Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Arthritis Rheumatol. 2020 Jul 8;72(8):1266–1277. doi: 10.1002/art.41254

Decorin Mediates Cartilage Matrix Degeneration and Fibrillation in Post-Traumatic Osteoarthritis

Qing Li 1, Biao Han 1, Chao Wang 1, Wei Tong 2, Yulong Wei 2, Wei-Ju Tseng 2, Li-Hsin Han 3, X Sherry Liu 2, Motomi Enomoto-Iwamoto 4, Robert L Mauck 2,5, Ling Qin 2, Renato V Iozzo 6, David E Birk 7, Lin Han 1,*
PMCID: PMC7486252  NIHMSID: NIHMS1576759  PMID: 32162789

Abstract

Objective

This study aimed to elucidate the role of decorin, a small leucine-rich proteoglycan, in the degradation of cartilage matrix during the progression of post-traumatic osteoarthritis (PTOA).

Methods

The destabilization of the medial meniscus (DMM) surgery was applied to 3-month-old decorin-null and inducible decorin knockout mice to induce PTOA. The resulted OA phenotype was evaluated by assessing joint morphology and sulfated glycosaminoglycan (sGAG) staining via histology (n = 6/group), surface collagen fibril nanostructure via scanning electron microscopy (n = 4), tissue modulus via atomic force microscopy-nanoindentation (n ≥ 5) and subchondral bone structure via micro-computed tomography (n = 5). Femoral head cartilage explants from wild-type and decorin-null mice were subjected to the stimuli of inflammatory cytokine interleukin-1β (IL-1β) in vitro (n = 6). The resulting chondrocyte response to IL-1β and release of sGAGs were quantified.

Results

In both decorin-null and inducible decorin knockout mice, the absence of decorin results in accelerated sGAG loss and formation of highly aligned collagen fibrils on cartilage surface relative to the control (p < 0.05). Also, decorin-null mice developed more salient osteophytes, illustrating more severe OA. In cartilage explants, with IL-1β treatment, loss of decorin did not alter the expression of either anabolic or catabolic genes. However, a greater proportion of sGAGs was released to the media from decorin-null explants, in both live and devitalized conditions (p < 0.05).

Conclusion

In PTOA, decorin delays the loss of fragmented aggrecan and fibrillation of cartilage surface, and thus, plays a protective role in ameliorating cartilage degeneration.

Keywords: decorin, extracellular matrix, aggrecan, cartilage fibrillation, post-traumatic osteoarthritis

1. INTRODUCTION

Post-traumatic osteoarthritis (PTOA) is the most prevalent form of arthritis in young adults, and often results in long term detrimental influence on the quality of life (1). One hallmark of PTOA is the irreversible degradation of articular cartilage following traumatic injuries and/or aberrant joint loading, leading to joint dysfunction, pain and limited locomotion (2). In PTOA, elevated chondrocyte catabolism results in aggravated proteolysis of cartilage extracellular matrix (ECM) (3). Aggrecan, the major proteoglycan, is one of the first ECM constituents undergoing fragmentation due to enzymatic cleavage by aggrecanases and matrix metallopeptidases (MMPs). This leads to the disassembly of the aggrecan-hyaluronan (HA) supramolecular network and the loss of aggrecan from the ECM (4). In turn, the loss of aggrecan impairs cartilage biomechanical functions (5), disrupts chondrocyte mechanotransduction (6), accelerates the damage of collagen fibrils (7) and formation of fibrocartilage (8), and so, contributes to the vicious loop of irreversible cartilage breakdown. Inhibition of aggrecan depletion from degenerative tissue has the potential to delay cartilage degradation, attenuate OA progression and prolong joint use.

Our recent study suggests that decorin, a small leucine-rich proteoglycan (SLRP), could play such a role in regulating cartilage degradation in OA (9). Decorin is a class I SLRP characterized by ≈ 36 kDa leucine-rich protein core harboring one chondroitin sulfate or dermatan sulfate glycosaminoglycan chain at its N-terminus (10). One canonical structural function of decorin is to regulate collagen fibril diameter and interfibrillar spacing during fibril assembly in tension-bearing tissues such as tendon, cornea and skin (11). In cartilage, decorin is actively expressed from newborn to adulthood (12), and is one of the most abundant small proteoglycan in the ECM, with a molar concentration (≈ 15 nmol/ml) similar to that of aggrecan (≈ 20 nmol/ml) (13). In early human OA, decorin is significantly up-regulated (14,15). Despite this up-regulation, decorin is not released into the synovial fluid at higher levels (16), suggesting that decorin may participate in stabilizing cartilage matrix. It is postulated that this up-regulation may be a compensatory response by chondrocytes to ameliorate cartilage damage (17). This hypothesis is supported by our recent study showing that decorin increases the retention of aggrecan in healthy cartilage ECM (9). However, one recent study showed that decorin-null mice (Dcn−/−) develop higher resistance to forced exercise-induced OA, which was attributed to the enhanced transforming growth factor-β (TGF-β) signaling in the absence of decorin, indicating a detrimental role of decorin (18). Taken together, the role of decorin in OA remains inconclusive.

The objective of this study was to elucidate the role of decorin in cartilage degradation and PTOA progression in vivo. Here, mild-to-moderate PTOA was induced in young adult mice via the destabilization of the medial meniscus (DMM) surgery (19). Given the crucial role of decorin in cartilage development (9), we first queried if absence of decorin increased the susceptibility to OA in decorin-null (Dcn−/−) mice (20). Next, to separate decorin activities in OA from those in normal joint growth, using the recently established decorin inducible knockout mice (DcniKO) (21), we allowed for normal joint growth, then induced the knockout of decorin expression at the time of DMM surgery. Thus, the resulting phenotype represents the impact of decorin loss during OA progression, with developmental defects being minimized. In these models, we assessed cartilage damage and sulfated glycosaminoglycan (sGAG) loss, cartilage surface fibrillation and tissue modulus changes, as well as alterations in subchondral bone structure. Further, we tested if decorin alters chondrocyte catabolism, or the retention of fragmented aggrecan in the matrix, or both, using cartilage explants under inflammatory stimuli. Collectively, outcomes pointed to a crucial protective role of decorin in increasing aggrecan retention and inhibiting cartilage surface fibrillation in PTOA.

2. MATERIALS AND METHODS

Animal models

Decorin-null (Dcn−/−) mice (20) and the inducible decorin knockout mice (Dcnflox/flox/Rosa26CreER, or DcniKO) (21) in the C57BL/6 strain were generated as previously described, and were housed in the Calhoun animal facility at Drexel University. To induce the homozygous knockout of Dcn gene in 3-month-old mice, tamoxifen was injected intraperitoneally (i.p.) on 3 consecutive days beginning 1 week prior to DMM surgery at a dose of 3 mg/40 g body weight in the form of 20 mg/mL suspended in sesame oil (S3547, Sigma, St. Louis, MO) with 1% v/v benzyl alcohol (305197, Sigma). Quantitative polymerase chain reaction (qPCR) was performed on day 5 to confirm that TM-induced gene excision reduced the expression of Dcn to the baseline level (Fig. S1). For Dcn−/− mice, littermates of age-matched wild-type (WT) mice from the breeding of decorin heterozygous mice (Dcn+/−) were used as the control. For DcniKO mice, two control samples were used, including the DcniKO mice injected with vehicle (the same amount of sesame oil and benzyl alcohol but without tamoxifen), and WT mice injected with tamoxifen at the same dose and frequency. All the mice used here were genotyped, following standard procedures (20,21). The animal work was approved by the Institutional Animal Care and Use Committee (IACUC) at Drexel University.

Destabilization of medial meniscus (DMM) surgery was performed on the right hind knees of 3-month-old male mice for all genotypes, following an established procedure (19), with the Sham surgery performed on the contralateral left knees. Briefly, after anesthesia, the joint capsule was opened and medial meniscotibial ligament was cut to destabilize the medial meniscus. The Sham surgery was performed by opening the joint capsule in the same fashion to expose the ligament, but without further damage. Mice were euthanized at 2 or 8 weeks after surgery for Dcn−/− and WT mice, and at 8 weeks after surgery for DcniKO mice and their controls for further analyses.

Histology and immunofluorescence imaging

Whole murine hind knee joints (n = 6/group) were harvested and fixed in 4% paraformaldehyde, first used for μCT analysis, and then, decalcified in 10% EDTA for 4 weeks before embedded in paraffin for histology. Serial 6-μm-thick sagittal sections were prepared, and two sections with every consecutive six sections of the medial side of the Sham and DMM knees were stained with Safranin-O/Fast Green. For each joint, approximately 15 sections were obtained and scored by two masked observers (QL and CW) using the modified Mankin method (22). Thicknesses of uncalcified and total cartilage were determined by averaging six thickness values evenly distributed across the entire cartilage, following an established procedure (23). For immunofluorescence (IF) imaging of decorin, additional paraffin sections were treated with 0.1% pepsin (P7000, Sigma) for antigen retrieval, and blocked with 5% BSA in PBS for 1 hour at room temperature. Sections were first incubated with primary antibody (LF-114, gift from Dr. Larry W. Fisher, NIDCR, 1:100 dilution) overnight at 4°C, and then, with secondary antibody (Alexa Fluor 594, ThermoFisher, Waltham, MA, 1:500) for 2 hours at room temperature. The sections were washed with PBS, counterstained and mounted with DAPI (Fluoromount-G, 0100–20, SouthernBiotech, Birmingham, AL), and imaged with a Zeiss Axio Observer Microscope (Carl Zeiss, Oberkochen, Germany). Additional IF imaging was performed on healthy and OA human cartilage specimens obtained from de-identified donors with total arthroplasty (n = 3). Similarly, serial paraffin sections were treated with 0.1% pepsin, blocked with 5% BSA in PBS, incubated with primary antibody (AF-143, 20 μg/ml, R&D Systems, Minneapolis, MN) and then secondary antibody (Alexa Fluor 594, ThermoFisher). Specificity of decorin antibodies were confirmed by the staining of isotype controls (mouse: AB37415, Abcam, Cambridge, MA; human: AB-108-C, Novus Biologicals, Centennial, CO, 1:100) (Fig. S2).

AFM-based nanoindentation was applied to freshly dissected femoral condyle cartilage (n ≥ 5), following an established procedure (23). The indentation tests were performed using borosilicate microspherical colloidal tips (R ≈ 5 μm, nominal k ≈ 8.9 N/m, HQ:NSC35/tipless/Cr-Au, cantilever A, NanoAndMore, Lady’s Island, SC) and a Dimension Icon AFM (BrukerNano, Santa Barbara, CA) at 10 μm/s rate up to ≈ 1 μN maximum load in PBS with protease inhibitors (Pierce 88266, ThermoFisher). For each joint, at least 10–15 locations were tested on the load-bearing region of medial condyle to account for spatial heterogeneity. The effective indentation modulus, Eind, was calculated by fitting each force-indentation depth (F-D) loading curve with the Hertz model.

Scanning electron microscopy (SEM) was used to quantify the fibril nanostructure on condyle cartilage surfaces, following an established procedure (24). Immediately after the AFM tests, joints were treated with 0.1% trypsin (T7409, Sigma) and 20 U/ml hyaluronidase (H3506, Sigma) at 37°C for 24 hours each to remove proteoglycans, fixed with Karnovsky’s fixative at room temperature for 3 hours, sequentially dehydrated in graded ethanol-water and ethanol-hexamethyldisilazane (HMDS) mixtures, and air dried overnight (n = 4). Samples were then coated with ≈ 6 nm thick platinum and imaged using a Supra 50 VP SEM (Carl Zeiss) (Fig. S3). Collagen fibril alignment angles were measured using ImageJ, and fitted with von Mises probability density function to calculate the von Mises concentration parameter, κ, a quantitative measure of the degree of fibril alignment (25).

Micro-computed tomography (μCT) scanning was performed to assess concurrent changes of subchondral bone at 8 weeks post DMM. For mice purposed for histology, prior to demineralization, knee joints (n = 5) were scanned ex vivo using MicroCT 35 (Scanco Medical AG, Switzerland) at 6 μm isotropic voxel size and smoothed by a Gaussian filter (sigma = 1.2, support = 2.0). Each region of interest (ROI) for subchondral bone plate (SBP), subchondral trabecular bone (STB) and medial meniscal ossicles was contoured at a threshold corresponding to 30% of the maximum image gray scale. SBP of the tibia plateau on the medial side central loading region was contoured to calculate the SBP thickness (SBP.Th), following an established procedure (26). STB was contoured on the entire load bearing ROI on the medial side (27) to calculate structure parameters, including bone volume/total volume fraction (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) via Scanco’s trabecular bone 3D standard microstructural analysis. In addition, meniscal ossicle bone volume was directly measured via Scanco’s standard microstructural analysis.

In vitro cartilage explant model

To assess the impact of decorin loss on sGAG release from degenerative cartilage, femoral head cartilage explants were isolated from 3-week-old WT and Dcn−/− mice (n = 6), following an established procedure (28). For live explants, immediately after harvesting, explants were sterilized and pre-cultured at 37°C with 5% CO2 for 2 days in the Dulbecco’s modified Eagle’s medium (DMEM, 11960, ThermoFisher) mixed with 10% fetal bovine serum (FBS), 1× insulin-transferrin-selenium-sodium pyruvate (ITS-A, 51300, ThermoFisher), 2 mM L-glutamine (25030, ThermoFisher), 250 μM L-ascorbic acid 2-phosphate (AA2P, A8960, Sigma), and 2× penicillin-streptomycin (Pen-Strep, 15140, ThermoFisher). The explants were then cultured in the same media but with 1× Pen-Strep, supplemented with 10 ng/ml recombinant murine interleukin-1β (IL-1β, 211–11B, Peprotech, Rocky Hill, NJ) for 3 days, with the control group cultured in the same manner but without IL-1β. On day 3, the amounts of sGAGs released to the media and retained in cartilage were assessed via DMMB after the papain digestion, and qPCR was performed on additional explants to measure the expressions of anabolic and catabolic genes. Total RNA (250 ng per well) was subjected to reverse transcription using the TaqMan Reverse Transcription Kit (N8080234, ThermoFisher) with amplification carried out via the PowerUp SYBR Green Master Mix (A25742, ThermoFisher) on a RealPlex 4S master cycler (Eppendorf AG, Hamburg, Germany). Tested genes and associated primer sequences are listed in Table S1. For devitalized explants, after extraction and pre-culture, explants underwent three freeze-thaw cycles between −80°C for 2 h and at 37°C for 45 min (29), and cultured in the same medium supplemented with 20 nM recombinant human ADAMTS-5 (30) (2198-AD, R&D Systems) or MMP-13 (31) (511-MM-010, R&D Systems) for 4 days. The amounts of sGAGs released to media and retained in explant were assessed following the same procedure. For both live and devitalized explants, cell viability was assessed via fluorescein diacetate (FDA) and propidium iodide (PI) staining (Fig. S4) (32).

Statistical analysis

To avoid the assumption of normal distribution of the data, nonparametric statistical tests were applied. To test the significance between genotypes within each surgery type or treatment condition, Mann-Whitney U test was applied to compare ttotal, tuncalcified, Eind, modified Mankin score, μCT outcomes, gene expression and ratio of sGAG release. Wilcoxon signed-rank test was applied to compare these parameters between surgery types within each genotype. To compare the degree of fibril alignment, θ, Mardia and Jupp test of concentration equality (33) was applied to compare the von Mises concentration parameter κ between genotypes and surgery types. All the quantitative outcomes and statistical analysis results were summarized in Tables S2–S5. In all the tests, the significance level was set at α = 0.05.

3. RESULTS

Dcn−/− mice undergo accelerated cartilage degradation after DMM

In healthy human cartilage matrix, decorin was present in both the pericellular and further-removed territorial/interterritorial domains of the ECM (Fig. 1A). In OA specimens, decorin was significantly up-regulated and present throughout the damaged cartilage matrix. This observation was consistent with literature reporting the increase of decorin in human OA cartilage (14,15). In WT mice, for the Sham group, decorin was also distributed throughout the ECM. By 8 weeks after DMM, we detected increased staining of decorin (Fig. 1B), which validated DMM as an appropriate model for querying the role of decorin and its up-regulation in PTOA.

Figure 1. Decorin-null (Dcn−/−) mice develop accelerated progression of PTOA after DMM.

Figure 1.

Open in a new tab

A,B) Immunofluorescence (IF) images of decorin show the up-regulation of decorin A) in human OA cartilage, B) in wild-type (WT) murine knee cartilage after Sham and DMM surgeries, and Dcn−/− cartilage in the Sham group is shown as negative control (blue: DAPI). C) Representative Safranin-O/Fast Green histological images at 2 and 8 weeks after Sham and DMM surgeries show more severe cartilage damage in the Dcn−/− mice. D) Modified Mankin score and E) uncalcified cartilage thickness of the medial femoral condyle, tuncalcified, show signs of more severe OA in Dcn−/− mice relative to the WT (mean ± 95% CI, n = 6, *: p < 0.05 between genotypes, #: p < 0.05 between surgeries within each genotype). Panels D, E: Each data point represents the average value measured from one animal.

In the DMM model, Dcn−/− mice developed accelerated cartilage degradation compared to WT, as shown by histology (Fig. 1C). At 2 weeks post-surgery, while WT cartilage did not show appreciable damage, Dcn−/− cartilage started to develop loss of sGAG staining on the surface, contributing to higher modified Mankin scores (Fig. 1D). By 8 weeks, both genotypes exhibited salient OA signs, which were more pronounced in Dcn−/− mice. Specifically, Dcn−/− cartilage was characterized by the formation of surface fissures, further reduced sGAG staining, more substantial cartilage thinning (lower tuncalcified, Fig. 1E), and thus, significantly higher Mankin scores (Fig. 1D).

Dcn−/− cartilage develops pronounced collagen fibrillation on the surface after DMM

One distinctive OA phenotype of Dcn−/− cartilage was the surface fibrillation at the nanoscale, as observed by SEM (Fig. 2A). In healthy joints, the cartilage surface is covered by a transversely random mesh of collagen fibrils (34), as present in both WT and Dcn−/− cartilage in adulthood (Fig. S3) (9). In the Sham group, both genotypes retained this random fibrillar architecture. In the DMM group, while this feature was retained in WT mice up to 8 weeks post-surgery, Dcn−/− cartilage surface started to develop aligned fibrils as early as 2 weeks post-surgery, and became dominated by highly aligned, densely packed collagen fibrils at 8 weeks. These changes were signified by the much higher von Mises concentration, κ, in Dcn−/− cartilage (Fig. 2B). These fibrils were aligned along the mediolateral orientation (Figs. 2A, S3), suggesting that the surface fibrillation could be induced by extensive shearing of the destabilized medial meniscus during joint loading. In both genotypes, cartilage damage was associated with marked reduction in modulus, Eind (Fig. 2C). This reduction was attributed to the loss of cartilage ECM structural integrity, and illustrated the impaired cartilage load-bearing function in OA (23). In both Sham and DMM groups, Dcn−/− cartilage showed lower modulus compared to WT, suggesting that loss of decorin impairs cartilage load-bearing function both during normal skeletal growth, and in DMM-induced cartilage degradation.

Figure 2. Decorin-null (Dcn−/−) cartilage develops pronounced surface fibrillation after DMM.

Figure 2.

Open in a new tab

A) Nanostructure of collagen fibrils on condyle cartilage surfaces at 2 and 8 weeks post-surgery via SEM. Red arrows denote the mediolateral direction. B) Dcn−/− cartilage surface shows significantly higher von Mises concentration, κ, than wild-type (WT) after DMM (mean ± 95% CI, estimated from ≥ 300 fibrils pooled from n = 4 animals for each group). C) Cartilage tissue modulus measured via AFM-nanoindentation on medial condyles (mean ± 95% CI, n ≥ 5). Panels B, C:*: p < 0.05 between genotypes, #: p < 0.05 between surgeries within each genotype). Panel C: Each data point represents the average value of ≥ 10 locations measured from one animal.

Dcn−/− mice do not show an appreciable subchondral bone phenotype after DMM

Given the crucial interplay between cartilage and subchondral bone in OA development (2), we queried if loss of decorin also impacted subchondral bone after DMM. At 8 weeks post-surgery, for both genotypes, the DMM group showed no significant structural changes in either the SBP or STB relative to the Sham group (Fig. 3A–C). This is consistent with the literature showing that in the DMM model, subchondral bone changes only occur after the erosion of cartilage in late OA (35). In both DMM and Sham groups, no differences were detected between the WT and Dcn−/− SBP or STB structure, indicating that loss of decorin does not impact the remodeling of subchondral bone in the DMM model. On the other hand, at 8 weeks post-surgery, we detected the formation of osteophyte in Dcn−/−, but not WT joints (Fig. 3D), which illustrated more advanced OA (36). Meanwhile, DMM increased meniscal ossification at the horns in both genotypes, and Dcn−/− joints showed a marginally higher degree of ossification at the posterior, but not the anterior end (Fig. 3E). Collectively, μCT outcomes suggested that the accelerated OA progression in Dcn−/− mice is more likely to be a direct impact of decorin loss in cartilage, rather than a secondary effect arising from changes in underlining subchondral bone.

Figure 3. Comparison of subchondral bone structure in wild-type (WT) and Dcn−/− mice at 8 weeks after surgery.

Figure 3.

Open in a new tab

A) Representative 2D μCT frontal plane images of the knee joint (L: lateral, M: medial). B) Subchondral bone plate thickness (SBP.Th) and C) subchondral trabecular bone (STB) structural parameters (BV/TV: bone volume/total volume, Tb.N: trabecular number, Tb.Th: trabecular thickness) of the medial tibia analyzed from μCT images. D) Representative reconstructed 3D μCT images show the presence of osteophyte in Dcn−/− mice after DMM (red ellipses), but not in other groups. E) Meniscal ossification. Left panel: Representative reconstructed 3D μCT images (top view) of medial meniscal ossicles show increased ossification after DMM in both genotypes (A: anterior, P: posterior). Right panel: medial meniscal ossicle volume at both anterior and posterior ends. Panels B, C, E: mean ± 95% CI, n = 5, *: p < 0.05 between genotypes, p-values are reported if a trend (p < 0.10) is detected; #: p < 0.05 between surgeries within each genotype. Each data point represents the average value measured from one animal.

Ablation of decorin in DcniKO mice at the time of DMM also accelerates OA progression

The more severe OA phenotype in Dcn−/− mice could be attributed to both altered OA pathology and impaired joint growth prior to surgery in the absence of decorin. In the Sham group, Dcn−/− cartilage showed moderate Mankin scores (Fig. 1D) and lower modulus relative to WT (Fig. 2C), which was due to the lower sGAG content in Dcn−/− cartilage even without surgery (9). To separate the roles of decorin in OA from that in joint growth, we studied the OA progression in the DcniKO mice at 8 weeks post-surgery, the expression of decorin was maintained up to maturity, and only ablated at the time of DMM. In these mice, we also detected accelerated OA, with features comparable to those seen in Dcn−/− mice. In comparison to the control, DcniKO mice developed higher Mankin scores, more pronounced reduction in sGAG staining, salient surface fibrillation (Fig. 4A–E), the formation of osteophytes (Fig. S5), but no appreciable subchondral bone phenotype (Fig. 4G). Thus, the accelerated OA phenotype in Dcn−/− and DcniKO mice was primarily due to the loss of decorin’s protective roles in DMM-induced cartilage degradation.

Figure 4. Deletion of decorin at the time of surgery results in accelerated progression of PTOA at 8 weeks after DMM.

Figure 4.

Open in a new tab

A) Representative Safranin-O/Fast Green histological images show more severe cartilage damage in the DcniKO mice. B) Modified Mankin score and C) uncalcified cartilage thickness of medial femoral condyle, tuncalcified, show signs of more severe OA in DcniKO mice relative to the control (mean ± 95% CI, n = 6). D) Representative SEM images of collagen fibril nanostructure on condyle cartilage surfaces for the DMM group. Red arrow denotes the mediolateral direction. E) DcniKO cartilage surface shows significantly higher von Mises concentration, κ, than the control after DMM (mean ± 95% CI, ≥ 300 fibrils pooled from n = 4 animals). F) Cartilage tissue modulus measured via AFM-nanoindentation on medial condyles (mean ± 95% CI, n = 5). G) Subchondral bone plate thickness (SBP.Th) and subchondral trabecular bone structural parameters (BV/TV, Tb.N and Tb.Th) of the medial tibia analyzed from μCT images (mean ± 95% CI, n = 5). Panels B, C, F, G: *: p < 0.05 between genotypes, #: p < 0.05 between surgeries within each genotype. Each data point represents the average value measured from one animal.

In comparison to Dcn−/− mice, DcniKO mice did not demonstrate more severe cartilage thinning (Fig. 4C) and had moderately lower Mankin scores (Fig. 4B). This confirmed that the OA phenotype in Dcn−/− mice is resulted from the combined effects of both altered OA pathology and impaired joint growth. Further, while the modulus of DcniKO cartilage was also reduced by DMM, it was similar to that of the control (Fig. 4F) and higher than that of Dcn−/− cartilage. The modulus of DcniKO cartilage could reflect the properties of newly formed fibrous tissues on the surface, which lack aggrecan and associated sGAGs, and thus, are incapable of dissipating energy through poroelasticity as normal hyaline cartilage.

Loss of decorin accelerates aggrecan release from degenerative cartilage explants

To determine if loss of decorin directly impacts chondrocyte catabolism, we analyzed chondrocyte gene expressions in femoral head cartilage explants (Fig. 5A). Under the IL-1β stimuli, the anabolic genes, aggrecan (Acan) and collagen II (Col2a1), exhibited a mild decrease in the expression in WT, but not in Dcn−/− explants (Fig. 5A). In contrast, major catabolic genes, including aggrecanases and MMPs, were significantly up-regulated by 10–100 folds in both genotypes. Comparing the two genotypes, we did not detect significant differences in all tested genes except for decorin (Dcn). This suggests that decorin does not directly regulate the anabolic or catabolic activities of chondrocytes, either with or without the stimuli of IL-1β.

Figure 5. Loss of decorin accelerates the release of sGAGs from degenerative murine cartilage explants, but does not alter chondrocyte anabolism or catabolism.

Figure 5.

Open in a new tab

A) Expressions of anabolic and catabolic genes from chondrocytes in femoral head cartilage explants cultured in DMEM for 3 days, with or without the stimuli of inflammatory cytokine, interleukin-1β (IL-1β), measured by qPCR (mean ± SEM, n = 6, *: p < 0.01 between genotypes. Different letters indicate significant differences between the untreated and IL-1β-treated groups within each genotype). B) Relative percentage of sGAGs released from live cartilage explants cultured in DMEM for 3 days, with or without IL-1β, measured by DMMB (mean ± 95%, n = 6). C) Relative percentage of sGAGs released from devitalized cartilage explants cultured in DMEM for 4 days, measured by DMMB. Panels B, C: mean ± 95% CI, n = 6, *: p < 0.05 between genotypes, #: p < 0.05 between untreated and treated groups with each genotype. Each data point represents the value from one biological repeat.

Finally, we compared the amount of sGAGs released from explants, in both live and devitalized conditions. Dcn−/− cartilage has lower amount of aggrecan and sGAGs than the WT, which indicates a lower concentration gradient for diffusion-driven release of sGAGs. Despite this, in live explants, when IL-1β aggravated chondrocyte catabolism, a greater proportion of sGAGs was released from Dcn−/− explants relative to WT (Fig. 5B). Further, in devitalized explants, when chondrocyte metabolism was abolished, Dcn−/− explants still experienced a higher degree of sGAG release upon exogenous proteolysis of aggrecan by ADAMTS-5 or MMP-13 (Fig. 5C). Therefore, in the explant model, absence of decorin accelerates the loss of fragmented aggrecan from cartilage matrix, but does not directly alter chondrocyte metabolism under IL-1β stimuli.

4. DISCUSSION

This study highlights the crucial role of decorin in mediating cartilage degradation in PTOA, as evidenced by the accelerated OA phenotype in both Dcn−/− and DcniKO mice (Figs. 1–4). We hypothesize that in degenerative cartilage, decorin increases the retention of fragmented aggrecan within degrading matrix, thereby delaying aggrecan loss and cartilage damage (Fig. 6A). This hypothesis extends findings from our recent study on the regulatory roles of decorin in cartilage post-natal growth (9). Specifically, we showed that in healthy cartilage, decorin functions as a “physical linker” (Fig. 6B) to increase molecular adhesion between aggrecan-aggrecan and aggrecan-collagen II fibrils. The canonical assembly mechanism of aggrecan network in the ECM is the aggregation of aggrecan-HA via the G1 domain (37), but this mechanism does not fully explain the integrity of aggrecan across development and disease states (38). While aggrecan can also form networks through interacting with tenascins (39) and fibulin-2 (40) via its G3 domain, the presence of G3 domain decreases markedly with age (41). To this end, the decorin-mediated aggrecan network assembly could be a crucial mechanism in maintaining the integrity of aggrecan network in the ECM, as supported by the markedly impaired biomechanical functions of Dcn−/− cartilage (9). In OA, when aggrecan molecules become increasingly fragmented and dissociated from the aggrecan-HA aggregates (4), the up-regulation of decorin could be a reparative attempt to increase the retention of fragmented aggrecan, and so, attenuate aggrecan depletion (Fig. 6A). Indeed, this hypothesis is supported by outcomes from explant models, in which, loss of decorin accelerates the release of fragmented aggrecan from both live and devitalized cartilage (Fig. 5B,C).

Figure 6.

Figure 6.

Open in a new tab

A) Schematic illustration of the working hypothesis on the structural role of decorin in degenerative cartilage matrix. Left panel: Decorin binds to aggrecan to increase its adhesion with other aggrecan molecules and with collagen II/IX/XI fibrils, thereby enhancing the retention of fragmented aggrecan in the ECM. Right panel: Flow diagram illustrates the compensatory effects of decorin up-regulation to slow down the depletion of aggrecan, and thus, attenuate cartilage degradation and onset of OA. B) Illustration of decorin role in increasing the aggregation of aggrecan. Tapping mode AFM images show that aggrecan molecules remain as individual monomers when reconstituted on mica surface in vitro, but form interconnected networks with the addition of free decorin protein (adapted from (9) with permission).

In the DMM model, both Dcn−/− and DcniKO cartilage surfaces develop extensive surface fibrillation (Figs. 2A,B, 4D,E). We attribute this effect to the loss of aggrecan’s protection against collagen remodeling. In the ECM, the densely packed, aggrecan-HA aggregates occupy the ~ 100 nm-sized inter-fibrillar spacing within the porous collagen II/IX/XI network (42), limiting aberrant collagen fibril lateral fusion and overgrowth. When loss of decorin leads to accelerated aggrecan depletion and impairs aggrecan’s protection of collagen fibrillar network following DMM, the extensive shear and frictional forces from the destabilized meniscus can induce the alignment of collagen fibrils along the shearing direction (Figs. 2A, 4D). In addition, decorin could also directly inhibit fibril lateral fusion given its capability of binding to collagen II (43), but such contribution may be less important due to its low mass concentration relative to aggrecan in cartilage (13). The presence of aligned fibrils suggests that hyaline cartilage now loses its integrity and transforms into fibrocartilage, which does not possess the energy dissipation function endowed by aggrecan (8). These results thus support a critical protective role of decorin in inhibiting cartilage fibrillation, an irreversible degenerative step of OA progression (8).

Integrating this study with our recent work on young adult Dcn−/− cartilage (9), we show that in both healthy and degenerative cartilage, the primary role of decorin is to mediate ECM assembly, rather than to influence chondrocyte metabolism. The absence of decorin’s impact on chondrocyte response to IL-1β (Fig. 5A) is similar to its lack of impact on the response to growth factor TGF-β1 (9). In both scenarios, although loss of decorin does not alter chondrocyte metabolism, it significantly reduces the retention of aggrecan in cartilage matrix, both during degeneration in situ (Fig. 5B,C) and during regeneration in alginate culture (9). In vivo, the role of decorin in increasing aggrecan retention could be more crucial, as the extensive physiological joint loading and associated interstitial fluid flow can further promote aggrecan depletion when its assembly is impaired. This finding is different from the literature reporting higher resistance of Dcn−/− mice to forced exercise-induced OA (18). We attribute this contrast mainly to differences in the OA models used. The forced exercise model represents joint overuse (44), while DMM induces joint instability and inflammation (19), and so, the two could have different disease etiology. For example, in WT cartilage, the expression of decorin is suppressed by forced exercise (18), but elevated by DMM (Fig. 1B). This increase in decorin expression corroborates observations in both murine (Fig. 5A) and bovine (45) cartilage explants under IL-1β, as well as cartilage specimens from OA patients (14,15). To this end, the protective role of decorin in PTOA is affirmed by the consistency of accelerated OA phenotype in Dcn−/− and DcniKO mice (Figs. 1–4), which were established in different manners (20,21).

This study has several limitations. First, while we showed that decorin increases molecular adhesion of aggrecan (9), we did not identify how decorin specifically interacts with aggrecan core protein or its sGAGs, and how such interaction is altered when decorin becomes increasingly fragmented as OA advances (46–48). To address this, our ongoing studies sought to test the interactions between purified decorin, aggrecan and collagen II at the single molecular level. Second, the half-life of decorin in adult murine cartilage is unknown. The lack of phenotype in the Sham group of DcniKO mice (Fig. 4) is in stark contrast with the pronounced phenotype resulted from induced decorin knockout at 1 month of age (9). This could be due to either the presence of residual decorin, or that decorin plays a less essential role in adulthood (≥ 3 months of age). However, while we cannot delineate the influence of residual decorin in DMM, the accelerated OA phenotype in DcniKO mice (Fig. 4) clearly demonstrated the impact of ablating the decorin up-regulation on DMM-induced cartilage degradation. Third, although we showed that decorin does not significantly impact chondrocyte catabolism under IL-1β, given its versatile interactomes (10), it is possible that decorin may affect chondrocyte signaling through its bindings with cell surface receptors and cytokines in other OA models. For example, spontaneous OA is associated with reduced autophagy in cartilage (49), and decorin may also ameliorate OA through evoking autophagy (50), which will be a topic of our future work.

In summary, this study identifies decorin as a central player in cartilage degradation in PTOA. Loss of decorin aggravates aggrecan depletion and surface fibrillation, leading to accelerated cartilage damage. We hypothesize that up-regulation of decorin in early OA acts as a reparative attempt to increase the retention of fragmented aggrecan in the ECM, thereby delaying aggrecan loss, cartilage fibrillation and irreversible cartilage breakdown (Fig. 6A). This role is mediated by the interactions of decorin with aggrecan molecules and collagen II fibrils. Therefore, modulating decorin activities through decorin-targeting gene therapies or decorin-based biomaterials has the potential to attenuate OA progression and prolong joint use.

Supplementary Material

Supp info

ACKNOWLEDGEMENTS

This work was supported by the National Institutes of Health (NIH) Grant AR066824 and AR074490 to Dr. L. Han, the National Science Foundation (NSF) Grant CMMI-1662544 to Dr. L. Han, as well as NIH Grant P30 AR069619 to the Penn Center for Musculoskeletal Disorders (PCMD). We thank Dr. L. W. Fisher (NIDCR) for the generous gift of decorin antibodies and Dr. S. Wakitani (Hiroshima University) for kindly providing human articular cartilage sections.

Footnotes

Financial Support Information:

There is no financial support or other benefits from commercial sources for the work reported on in the manuscript, or any other financial interests that any of the authors may have, which could create a potential conflict of interest or the appearance of a conflict of interest with regard to the work.

REFERENCES

  • 1.Helmick CG, Felson DT, Lawrence RC, Gabriel S, Hirsch R, Kwoh CK, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part I. Arthritis Rheum. 2008;58:15–25. [DOI] [PubMed] [Google Scholar]
  • 2.Goldring MB, Goldring SR. Articular cartilage and subchondral bone in the pathogenesis of osteoarthritis. Ann N Y Acad Sci. 2010;1192:230–7. [DOI] [PubMed] [Google Scholar]
  • 3.Roughley PJ, Mort JS. The role of aggrecan in normal and osteoarthritic cartilage. J Exp Orthop. 2014;1:8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Lohmander LS, Neame PJ, Sandy JD. The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis. Arthritis Rheum. 1993;36:1214–22. [DOI] [PubMed] [Google Scholar]
  • 5.Krishnan Y, Grodzinsky AJ. Cartilage diseases. Matrix Biol. 2018;71–72:51–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Guilak F, Nims RJ, Dicks A, Wu CL, Meulenbelt I. Osteoarthritis as a disease of the cartilage pericellular matrix. Matrix Biol. 2018;71–72:40–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Pratta MA, Yao W, Decicco C, Tortorella MD, Liu R-Q, Copeland RA, et al. Aggrecan protects cartilage collagen from proteolytic cleavage. J Biol Chem. 2003;278:45539–45. [DOI] [PubMed] [Google Scholar]
  • 8.Stoop R, Buma P, van der Kraan PM, Hollander AP, Billinghurst RC, Meijers TH, et al. Type II collagen degradation in articular cartilage fibrillation after anterior cruciate ligament transection in rats. Osteoarthritis Cartilage. 2001;9:308–15. [DOI] [PubMed] [Google Scholar]
  • 9.Han B, Li Q, Wang C, Patel P, Adams SM, Doyran B, et al. Decorin regulates the aggrecan network integrity and biomechanical functions of cartilage extracellular matrix. ACS Nano. 2019;13:11320–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Gubbiotti MA, Vallet SD, Ricard-Blum S, Iozzo RV. Decorin interacting network: A comprehensive analysis of decorin-binding partners and their versatile functions. Matrix Biol. 2016;55:7–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Chen S, Birk DE. The regulatory roles of small leucine-rich proteoglycans in extracellular matrix assembly. FEBS J. 2013;280:2120–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.McAlinden A, Dudhia J, Bolton MC, Lorenzo P, Heinegård D, Bayliss MT. Age-related changes in the synthesis and mRNA expression of decorin and aggrecan in human meniscus and articular cartilage. Osteoarthritis Cartilage. 2001;9:33–41. [DOI] [PubMed] [Google Scholar]
  • 13.Poole AR, Rosenberg LC, Reiner A, Ionescu M, Bogoch E, Roughley PJ. Contents and distributions of the proteoglycans decorin and biglycan in normal and osteoarthritic human articular cartilage. J Orthop Res. 1996;14:681–9. [DOI] [PubMed] [Google Scholar]
  • 14.Cs-Szabó G, Roughley PJ, Plaas AHK, Glant TT. Large and small proteoglycans of osteoarthritic and rheumatoid articular-cartilage. Arthritis Rheum. 1995;38:660–8. [DOI] [PubMed] [Google Scholar]
  • 15.Cs-Szabó G, Melching LI, Roughley PJ, Glant TT. Changes in messenger RNA and protein levels of proteoglycans and link protein in human osteoarthritic cartilage samples. Arthritis Rheum. 1997;40:1037–45. [DOI] [PubMed] [Google Scholar]
  • 16.Barreto G, Soininen A, Ylinen P, Sandelin J, Konttinen YT, Nordstrom DC, et al. Soluble biglycan: a potential mediator of cartilage degradation in osteoarthritis. Arthritis Res Ther. 2015;17:379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Ni GX, Li Z, Zhou YZ. The role of small leucine-rich proteoglycans in osteoarthritis pathogenesis. Osteoarthritis Cartilage. 2014;22:896–903. [DOI] [PubMed] [Google Scholar]
  • 18.Gronau T, Kruger K, Prein C, Aszodi A, Gronau I, Iozzo RV, et al. Forced exercise-induced osteoarthritis is attenuated in mice lacking the small leucine-rich proteoglycan decorin. Ann Rheum Dis. 2017;76:442–9. [DOI] [PubMed] [Google Scholar]
  • 19.Glasson SS, Blanchet TJ, Morris EA. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis Cartilage. 2007;15:1061–9. [DOI] [PubMed] [Google Scholar]
  • 20.Danielson KG, Baribault H, Holmes DF, Graham H, Kadler KE, Iozzo RV. Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. J Cell Biol. 1997;136:729–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Robinson KA, Sun M, Barnum CE, Weiss SN, Huegel J, Shetye SS, et al. Decorin and biglycan are necessary for maintaining collagen fibril structure, fiber realignment, and mechanical properties of mature tendons. Matrix Biol. 2017;64:81–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.McNulty MA, Loeser RF, Davey C, Callahan MF, Ferguson CM, Carlson CS. Histopathology of naturally occurring and surgically induced osteoarthritis in mice. Osteoarthritis Cartilage. 2012;20:949–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Doyran B, Tong W, Li Q, Jia H, Zhang X, Chen C, et al. Nanoindentation modulus of murine cartilage: a sensitive indicator of the initiation and progression of post-traumatic osteoarthritis. Osteoarthritis Cartilage. 2017;25:108–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Wang C, Brisson BK, Terajima M, Li Q, Hoxha K, Han B, et al. Type III collagen is a key regulator of the collagen fibrillar structure and biomechanics of articular cartilage and meniscus. Matrix Biol. 2020: In press, doi: 10.1016/j.matbio.2019.10.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Li Q, Qu F, Han B, Wang C, Li H, Mauck RL, et al. Micromechanical anisotropy and heterogeneity of the meniscus extracellular matrix. Acta Biomater. 2017;54:356–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Huang H, Skelly JD, Ayers DC, Song J. Age-dependent changes in the articular cartilage and subchondral bone of C57BL/6 mice after surgical destabilization of medial meniscus. Sci Rep. 2017;7:42294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Moodie JP, Stok KS, Muller R, Vincent TL, Shefelbine SJ. Multimodal imaging demonstrates concomitant changes in bone and cartilage after destabilisation of the medial meniscus and increased joint laxity. Osteoarthritis Cartilage. 2011;19:163–70. [DOI] [PubMed] [Google Scholar]
  • 28.Stanton H, Golub SB, Rogerson FM, Last K, Little CB, Fosang AJ. Investigating ADAMTS-mediated aggrecanolysis in mouse cartilage. Nat Protoc. 2011;6:388–404. [DOI] [PubMed] [Google Scholar]
  • 29.Clements KM, Bee ZC, Crossingham GV, Adams MA, Sharif M. How severe must repetitive loading be to kill chondrocytes in articular cartilage? Osteoarthritis Cartilage. 2001;9:499–507. [DOI] [PubMed] [Google Scholar]
  • 30.Seuffert F, Weidner D, Baum W, Schett G, Stock M. Upper zone of growth plate and cartilage matrix associated protein protects cartilage during inflammatory arthritis. Arthrit Res Ther. 2018;20:88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Rojas FP, Batista MA, Lindburg CA, Dean D, Grodzinsky AJ, Ortiz C, et al. Molecular adhesion between cartilage extracellular matrix macromolecules. Biomacromolecules. 2014;15:772–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Clements KM, Bee ZC, Crossingham GV, Adams MA, Sharif M. How severe must repetitive loading be to kill chondrocytes in articular cartilage? Osteoarthritis Cartilage. 2001;9:499–507. [DOI] [PubMed] [Google Scholar]
  • 33.Mardia KV, Jupp PE. Tests of von Mises distributions In Directional Statistics. London, UK: John Wiley & Sons, Ltd.; 2000. p. 119–43. [Google Scholar]
  • 34.Vanden Berg-Foels WS, Scipioni L, Huynh C, Wen X. Helium ion microscopy for high-resolution visualization of the articular cartilage collagen network. J Microsc. 2012;246:168–76. [DOI] [PubMed] [Google Scholar]
  • 35.Jia H, Ma X, Wei Y, Tong W, Tower RJ, Chandra A, et al. Loading-induced reduction in Sclerostin as a mechanism of subchondral bone plate sclerosis in mouse knee joints during late-stage osteoarthritis. Arthritis Rheumatol. 2018;70:230–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Felson DT, Gale DR, Elon Gale M, Niu J, Hunter DJ, Goggins J, et al. Osteophytes and progression of knee osteoarthritis. Rheumatology (Oxford). 2005;44:100–4. [DOI] [PubMed] [Google Scholar]
  • 37.Garantziotis S, Savani RC. Hyaluronan biology: a complex balancing act of structure, function, location and context. Matrix Biol. 2019;78–79:1–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Bayliss MT, Howat S, Davidson C, Dudhia J. The organization of aggrecan in human articular cartilage. Evidence for age-related changes in the rate of aggregation of newly synthesized molecules. J Biol Chem. 2000;275:6321–7. [DOI] [PubMed] [Google Scholar]
  • 39.Lundell A, Olin AI, Morgelin M, al-Karadaghi S, Aspberg A, Logan DT. Structural basis for interactions between tenascins and lectican C-type lectin domains: evidence for a crosslinking role for tenascins. Structure. 2004;12:1495–506. [DOI] [PubMed] [Google Scholar]
  • 40.Olin AI, Morgelin M, Sasaki T, Timpl R, Heinegard D, Aspberg A. The proteoglycans aggrecan and versican form networks with fibulin-2 through their lectin domain binding. J Biol Chem. 2001;276:1253–61. [DOI] [PubMed] [Google Scholar]
  • 41.Dudhia J, Davidson CM, Wells TM, Hardingham TE, Bayliss MT. Studies on the G3 domain of aggrecan from human cartilage. Ann N Y Acad Sci. 1996;785:245–7. [DOI] [PubMed] [Google Scholar]
  • 42.Clark IC. Articular cartilage: a review and scanning electron microscopy study. I. The interterritorial fibrillar architecture. J Bone Joint Surg Br. 1971;53B:732–50. [PubMed] [Google Scholar]
  • 43.Douglas T, Heinemann S, Bierbaum S, Scharnweber D, Worch H. Fibrillogenesis of collagen types I, II, and III with small leucine-rich proteoglycans decorin and biglycan. Biomacromolecules. 2006;7:2388–93. [DOI] [PubMed] [Google Scholar]
  • 44.Bomer N, Cornelis FM, Ramos YF, den Hollander W, Storms L, van der Breggen R, et al. The effect of forced exercise on knee joints in Dio2−/− mice: type II iodothyronine deiodinase-deficient mice are less prone to develop OA-like cartilage damage upon excessive mechanical stress. Ann Rheum Dis. 2016;75:571–7. [DOI] [PubMed] [Google Scholar]
  • 45.Lv M, Zhou Y, Polson SW, Wan LQ, Wang M, Han L, et al. Identification of chondrocyte genes and signaling pathways in response to acute joint inflammation. Sci Rep. 2019;9:93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Bock HC, Michaeli P, Bode C, Schultz W, Kresse H, Herken R, et al. The small proteoglycans decorin and biglycan in human articular cartilage of late-stage osteoarthritis. Osteoarthritis Cartilage. 2001;9:654–63. [DOI] [PubMed] [Google Scholar]
  • 47.Young AA, Smith MM, Smith SM, Cake MA, Ghosh P, Read RA, et al. Regional assessment of articular cartilage gene expression and small proteoglycan metabolism in an animal model of osteoarthritis. Arthritis Res Ther. 2005;7:R852–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Melrose J, Fuller ES, Roughley PJ, Smith MM, Kerr B, Hughes CE, et al. Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues. Arthrit Res Ther. 2008;10:R79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Carames B, Olmer M, Kiosses WB, Lotz MK. The relationship of autophagy defects to cartilage damage during joint aging in a mouse model. Arthritis Rheumatol. 2015;67:1568–76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Gubbiotti MA, Neill T, Frey H, Schaefer L, Iozzo RV. Decorin is an autophagy-inducible proteoglycan and is required for proper in vivo autophagy. Matrix Biol. 2015;48:14–25. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp info
NIHMS1576759-supplement-Supp_info.docx (42.8MB, docx)

RESOURCES