, and orientation across the BCU.
Bar plots of the (A) fibril D-period (pre-strain), (B) degree of alignment (ρ) and (C) fibril orientation direction. The SAXS ultrastructural measures are calculated on a per-zone basis for each sample (small symbols, grey outline), which are averaged across the regular scan (n = 6) samples and shown as white squares; error bars: standard deviation across samples.
Fig 5.
Variation of SAXS intensity profiles across zones.
(A) I(q) plot, showing 3rd order and 5th order peaks due to collagen D-period, after azimuthally integration around the diffraction pattern for the q-radial range 0.2–0.6nm-1. (Red: SZ, Green: TZ, Blue: DZ, Cyan: CP and Magenta: TB). (B) The 3rd order collagen peaks across the BCU after the diffuse SAXS background subtraction.
Fig 6.
High spatial resolution map of fibril pre-strain and ultrastructural SAXS measures at the calcified/uncalcified boundary in the BCU.
(A) D-period, (B) SAXS total intensity and (C) SAXS collagen intensity. The uncalcified articular cartilage is at the top of the image (blue shaded region near the ordinate value of 0.0mm for B); the calcified plate (CP) is from ordinal values of 0.0–0.2mm (yellow-red regions in B), and the regions from 0.2mm and below are the trabecular bone (TB) region. In A), the D-period shows a lower average value near the interface. SAXS collagen intensity is higher toward the interface.
Fig 7.
High spatial resolution map of fibril degree of alignment and orientation at the calcified/uncalcified boundary in the BCU.
(Same sample as in Fig 6) (A) degree of alignment ρ and (B) collagen fibril orientation. From A) the tissue shows a high degree of fibrillar alignment when transitioning from the deep zone of articular cartilage to the calcified plate, and in B) we see fibres predominantly orientated in the angle of 90° (green colour), perpendicular to the articulating surface. In the lower part of the calcified plate (dashed rectangle; ordinal values from 0.15mm to 0.3mm) the ρ values are lower than in the upper part; it is suggested this is subchondral bone as opposed to calcified cartilage. From 0.3 mm downward, the degree of orientation is much lower, and orientation is more random, characteristic of trabecular bone.
Fig 8.
Variation of fibril D-period, degree of orientation and direction of orientation across the bone-cartilage interface.
1D line plots, averaged in the horizontal direction, for the high resolution 2D scans in Figs 6 and 7. (A) The mean D-period (B) mean ρ and (C) mean orientation, averaged for each y-coordinate, displayed with error bars representing standard error of the mean. Horizontal (abscissa)-axis is in microns. Interface between calcified and uncalcified cartilage is on the left, indicated by bone-cartilage interface with left-arrow. In A), it is noted that D-period rises from a local minimum toward the interface between the calcified plate and articular cartilage (on the left); it stabilises at the first vertical dashed line ~100 μm to the right of the interface. The right two vertical dashed lines indicate a proposed demarcation of the calcified plate (CP) into calcified cartilage (CC) and subchondral bone (SCB), at the same points as the dashed rectangle in Fig 7A.
Fig 9.
Schematic overview and X-ray fibrillar model.
(A) Correlation between nanoscale fibrillar pre-strain and microscale residual compressive strain. Left: schematic of fibrillar pre-strain gradients (this work; Figs 2, 3 and 6) across the AC, CP, and TB in the bovine MCP joint. Right: From independent work on the medial condyle of bovine tibiae [49], residual compressive strain (after loading) visualised using microcomputed tomography (CT) digital volume correlation (DVC). The calcified plate (both CC and SCB) are seen as regions with both high fibrillar tensile pre-strain (in unloaded state) and high compressive strain (energy storage) after loading. (B) Fibre-diffraction simulations showing how increasing fibril radius affects the skewness of the peak and estimated D-period. Left: schematic of meridional SAXS ellipsoidal peaks from a single fibril for different ratios of parallel to axial peak widths wp/wa. Blue: wp/wa = 1; red: wp/wa = 5; green: wp/wa = 10. Middle: Simulated I(q) plots for 3rd order meridional peak for these ratios (with D-period kept constant), showing increasing skewness as wp/wa increases, along with a slight rightward peak shift. Right: effect of increasing wp/wa from 1 to 10 on (top): D-period calculated by moments method and (bottom) peak skewness, showing that the moment method underestimates the D-period as fibril radius decreases (1→2→3).