Collagen pre-strain discontinuity at the bone—Cartilage interface

The bone-cartilage unit (BCU) is a universal feature in diarthrodial joints, which is mechanically-graded and subjected to shear and compressive strains. Changes in the BCU have been linked to osteoarthritis (OA) progression. Here we report existence of a physiological internal strain gradient (pre-strain) across the BCU at the ultrastructural scale of the extracellular matrix (ECM) constituents, specifically the collagen fibril. We use X-ray scattering that probes changes in the axial periodicity of fibril-level D-stagger of tropocollagen molecules in the matrix fibrils, as a measure of microscopic pre-strain. We find that mineralized collagen nanofibrils in the calcified plate are in tensile pre-strain relative to the underlying trabecular bone. This behaviour contrasts with the previously accepted notion that fibrillar pre-strain (or D-stagger) in collagenous tissues always reduces with mineralization, via reduced hydration and associated swelling pressure. Within the calcified part of the BCU, a finer-scale gradient in pre-strain (0.6% increase over ~50μm) is observed. The increased fibrillar pre-strain is linked to prior research reporting large tissue-level residual strains under compression. The findings may have biomechanical adaptative significance: higher in-built molecular level resilience/damage resistance to physiological compression, and disruption of the molecular-level pre-strains during remodelling of the bone-cartilage interface may be potential factors in osteoarthritis-based degeneration

Hes5 Expression in the Postnatal and Adult Mouse Inner Ear and the Drug-Damaged Cochlea

The Notch signaling pathway is known to have multiple roles during development of the inner ear. Notch signaling activates transcription of Hes5, a homologue of Drosophila hairy and enhancer of split, which encodes a basic helix-loop-helix transcriptional repressor. Previous studies have shown that Hes5 is expressed in the cochlea during embryonic development, and loss of Hes5 leads to overproduction of auditory and vestibular hair cells. However, due to technical limitations and inconsistency between previous reports, the precise spatial and temporal pattern of Hes5 expression in the postnatal and adult inner ear has remained unclear. In this study, we use Hes5-GFP transgenic mice and in situ hybridization to report the expression pattern of Hes5 in the inner ear. We find that Hes5 is expressed in the developing auditory epithelium of the cochlea beginning at embryonic day 14.5 (E14.5), becomes restricted to a particular subset of cochlear supporting cells, is downregulated in the postnatal cochlea, and is not present in adults. In the vestibular system, we detect Hes5 in developing supporting cells as early as E12.5 and find that Hes5 expression is maintained in some adult vestibular supporting cells. In order to determine the effect of hair cell damage on Notch signaling in the cochlea, we damaged cochlear hair cells of adult Hes5-GFP mice in vivo using injection of kanamycin and furosemide. Although outer hair cells were killed in treated animals and supporting cells were still present after damage, supporting cells did not upregulate Hes5-GFP in the damaged cochlea. Therefore, absence of Notch-Hes5 signaling in the normal and damaged adult cochlea is correlated with lack of regeneration potential, while its presence in the neonatal cochlea and adult vestibular epithelia is associated with greater capacity for plasticity or regeneration in these tissues; which suggests that this pathway may be involved in regulating regenerative potential

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).</p

Fibre diffraction modelling B.

Blue bars: D-period (in nm) for the different I(q) curves in S4 Fig, calculated using the first moment of area method used in the main text. Black bars: True D-period, obtained by correcting for the artificial increase in D-period due to the skew, demonstrated in S5B Fig. (TIF)</p

SAXS scans of full-length scan.

Colour map of the depth-wise variation in SAXS derived parameters across bovine bone-cartilage core of 5mm length and 2mm diameter for a full-length scan. This single sample was scanned across a greater depth in the trabecular bone but is otherwise similar to the samples imaged in Figs 2 and 3 in the main text. Step size of 20 microns, sample size ~0.38mm width, 5mm length. In this sample we were unable to resolve the thin superficial zone (SZ) at the top. Colour plots display: (A) Regions TZ: transitional zone, DZ: deep zone, CP: calcified plate, and TB: trabecular bone (as in Fig 1A, main text), (B) D-period (nm), reflecting collagen pre-strain, (C) total SAXS intensity (a.u.); areas of high intensity correspond to mineral-dense regions, (D) Total SAXS intensity from the background-corrected meridional collagen peak intensity; here, high intensity is observed in articular cartilage and (E) degree of orientation ρ (a.u.), showing high values in the deep zone (DZ), intermediate values in the calcified plate (CP) and transitional zone (TZ), and low values in trabecular bone (TB). (TIF)</p

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.</p

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.</p

Asymmetry in I(q) plots.

(A) Azimuthally integrated I(q) plots from the different tissue zones (SZ, TZ, DZ, CP and TB), corrected for diffuse background. I(q) plots are laterally averaged across sample width at specific vertical depths from cartilage surface; x-axis wavevector q is in nm-1 (B) Plots from (A) normalized to maximum peak intensity, to show the peak shape variations more clearly. (TIF)</p

Region classification of samples.

Representation of the region classification into SZ, TZ, DZ, CP and TB, for the 6 samples used in the analysis. Note that Sample 2 does not have an observable superficial (SZ) zone. The colour indicates the type of tissue region across BCU. (TIF)</p

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.</p