Matrix disruptions, growth, and degradation of cartilage with impaired sulfation

Abstract

Diastrophic dysplasia (DTD) is an incurable recessive chondrodysplasia caused by mutations in the SLC26A2 transporter responsible for sulfate uptake by chondrocytes. The mutations cause undersulfation of glycosaminoglycans in cartilage. Studies of dtd mice with a knock-in Slc26a2 mutation showed an unusual progression of the disorder: net undersulfation is mild and normalizing with age, but the articular cartilage degrades with age and bones develop abnormally. To understand underlying mechanisms, we studied newborn dtd mice. We developed, verified and used high-definition infrared hyperspectral imaging of cartilage sections at physiological conditions, to quantify collagen and its orientation, noncollagenous proteins, and chondroitin chains, and their sulfation with 6-μm spatial resolution and without labeling. We found that chondroitin sulfation across the proximal femur cartilage varied dramatically in dtd, but not in the wild type. Corresponding undersulfation of dtd was mild in most regions, but strong in narrow articular and growth plate regions crucial for bone development. This undersulfation correlated with the chondroitin synthesis rate measured via radioactive sulfate incorporation, explaining the sulfation normalization with age. Collagen orientation was reduced, and the reduction correlated with chondroitin undersulfation. Such disorientation involved the layer of collagen covering the articular surface and protecting cartilage from degradation. Malformation of this layer may contribute to the degradation progression with age and to collagen and proteoglycan depletion from the articular region, which we observed in mice already at birth. The results provide clues to in vivo sulfation, DTD treatment, and cartilage growth.

FIGURE 1.

Visible and ³⁵S-autoradiographic images of mid-coronal cryosections of proximal femur cartilage explants from 0.5-day-old wt and dtd mice. Left, Black lines mark regions (within ∼50 μm from the lines) where matrix composition and sulfate incorporation were sampled by HDIR imaging and autoradiography. Squares at the articular and growth plate regions illustrate some of the areas captured by HDIR. Ticks mark boundary between articular and subarticular zones and beginnings of columnar, enlargement, hypertrophic, mineralizing cartilage, and bone marrow zones defined morphologically under “Materials and Methods.” Right, autoradiographic images of the sections that are shown on the left and contain [³⁵S]sulfate incorporated into the explants. The black lines, the zone ticks, and articular surface lines from the visible images were superimposed onto the ³⁵S images. Pixel darkness is proportional to the ³⁵S signal, with the darkness scale being the same for wt and dtd. Bottom, profiles of the ³⁵S-intensity divided by areal cell density along the black lines shown on left images. Curves are for single representative sections. The hatched band shows variability of profile shape for 3 wt and 3 normal, heterozygous littermates scaled to have the same average value in the middle of the columnar zone. The band is confined by mean ± S.D. curves. The error bar is S.D. of the unscaled intensity/density ratio at the middle of the columnar zone. The zigzag line marks shift of the E zone in dtd with respect to wt.

FIGURE 2.

HDIR absorption spectra of model compounds of cartilage extracellular matrix. A, isotropic spectra of fibers reconstituted from purified bovine type II collagen and BSA modeling noncollagenous core proteins (cf. supplemental Figs. S2 and S3 and Table S2). B, spectra of chondroitins with C0S, C4S, and C6S forms of sulfation were derived by decomposition of spectra of highly enriched chondroitins. The available dermatan sulfate sample (DS) appeared contaminated by ∼30% of GAGs other than HA or chondroitins (cf. supplemental Table S1). HA and C0S spectra were shifted down for clarity. Bold horizontal segments mark integration ranges of peaks used to analyze matrix composition.

FIGURE 3.

HDIR absorption spectra of cartilage extracellular matrix and a collagen fiber. A, matrix spectra from 5 × 5 μm² area 80 μm below the articular surface in wt and dtd mice are compared with spectra reconstructed from the model chondroitins, collagen type II, and BSA spectra based on decomposition of 4 spectral peaks marked by horizontal segments: collagen prolines 1340 cm⁻¹ band, which is weak in NCPs due to low proline content; the CH₂ bending of NCP of the 1401 cm⁻¹ band, which is shifted to 1405 cm⁻¹ and is weaker in collagen due to the high glycine content; sugar ring bands between 960–1188 cm⁻¹, where the intensity of protein cores is weak; and GAG sulfate vibrations at 1235 cm⁻¹ , where collagen also has a sizable contribution. B, polarized HDIR spectra of wt articular extracellular matrix and washed, stretched tendon fiber. The fiber spectra were normalized to have collagen content equivalent to that of the matrix. IR light polarization was parallel ‖ and perpendicular ⊥ to the articular surface or to the fiber axis. C, dichroic difference ‖ − ⊥ of the spectra shown in B. The fiber spectra at 1550–1700 cm⁻¹ were distorted due to high absorbance.

FIGURE 4.

Densities of GAGs, noncollagenous core proteins, and collagen and sulfate/GAG ratio in proximal femur cartilage of 0.5-day-old wt and dtd mice measured by HDIR hyperspectral imaging. Images, hyperspectral images of articular and growth plate regions marked in Fig. 1. Pixel brightness is proportional to the densities and ratio according to vertical gray scale bars at y axes of graphs under the images. Pixels at cells appear darker in D or brighter in C than pixels at matrix due to, respectively, lower collagen or higher noncollagenous protein content at cells. The brightness of the cell pixels is quantitatively inaccurate. Graphs, the densities and ratio at the extracellular matrix areas near the anatomical lines (shown in Images and in Fig. 1) versus distance from the bone marrow and the articular surface. Shaded areas are confined by curves whose y values are mean ± S.D. at each distance for a single section (cf. supplemental Fig. S7). Points are mean ± S.D. for 6 mice at selected distances. *, **, ***, and **** denote p values ≤ 0.05, 0.005, 0.0005, and 0.00005, respectively. Letters and ticks mark the cartilage zones shown in Fig. 1. Zigzag line marks shift of the E zone in dtd with respect to wt. One unit of the y scales corresponds to: A, 1 ± 0.1 fmol/picoliter of chondroitins disaccharides or 0.5 ± 0.05 pg/picoliter of chondroitin sulfate disodium salt; B, 1 ± 0.07 sulfate/chondroitin disaccharide molar ratio; C, 1 ± 0.1 pg/picoliter of noncollagenous core proteins; D, 1 ± 0.1 pg/picoliter of triple helical collagens.

FIGURE 5.

Collagen orientation order parameters of matrix of proximal femur cartilage from 0.5-day-old wt and dtd mice measured by polarized HDIR hyperspectral imaging. Images, parameter (P) uncorrected for collagen concentration. Cyan and magenta pixels mark areas with preferred collagen alignment indicated by orthogonal arrows of the corresponding color. The cyan-black-magenta bar corresponds pixel brightness to P values with 1 unit equal to 1 ± 0.1 pg/picoliter. Bottom, parameter (p) corrected for collagen concentration sampled at extracellular matrix near the lines (shown in these images and in Fig. 1) versus distance from the bone marrow and articular surface. Shaded areas represent mean ± S.D. for a single section (cf. supplemental Fig. S7E). Letters and ticks mark the cartilage zones shown in Fig. 1. Right, parameter (P) and its coefficient of variation CV at the articular surface. * and ** denote p values ≤ 0.05 and 0.005, respectively.

FIGURE 6.

Correlation between chondroitin synthesis rate per cell, chondroitin undersulfation in dtd, and collagen disorientation in dtd at selected distances. Top, [³⁵S]sulfate incorporation rate per cell for 6 normal (3 wt and 3 heterozygous, denoted wt) and 1 dtd littermates. For normal mice, the rate profiles were scaled to have the same average value at the middle of the columnar (C) zone. The vertical bar at the C zone is standard error of the unscaled rate. Bars at the other positions are S.E. of the scaled values. The incorporation rate reflects the chondroitin synthesis rate per cell in wt, but underestimates the synthesis rate in dtd. Bottom, differences between wt and dtd for chondroitin sulfation ratio cs and collagen orientation order parameter p replotted from Figs. 4B and 5. In our definition, the chondroitin undersulfation and collagen disorientation decrease with cs_dtd and p_dtd getting closer to cs_wt and p_wt. Error bars are S.E. of the cs and p differences. All data were measured at similar anatomical positions and plotted at the positions of a wt sample. * denotes p values ≤ 0.05. § indicates statistically significant (p values ≤ 0.05) peaks.