Transcriptional profiling differences for articular cartilage and repair tissue in equine joint surface lesions
© Mienaltowski et al; licensee BioMed Central Ltd. 2009
Received: 28 April 2009
Accepted: 14 September 2009
Published: 14 September 2009
Full-thickness articular cartilage lesions that reach to the subchondral bone yet are restricted to the chondral compartment usually fill with a fibrocartilage-like repair tissue which is structurally and biomechanically compromised relative to normal articular cartilage. The objective of this study was to evaluate transcriptional differences between chondrocytes of normal articular cartilage and repair tissue cells four months post-microfracture.
Bilateral one-cm2 full-thickness defects were made in the articular surface of both distal femurs of four adult horses followed by subchondral microfracture. Four months postoperatively, repair tissue from the lesion site and grossly normal articular cartilage from within the same femorotibial joint were collected. Total RNA was isolated from the tissue samples, linearly amplified, and applied to a 9,413-probe set equine-specific cDNA microarray. Eight paired comparisons matched by limb and horse were made with a dye-swap experimental design with validation by histological analyses and quantitative real-time polymerase chain reaction (RT-qPCR).
Statistical analyses revealed 3,327 (35.3%) differentially expressed probe sets. Expression of biomarkers typically associated with normal articular cartilage and fibrocartilage repair tissue corroborate earlier studies. Other changes in gene expression previously unassociated with cartilage repair were also revealed and validated by RT-qPCR.
The magnitude of divergence in transcriptional profiles between normal chondrocytes and the cells that populate repair tissue reveal substantial functional differences between these two cell populations. At the four-month postoperative time point, the relative deficiency within repair tissue of gene transcripts which typically define articular cartilage indicate that while cells occupying the lesion might be of mesenchymal origin, they have not recapitulated differentiation to the chondrogenic phenotype of normal articular chondrocytes.
Full-thickness articular cartilage defects that penetrate into the subchondral bone undergo a repair process characterized by the in-growth of fibrous tissue within the lesion [1, 2]. Initially, blood from the bone marrow below the articular cartilage fills the defect and forms a fibrin clot [2, 3]. Subsequent to vascularization of the defect is the proliferation of granulation tissue over the first 10 days as the clot scleroses [2, 3]. The granulation tissue is rich in type I collagen fibers and the cells within the tissue have been traced to a mesenchymal origin [2, 4–6]. Within full-thickness defects generated by arthrotomy and controlled drilling into the subchondral bone, not more than 30% of total collagen content is type II four months after surgery . Type I fibrillar collagen predominates the extracellular matrix in repair tissue of most full-thickness defects without graft or transplant [4, 7]. Decreases in proteoglycan content also occur which render the repair tissue more rigid and unable to fully protect the joint from biomechanical stress [1, 4, 5, 7]. In addition, morphological differences exist between the cells in repair tissue and the chondrocytes of skeletally mature articular cartilage . Repair tissue anchors incompletely to the surrounding articular cartilage matrix adjacent to the lesion . While repair tissue seems to be primarily derived from stromal cells of mesenchymal origin, the functional similarity of these cells to articular chondrocytes is not completely described. Repair tissue is often called fibrocartilage or hyaline-like repair cartilage, though it does not necessarily contain an actual chondrocyte cell population.
The engineering of repair tissue cells is widely investigated in an attempt to improve the chondral surface within injured joints. Techniques like microfracture have been developed in an effort to facilitate healing of the articular surface with cells from the subchondral bone [6, 8–13]. There is also a focus on manipulating repair tissue, implanted stem cells, and even autologous chondrocyte transplants in an effort to generate more hyaline-like phenotypes [14, 15]. Assessment of the similarity of repair tissue to cartilage is typically done by monitoring established matrix biomarkers, such as type I collagen, type II collagen, and aggrecan core protein. Even with the introduction of growth factors or scaffolds of maintenance proteins associated with the chondrocyte phenotype, the repair tissue is still unable to completely restore the structural and biomechanical integrity of the joint surface, consistent with the limited capacity of articular cartilage to heal.
In this study, we used an equine cDNA microarray containing 9,413 probe sets to compare gene expression profiles of grossly normal articular cartilage and repair tissue occupying medial femoral condyle full-thickness defects in the femorotibial joints of skeletally mature horses four months after a microfracture surgical procedure. The hypothesis tested was that the cells occupying repair tissue four months postoperatively are not identical to articular chondrocytes. Consequently, we would expect the transcriptomes of cells from each tissue to have substantial differences, especially with respect to the expression of cartilage matrix biomarkers.
Articular cartilage defects were made in the axial weightbearing portion of the medial femoral condyles of four adult Quarterhorses (2-3 years) as previously described by Frisbie et al. [6, 16] within the guidelines set forth in an Institutional Animal Care and Use Committee-approved protocol at Colorado State University. Briefly, one-cm2 full-thickness articular cartilage lesions were arthroscopically made bilaterally which included the removal of the calcified cartilage layer. This was followed by microfracture penetration of the subchondral bone to create perforations with an approximate spacing of 2-3 mm and depth of 3 mm uniformly within the defect site. The horses were maintained for four months in box stalls (3.65 m × 3.65 m) with controlled hand walking. After euthanasia, repair tissue from the lesions and full-thickness grossly normal articular cartilage from within the same joint were collected from each stifle, rinsed in sterile phosphate-buffered saline, snap-frozen in liquid nitrogen, and stored at -80°C.
Samples were also collected and prepared for histological analyses as described in Frisbie et al. . Briefly, repair tissue and adjacent cartilage were trimmed with a standard bone saw and Exakt bone saw with a diamond chip blade (Exakt Technologies, Oklahoma City, OK, USA), placed into histological cassettes, and then fixed in 10% neutral buffered formalin for a minimum of 2 days. Samples were then applied to 0.1% EDTA/3%HCl decalcification solution (Thermo Scientific Richard-Allan Decalcifying Solution, cat. no. 8340) which was replenished every three days until specimens were decalcified. Specimens were embedded in paraffin and sectioned at 5 μm. Sections were stained with hematoxylin and eosin or with Safranin-O.
Total RNA Isolation and Linear Amplification
Normal articular cartilage was reduced to powder with a BioPulverizer (BioSpec Products, Bartlesville, OK, USA) under liquid nitrogen and total RNA was isolated as described by MacLeod et al. [18, 19]. Briefly, total RNA was isolated in a buffer of 4 M guanidinium isothiocyanate, 0.1 M Tris-HCl, 25 mM EDTA (pH 7.5) with 1% (v/v) 2-mercaptoethanol, followed by differential alcohol and salt precipitations and then final purification using QIAGEN RNeasy columns [18–21]. Repair tissue sample sizes were minimal in size (10-50 mg). Repair tissue was placed in QIAzol reagent (QIAGEN), cut into 1-mm3 slices, and total RNA isolated using the QIAGEN RNeasy Lipid Tissue Mini Kit. RNA quantification and quality assessments were performed with a NanoDrop ND-1000 and a BioAnalyzer 2100 (Agilent, Eukaryotic Total RNA Nano Series II). Total RNA (1 μg) from each tissue sample received one round of linear amplification primed with oligo-dT (Invitrogen - SuperScript RNA Amplification System) [22, 23]. Two micrograms of amplified RNA were then used as a template to create fluorescent dye-coupled single-stranded aminoallyl-cDNA probes (Invitrogen - Superscript Indirect cDNA Labeling System, Molecular Probes - Alexa Fluor 555 and 647 Reactive Dyes). For each sample, probes were coupled to both Alexa Fluor dyes individually so that a dye swap comparison could be made.
Microarray slides were printed with clones selected from a cDNA library generated using equine articular cartilage mRNA from a 15-month old Thoroughbred . Microarray slides were pre- hybridized in 20% formamide, 5× Denhardt's, 6× SSC, 0.1% SDS, and 25 μg/ml tRNA for 45 minutes. The slides were then washed five times in deionized water, once in isopropanol, and spun dry at 700 g for 3 minutes . Two labeled cDNA samples, one repair tissue and the other normal cartilage from the same joint, were combined with 1× hybridization buffer (Ambion, 1× Slide Hybridization Buffer #1, cat. no. 8801), incubated for 2 minutes at 95°C, and then applied to the slide under a glass lifterslip for 48 hours at 42°C. All hybridizations were performed in duplicate with a dye swap to eliminate possible dye bias . Sequential post-hybridization washes were each for 5 minutes as follows: 1× SSC, 0.2% SDS at 42°C; 0.1× SSC, 0.2% SDS at room temperature; and twice with 0.1× SSC at room temperature. The slides were then spun dry under argon gas at 700 g for 3 minutes. Each slide was coated once in DyeSaver 2 (Genisphere) and allowed to dry for 10 minutes. Slides were scanned using a GenePix 4100A scanner and spot intensities were computed using GENEPIX 6.0 image analysis software (Axon Instruments/Molecular Devices).
Statistics and Analysis
with the components designated as follows: d, additive effects due to dye (red or green); c, chip effect (1-16); t, tissue (repair or normal); h, horse (1-4); l, leg (left or right); E, statistical error. The dye swap design yields two outcomes per location and tissue type. Thus, for each of the eight locations corresponding to a particular leg of a particular horse, a new aggregated quantity is calculated that takes into account all measurements related to this location. The only remaining systematic effect represents the expressional difference between tissues with remaining statistical error. Since there were 4 horses with 2 femorotibial joints per horse, eight such tissue differences were evaluated. Gene identity was assigned for each microarray ID from an internal annotation database through selection of either the best RNA RefSeq BLAST (E < 1 × 10-7) or Protein RefSeq BLAST (E < 1 × 10-5) result [29–31]. Gene ontology (GO) annotation was derived from batch queries of the Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics tool or manually through individual NCBI Entrez Gene queries [32, 33]. The human ortholog of each gene was predicted and used for the determination of overrepresentation of GO categories via Expression Analysis Systematic Explorer (EASE) standalone software [32, 34]. Statistical data, fold change quantities, and GO annotations were managed within an Excel spreadsheet (Microsoft, Redmond, WA). Microarray data are available at the NCBI Gene Expression Omnibus (GEO) under Series Accession GSE11760.
Validation of Microarray Hybridization Results with RT-qPCR
Primer nucleotide sequences used in RT-qPCR assays for genes described in the study.
Ribosomal protein, large, P0
Procollagen, type I, alpha 2
Procollagen, type II, alpha 1
Cartilage oligomeric matrix protein
Fibroblast activation protein
Repair tissue histology
Overall level of differential gene expression
Overrepresented ontological categories for transcripts with >2-fold difference in normal articular cartilage versus repair tissue.
signal transducer activity
calcium ion binding
regulation of transcription (3)
endopeptidase inhibitor activity (2)
protease inhibitor activity
steroid hormone receptor activity
insulin-like growth factor binding
Overrepresented ontological categories for transcripts with >2-fold difference in repair tissue versus normal articular cartilage.
response to biotic stimulus
organelle organization and biogenesis
cytoskeleton organization and biogenesis
response to external stimulus
calcium ion binding
protein complex assembly
cell adhesion (3)
hydrogen ion transporter activity (2)
Quantitative PCR validation
Histological analyses and transcriptional studies identified clear differences between chondrocytes of grossly normal articular cartilage and the cells present in repair tissue of full-thickness articular lesions following a microfracture surgical procedure. At four months post-surgery, repair tissue is morphologically discernible from normal cartilage. Type I collagen transcripts are detected in the repair tissue, and much of the repair tissue is proteoglycan-deficient. Moreover, a substantial transcriptional divergence is readily apparent between the two cell types even at a genomic level. Analyses of overrepresented gene categories for differentially expressed transcripts demonstrate broad functional differences.
Conventional biomarker transcripts used to characterize a chondrocytic phenotype indicated that the repair tissues in this sample set were quite different from the adjacent articular cartilage in the same joint. Increased transcript levels for types II and IX collagen were found in the articular cartilage (Figure 4). Quantitative RT-PCR indicated a 16.1-fold expression difference for COL2A1 in articular cartilage relative to repair tissue (p = 0.0090, Figure 6B). In contrast, abundance of transcripts associated with type I collagen-rich fibrous tissues were greater in repair tissue (Figure 4). Steady-state mRNA levels for COL1A2 were 77.1-fold higher in repair tissue relative to articular cartilage (p = 0.0485, Figure 6A). These transcriptional data directly support published biochemical results which demonstrated differing collagen type I: type II ratios for articular repair tissue and perilesional articular cartilage through detection of cleaved peptides [4, 7]. Differences in the magnitude of fold changes in microarray and RT-qPCR results can be explained by the differences in dynamic range of detection between hybridization-based assays and amplification-based assays . Notable differences for proteoglycans between repair tissue and the surrounding articular cartilage were observed with transcript levels and by Safranin-O staining (Figures 1, 4). Proteoglycan differences have also been noted through Safranin-O staining of articular repair tissue in the distal femur of the New Zealand White rabbit  and in the distal radial carpal bone of the horse , relative to proteoglycan content of perilesional articular cartilage in both studies.
Divergent characteristics between articular cartilage and repair tissue extend to transcripts of other matrix proteins. Transcripts encoding cartilage macromolecules believed to play a role in cell-cell and cell-matrix interactions were significantly less abundant in repair tissue relative to normal articular cartilage (Figure 4). Such transcripts included chondroadherin (CHAD), cartilage intermediate layer protein (CILP), cartilage oligomeric matrix protein (COMP), and fibronectin (FN1) [39–44]. COMP interacts with type II collagen for fibrillogenesis and has been shown to bind to the chondroitin sulfate glycosaminoglycans associated with aggrecan. COMP expression is initially up-regulated in chondrocytes exposed to increased dynamic compression [45, 46], those from the superficial zone in fibrillated OA cartilage , and chondrocytes adjacent to an OA lesion ; however, transcript levels in repair tissue at the four month time point were 30.5-fold lower (p = 0.0010, Figure 6C). Matrix molecules like CILP which are present in normal cartilage slow down the responsiveness of chondrocytes to insulin-like growth factor 1 (IGF-1) as a result of accumulation of calcium pyrophosphate dehydrate . Thus, CILP might inhibit the ability of the surrounding chondrocytes to expand and occupy the lesion . Transcript abundance for hypoxia inducible transcription factor 2α (HIF-2α) was up-regulated in normal cartilage (Figure 5) and has been found to support the cartilage phenotype by (SRY-box 9) SOX9 induction of matrix genes . In contrast, tenascin-C (TNC), which is typically found in provisional matrices throughout development and wound healing [49–52], demonstrated greater transcript levels in repair tissue by microarray analyses (Figure 4). While statistical significance was not confirmed by RT-qPCR (p = 0.0665, Figure 6F), upregulation of TNC has been noted in early stages of osteoarthritis and also during the repair process of many other tissues through in situ hybridization, immunohistochemistry, and knockout mouse studies [49, 53–55]. Based on its function in the expansion of provisional matrices, it is likely that analyses of earlier time points would have detected greater divergence of TNC mRNA levels.
Within the repair tissue, differential expression was noted for transcripts encoding proteins involved in wound healing and matrix synthesis. Shapiro et al. have shown that stromal cells of mesenchymal origin from the subchondral bone enter into the wound with the blood which fills the full-thickness lesion . With angiogenic cues such as vascular endothelial growth factor (Figure 5) and vascularization from the subchondral bone, these cells proliferate within the granulation tissue to occupy the lesion [2, 56, 57]. Increased transcript abundance of fibroblast activation protein (FAP) is consistent with the proliferative cellular response reported by Shapiro et al. (Figure 5) . RT-qPCR indicated a 2.6-fold relative expression difference for FAP in repair tissue four months post-microfracture relative to articular cartilage (p = 0.0415, Figure 6E). Assessment of FAP expression at additional time points during the repair process would further delineate its importance. Steady-state levels of dermatopontin (DPT) were also elevated in repair tissue (Figures 5, 6D). Fibrillogenesis of type I collagen is accelerated by DPT, which has previously been localized in skin fibroblasts, skeletal muscle, heart, lung, bone, and chondrocytes that de-differentiate while expanding in monolayer culture [58–60]. DPT interacts synergistically with decorin and transforming growth factor-β1 to bolster collagen synthesis and accelerate fibrillogenesis to the point of decreasing fibril diameters in proliferating skin fibroblast cultures . A wound healing process is further indicated by the 7.5-fold up-regulation of cyclooxygenase 2 (COX2, Figure 5), an inflammatory modulator shown to be essential in the repair of bone fractures and growth plate lesions . Transcript profiles for COX2 and S100 protein are compatible with chondrogenic differentiation of stromal cells [61, 62], but the consistent deficiency of cartilage matrix protein biomarkers highlighted by the switch of type I collagen (COL1A1, COL1A2) in place of type II collagen (COL2A1) as the primary fibrillar collagen document the failure of true hyaline cartilage restoration.
A limitation of this study must be noted. Tissues utilized in these experiments included repair tissue and grossly normal articular cartilage from within the same joint. Thus, any gene expression differences between grossly normal cartilage within the lesioned joint and cartilage from an intact articular surface from another joint were not assessed. Differences have been reported with intact cartilage from human OA joints . However, equine joints used for the current sample set had minimal OA and the defects were freshly created in the medial femoral condyles four months prior to tissue sample collection.
Transcriptional profiling data support the hypothesis and indicate that repair tissue cells following a microfracture surgical procedure are still very different from normal articular chondrocytes at the four month postoperative time point. The cell and matrix organizational phenotypes of repair tissue are substantially different from those of chondrocytes within mature articular cartilage that has developed and adapted to biomechanical strains from birth. Microarray data in the current study corroborate what has been reported previously at mRNA and protein levels for conventional cartilage biomarkers, but extends our understanding by documenting differences in transcript abundance across multiple ontology categories and genes not previously studied in these tissues. By directing further research toward factors which contribute to the transcriptome dissimilarities of repair tissue and normal articular cartilage phenotypes, we should advance our understanding of the repair process and improve upon therapeutic strategies directed at restoring the structural and biomechanical integrity of the joint surface.
List of Abbreviations
cartilage intermediate layer protein
collagen, type 1, alpha 2
collagen, type 2, alpha 1
cartilage oligomeric matrix protein
Expression Analysis Systematic Explorer
fibroblast activation protein
Gene Expression Omnibus
insulin-like growth factor-1
National Center for Biotechnology Information
quantitative polymerase chain reaction
ribosomal protein, large, P0
sex-determining region homeobox-9
Dr. Mark Band and the W.M. Keck Center for Comparative and Functional Genomics at the University of Illinois are graciously acknowledged. The authors also appreciate Rebekah Cosden for her insight into RNA isolation of small samples and Dr. Timothy McClintock for his advice in functional genomics analysis. Financial support was received from the Gluck Equine Research Foundation, The Geoffrey C. Hughes Foundation, The Morris Animal Foundation (Training Fellowship D06EQ-409 to M.M. and the Consortium for Equine Medical Genetics D07EQ-500), and the NIH (KY-INBRE P20 RR16481).
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