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. 2008 Jul 8;213(3):266–273. doi: 10.1111/j.1469-7580.2008.00942.x

Glycosaminoglycans in the pericellular matrix of chondrons and chondrocytes

Qi Guang Wang 1, Alicia J El Haj 1, Nicola J Kuiper 1
PMCID: PMC2732045  PMID: 18631286

Abstract

This is the first study to quantitate and profile the glycosaminoglycan (GAG) composition of the pericellular matrix (PCM) of chondrons and chondrocytes using the highly sensitive technique; fluorophore-assisted carbohydrate electrophoresis (FACE). Bovine articular chondrocytes and chondrons were isolated enzymatically. High cell yield and viability were obtained for both preparations. Chondrons had strong immunofluorescent labeling for keratan sulphate and chondroitin-6 sulphate but no labeling for hyaluronan. We compared the immunofluorescent data with FACE. The quantities of total keratan sulphate were determined to be 0.013±0.002 pg cell−1 and 0.032 ±0.003 pg cell−1 in the chondrocyte and chondron preparations, respectively. Four internal keratan sulphate sugars were detected (galβ1, 4glcNAc6S, gal6Sβ1, 4glcNAc6S, glcNAcβ1, 3gal and glcNAc6Sβ1, 3gal) for both preparations but they were present at significantly higher concentrations in chondron preparations (P<0.01). Total chondroitin sulphate (CS) was determined to be 0.054±0.004 pg cell−1 and 0.077±0.005 pg cell−1 for chondrocyte and chondron preparations, respectively. Unsulphated CS disaccharide levels were similar but chondrons had significantly more chondroitin-4 sulphated disaccharides and chondroitin-6 sulphated disaccharides (P<0.05). Hyaluronan acid was present at low concentrations (0.010±0.001 pg cell−1) in both chondrocytes and chondrons. In this study, enzyme digestion coupled with FACE separation revealed new information about the differences in GAGs from isolated chondrocyte and chondron preparations. Further investigation of the differences in GAGs from chondrocytes and chondrons from different zones of articular cartilage may be useful for tissue engineering approaches.

Keywords: cartilage, chondron, chondrocyte, fluorophore-assisted carbohydrate electrophoresis (FACE), glycosaminoglycans, tissue engineering

Introduction

The chondrocyte and its associated hydrated pericellular matrix (PCM) are termed a chondron (Smirzai, 1974). The chondron is widely considered to be the primary structural, functional and metabolic unit of articular cartilage (Poole, 1997). Currently, chondrocytes isolated without a PCM are the preferred choice for cartilage tissue engineering studies. However, the retention of the in vivo PCM has been reported to influence chondrocyte gene expression positively, to stabilize the chondrocyte phenotype, and to give rise to improved extracellular matrix production (Larson et al. 2002; Graff et al. 2003). Enzymatically isolated chondrocytes do produce a new PCM in a matter of hours, but it appears to interfere with the formation of further extracellular matrix around the cell (Lee & Loeser, 1998). The newly formed PCM is different to the PCM surrounding freshly enzymatically isolated chondrons and it is thought to take 2–3 weeks to fully mature (Lee & Loeser, 1998). The chondron PCM has been well characterized in tissue sections by techniques such as immunolocalization and guanidine extraction (Poole et al. 1990). It is rich in sulphated and non-sulphated glycosaminoglycans (GAGs), large and small proteoglycans, and collagens. In articular cartilage, the PCM contains many of the same molecular components as the extracellular matrix; however, there are some distinct differences. The PCM can be primarily defined by a layer of collagen type VI at the cell surface (Keene et al. 1988). Collagen type VI has been co-localized with fibronectin (Burton-Wurster et al. 1997). The PCM also contains high concentrations of collagen types II, XI and IX (Poole et al. 1988b; Wotton et al. 1991; Hagg et al. 1998). High concentrations of chondroitin-6 sulphate, keratan sulphate and hyaluronan have been reported (Poole et al. 1990; Poole, 1997). To date, studies have presented the total amount of each GAG but not the qualitative composition of each GAG, e.g. disaccharide units or sulphations.

Characterization of the components of the PCM has been predominantly based upon studies using mechanically isolated chondrons (Poole et al. 1991; Guilak et al. 2005). Their PCM is considered to be more intact than enzymatically prepared chondrons (Burton-Wurster et al. 1997; Lee et al. 1997). However, the mechanical isolation of chondrons has several inherent difficulties which make it hard to prepare reproducible chondron preparations. Mechanical isolation involves slow speed serial homogenization, which gives rise to a flocculent homogenate containing intact viable and non-viable chondrons, ‘empty’ PCM capsules, tissue debris and collagenous debris (Poole et al. 1988a). Generally, mechanically isolated chondron yields are low and the cells demonstrate poor viability (Poole et al. 1988a). Also, chondron preparations cannot be separated from the associated tissue and collagenous debris. Enzymatically prepared chondron techniques provide a higher cell yield, a better cell viability and preparations that are free from tissue debris (Burton-Wurster et al. 1997; Lee et al. 1997).

Herein, we compared two different enzymatic protocols for the preparation of chondrons and chondrocytes from bovine cartilage. The cell-associated matrix was analysed by immunohistochemistry. For the first time we profiled and quantitated GAGs using the highly sensitive technique of fluorophore-assisted carbohydrate electrophoresis (FACE) (Calabro et al. 2001; Sharma et al. 2007).

Materials and methods

Isolation of chondrocytes and chondrons

Full depth articular cartilage was dissected from the articulating surface of the trochleal humerus of 18-month-old cows. Four separate isolations were performed, each using one humerus. Chondrocyte isolation was performed as previously described (Kuettner et al. 1982). Briefly, diced cartilage was sequentially digested with 700UmL−1 Pronase E™ for 1h, then 200UmL−1 collagenase XI and 0.1mgmL−1 DNase 1 for 16h. Chondrocytes from the supernatant were strained through a 70-µm cell sieve, washed in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS) and centrifuged at 750g. The cells were washed three times.

For chondron isolation, we modified a previous published protocol (Lee et al. 1997). Again, four separate isolations were performed, each using one humerus. Diced cartilage was digested with 3.3UmL−1 dispase and 560UmL−1 collagenase type XI in DMEM for 5h. These conditions achieved the optimal cell viability and cell yield (data not shown). The cell suspension was filtered through a 70-µm cell sieve, washed in DMEM supplemented with 10% FCS and centrifuged at 400g. The cells were washed three times.

Assessment of cell viability

Viability of chondrocytes and chondrons was assessed using Trypan blue exclusion and Cellstain double staining (Live/Dead Cell Double Staining Kit, BioChemika) (Yoshida et al. 1998). The kit contains calcein-AM and propidium iodide, which stain viable and non-viable cells, respectively.

Assessment of cell number

Cell number was assessed by Picogreen™ DNA quantitation (Ahn et al. 1996). The previously reported value of 7.7pg of DNA per chondrocyte was used to calculate DNA content (Kim et al. 1988). Isolated cells were digested with 5UmL−1 pronase K in 100mm ammonium acetate, pH 7.8, at 65°C for 4h. Fluorescence was read against a standard curve of calf thymus DNA.

Immunohistochemistry

Immunohistochemistry was performed using primary antibodies against the following components of the PCM: collagen type VI (Rockland), collagen type II (Abcam), chondroitin-6 sulphate (Chemicon), keratan sulphate (Abcam), fibronectin (Sigma) and hyaluronan (biotinylated hyaluronic acid binding protein provided by Dr Raija Tammi, University of Kuopio, Finland). Isolated chondrons and chondrocytes were first immobilized onto glass slides in 3% (w/v) agarose gels (Poole et al. 1991). They were then fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS). For chondroitin-6 sulphate, keratan sulphate (KS) and hyaluronan (HA), cells were initially treated with 25mUmL−1 chondroitinase ABC to generate ‘stubs’ of unsaturated disaccharides on the proteoglycan core proteins and to unmask epitopes. For type VI collagen and type II collagen; cells were initially treated with 2mgmL−1 testicular hyaluronidase. After initial treatments, the cells were incubated with the primary antibody, then labeled with the FITC-conjugated secondary antibody and finally stained with propidium iodide. Cells were observed under a Leica DM IRB fluorescence microscope and images were captured using a Leica DC 200 digital camera. The negative control comprised chondrons or chondrocytes treated with PBS alone, in place of primary antibodies. The positive control comprised sections (10µm) of bovine intervertebral disc.

Preparation of glycosaminoglycan saccharides for FACE

The GAGs were extracted from chondron and chondrocyte preparations as described previously (Calabro et al. 2000b; Sharma et al. 2007). In brief, purified GAGs were depolymerized using GAG-specific enzymes (Seikagaku). Hyaluronic acid (HA) was digested into disaccharides (ΔDiHA) using 100mUmL−1 of hyaluronidase Streptococcus dysgalactiae (SD) for 1h at 37°C (Calabro et al. 2001; Karousou et al. 2004). CS was digested into disaccharides (chondroitin-6 sulphate, ΔDi6S; chondroitin-4 sulphate, ΔDi4S; and unsulphated, ΔDi0S) with 100mUmL−1 of chondroitinase ABC (cABC) for 3h at 37°C. Sulphation of CS was confirmed by incubation of cABC-digested samples with 100mUmL−1 of chondroitin-4ase and/or chondroitin-6ase for 12h at 37°C. The non-reducing terminal sugars of CS were identified by mercuric ion treatment (Calabro et al. 2000a).

Keratan sulphate (KS) was digested as previously described (Plaas et al. 2001; Sharma et al. 2007). Briefly, samples were incubated with 100mUmL−1 of keratanase II for 3h at 37°C or 100mUmL−1 of endo-β-galactosidase for 14h. KS digestion was confirmed by sequential digestion with 100mUmL−1 of keratanase II for 3h and 100mUmL−1 of endo-β-galactosidase for 14h and conversely with 100mUmL−1 of endo-β-galactosidase for 14h and 100mUmL−1 of keratanase II for 3h. This approach generated six major digestion products from the internal KS chain (unsulphated disaccharide, glcNAcβ1,3gal; monosulphated disaccharides, glcNAc6Sβ1, 3gal and galβ1, 4glcNAc6S; disulphated disaccharide, gal6Sβ1, 4glcNAc6S; disulphated tetrasaccharides, galβ1, 4glcNAc6Sβ1, 3galβ1, 4glcNAc6S; trisulphated tetrasaccharides, galβ1, 4glcNAc6Sβ1, 3gal6Sβ1, 4glcNAc6S) (Plaas et al. 2001).

Fluorotagging and FACE separation

Lyophilized enzyme-digested samples and pre-defined saccharide standards (Seikagaku) were reconstituted with the fluorescent tag, 5µL 12.5mm 2-aminoacridone, in glacial acetic acid/DMSO (3:17, v/v) and incubated at room temperature for 15min. Five µL of 1.25m sodium cyanoborohydride in distilled deionized water was added. Samples were incubated at 37°C for 16h. After tagging, 10µL of 25% glycerol (20% v/v final concentration) was used to quench excess sodium cyanoborohydride. Electrophoresis was carried out for 80min at 4°C as previously described (Grande-Allen et al. 2003; Sharma et al. 2007).

FACE gel imaging and quantitation

Gels were placed on a transilluminator light box fitted with a 312-nm light source. Fluorescent images were captured using a High CCD Camera (UVP, Cambridge, UK) and the mean pixel density for each product band was quantified using LabWorks Software (UVP). For each gel, FACE product bands were identified by their co-electrophoresis with a range of pre-defined fluorotagged saccharide standards. Two image exposures were captured. The first exposure was used for quantitation as it had all of the pixels within a linear 12-bit depth range to provide baseline data. The second exposure had an over-saturated pixel intensity allowing identification of less abundant structures. Accurate quantitation was achieved between 10 and 400 pmol of product.

Statistical analysis

Four separate preparations of chondrocytes and chondrons were made and analysed. Each preparation used one humerus. The tissue from each humerus was divided into two portions so that the results for chondrons could be compared directly with the results for chondrocytes. Where appropriate, results are represented as the mean±SD. Statistical differences between chondrocytes and chondrons were examined using the Mann–Whitney U-test with two significance levels; *P<0.05 and **P<0.01.

Results

Isolation of chondrocytes and chondrons

Homogeneous populations of chondrocytes and chondrons were obtained (data not shown). They had good viability (>99%) as assessed by Trypan blue exclusion. Chondrocytes were 12±2.5µm in diameter and chondrons were 14± 2.5µm in diameter. These data are comparable with previously published values (Lee et al. 1997). The yield of chondrocytes (13±2.5 million cells per g of wet tissue) and of chondrons (9±1.5 million cells per g wet tissue) were determined by Picogreen DNA quantitation.

Immunohistochemistry

Figure 1 shows the immunofluorescence data for markers of the PCM. Both chondrocytes and chondrons were evaluated using the same exposure time, gain and offset camera settings so that the immunofluorescence in both cell preparations was directly comparable for each given antibody. Strong immunofluorescence labeling for collagen type VI, collagen type II and KS was seen with chondrons but not with chondrocytes. Chondrons had more immunofluorescence for chondroitin-6 sulphate than did chondrocytes. Following enzymatic isolation, we did not detect immunofluorescence for HA or fibronectin with chondrons or chondrocytes. These data were confirmed by comparison with positive controls using bovine intervertebral disc.

Fig. 1.

Fig. 1

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Immunofluorescence microscopy of pericellular matrix markers. The red fluorescence indicates the locations of cell nuclei and green fluorescence indicates the presence of the pericellular matrix markers. Bar=10µm.

Profile of glycosaminoglycans

GAGs were profiled by FACE. Figure 2 is a representative FACE gel showing the co-electrophoresis of a known concentration of pre-defined fluorotagged saccharide standards alongside hyaluronidase (lane 1) and chondroitinase digestion (lane 2–6) products obtained from freshly isolated chondrons (1.5 million cells per lane) and chondrocytes (1.5million cells per lane). Lane 7 is the negative control. Images were captured within a linear 12-bit depth range to provide baseline data (Fig. 2A,B). Images were further exposed to over-saturate pixel intensity to allow identification of less abundant structures (Fig. 2C,D). Lane 2 shows the CS disaccharides (ΔDi0S, ΔDi4S and ΔDi6S) after cABC digestion. The CS disaccharides were digested using chondroitin-4ase (lane 4), chondroitin-6ase (lane 5) or chondrotinase-4ase/chondroitin-6ase (lane 6) to confirm the presence of sulphation. The identities of ΔDi0S, ΔDi4S and ΔDi6S bands were further confirmed by mercuric ion treatment (lane 3) (Calabro et al. 2000a). This also yields non-reducing termini in the appropriate molar amount. No non-reducing termini were detectable, suggesting that they were below the level of detection by FACE.

Fig. 2.

Fig. 2

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Representative FACE gels for analysis of HA and CS in preparations of chondrocytes and chondrons. The first exposure (A,B) was used for quantitation to provide baseline data. The second exposure (C,D) had over-saturated pixel intensity to allow identification of less abundant structures. Lanes S1 and S2 contain pre-defined fluorotagged saccharide standards. S1: N-acetyl galactosamine (GalNAc) (1), ΔDiOS (2), N-acetyl galactosamine-6 sulphate (GalNAc6S) (3), N-acetyl galactosamine-4 sulphate (GalNAc4S) (4) and 4-/6-sulphated N-acetyl galactosamine (GalNAc4,6S) (5). S2: DiHA (6); ΔDi6S (7), ΔDi4S (8); dermatan sulphate disaccharides ΔDi2S (9), ΔDi4,6S (10) and Di2,6S (11). Samples were digested with hyaluronidase Streptococcus dysgalactiae (SD) (lane 1), cABC (lanes 2), cABC+mercuric ion treatment (lane 3), cABC+chondroitin-4ase (lane 4), cABC+chondroitin-6ase (lane 5) and cABC+chondroitin-4ase/chondroitin-6ase (lane 6). Lane 7 is the negative control. Arrows indicate the migratory position of ΔDiHA (a), ΔDiOS (b), ΔDi6S (c), ΔDi4S (d), GalNAc (b’), GalNAc6S (c’), and GalNAc4S (d’).

The FACE data for HA and CS are summarized in Fig. 3. Total CS and HA in the chondrocyte preparation were determined to be 0.054±0.004 pg cell−1 (n = 4) and 0.010± 0.001 pg cell−1 (n = 4), respectively. For the chondron preparation, CS and HA were determined to be 0.077± 0.005 pg cell−1 (n = 4) and 0.010±0.001 pg cell−1 (n = 4), respectively. The total CS comprised ΔDi6S (37.9%), ΔDi4S (47.9%), and ΔDi0S (14.2%) for chondrocytes and ΔDi6S (46.1%), ΔDi4S (45.9%), and ΔDi0S (8.0%) for chondrons. No other disaccharide bands were observed. There were no significant differences for ΔDiHA and ΔDiOS for the preparations. By contrast, ΔDi4S and ΔDi6S were significantly increased in the chondron preparation (Fig. 4, *P< 0.05 and **P<0.01).

Fig. 3.

Fig. 3

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Summary of FACE data for HA and CS. The concentrations of the HA and CS disaccharides derived from chondrons and chondrocytes as assessed by FACE. Values were plotted as pg cell−1. Each bar represents the mean±SD from four separate experiments. Statistical differences were examined using the Mann–Whitney U-test with two significance levels: *P<0.05 and **P<0.01.

Fig. 4.

Fig. 4

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Representative FACE gels for KS in preparations of chondrocytes and chondrons. Lanes S1–S4 are the KS standards derived by digestion with keratanase II (S1), endo-β-galactosidase (S2), keratanase II/endo- β-galactosidase (S3) and endo- β- galactosidase/keratanase II (S4). S1: galβ1, 4glcNAc6S (1), galβ1, 4glcNAc6S β1, 3gal β1, 4glcNAc6S (2), gal β1, 4glcNAc6S β1, 3gal6S β1, 4glcNAc6S (3), gal6S β1, 4glcNAc6S (4). Lane S2: glcNAc β1, 3gal (5) and glcNAc6S β1, 3gal (6). Lanes 1–4 illustrate the samples digested with keratanase II (lane 1), endo-β-galactosidase (lane 2), KII/Endo (lane 3) and Endo/KII (lane 4) treatments. Lane 5 is the negative control. Arrows indicate the migratory position of galβ1, 4glcNAc6S (a), gal6Sβ1, 4glcNAc6S (b), glcNAcβ1, 3gal (c), and glcNAc6Sβ1, 3gal (d).

Figure 4 is a representative FACE gel showing the co-electrophoresis of a known concentration of pre-defined saccharide standards alongside keratanase II and endo-β-galactosidase digestion products obtained from freshly isolated chondrons (1.5 million cells per lane) and chondrocytes (1.5 million cells per lane). The quantities of total KS in the chondrocyte and chondron preparation were determined to be 0.013±0.002 pg cell−1 (n = 4) and 0.032±0.003 pg cell−1 (n = 4), respectively. For total KS in the chondrocyte preparation, FACE detected four internal sugars [galβ1, 4glcNAc6S (11.3%), gal6Sβ1, 4glcNAc6S (19.8%), glcNAcβ1, 3gal (21.4%) and glcNAc6Sβ1, 3gal (47.5%)]. For total KS in the chondron preparation, different preparations of the same four internal sugars were detected [galβ1, 4glcNAc6S (10.4%), gal6Sβ1, 4glcNAc6S (36.8%), glcNAcβ1, 3gal (19.8%), and glcNAc6Sβ1, 3gal (33.0%)]. No other saccharide bands were observed. Figure 5 summarizes the data obtained for the keratanase II and endo-β-galactosidase digestion products following FACE analysis of chondrocytes and chondrons. For KS analysis, there was a significant difference between the two preparations.

Fig. 5.

Fig. 5

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Summary of FACE data for KS. The concentrations of the KS saccharides derived from chondrons and chondrocytes as assessed by FACE. Values were plotted as pg cell−1. Each bar represents the mean±SD for four separate experiments. Statistical differences were examined using the Mann–Whitney U-test with two significance levels: *P<0.05 and **P<0.01.

To summarize the FACE data, Fig. 6 illustrates the proportions of the GAGs in each of the two preparations: HA (13.0%), CS (70.4%), KS (16.6%) for chondrocytes and HA (8.0%), CS (64.9%), KS (27.1%) for chondrons.

Fig. 6.

Fig. 6

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The percentage distribution of GAGs isolated from preparations of chondrocytes and chondrons. Each bar represents the mean±SD for four separate experiments. Statistical differences were examined using the Mann–Whitney U-test with two significance levels: *P<0.05 and **P<0.01.

Discussion

This is the first study to quantitate and profile the GAG composition of the PCM of chondrons using FACE separation. FACE data complemented the qualitative immunofluorescence data. FACE detected and quantitated GAG saccharides in the picomolar range. This is useful as immunofluorescence alone cannot provide true quantitation because staining intensity may be affected by antigen affinity, masking of epitopes, effectiveness of enzyme pre-treatment and stoichiometry.

The PCM surrounding mechanically isolated chondrons is reportedly rich in type VI collagen, fibronectin, type II collagen, CS, KS and HA (Poole et al. 1991; Poole et al. 1992). In our study we performed enzymatic digestion, using dispase and collagenase, to provide high yields of viable chondrons. With immunofluorescence, we detected the principal constituents, type VI and II collagen, but we did not identify any fibronectin. This result is similar to many previous studies using enzymatic digestion, which have shown that there is a substantial reduction in the size of the PCM microenvironment of enzymatically isolated chondrons compared with mechanically isolated chondrons (Lee et al. 1997; Knight et al. 2001; Hing et al. 2002). Our results are further supported by other studies. Ross et al. (2006) demonstrated the presence of type VI collagen, reduced amounts of type II collagen and aggrecan, and complete loss of fibronectin. As dispase has been demonstrated to cleave fibronectin (Stenn et al. 1989) we concluded that fibronectin was probably lost during enzymatic digestion.

FACE analysis detected 0.054±0.004 pg cell−1 and 0.077± 0.005 pg cell−1 total CS for chondrocytes and chondrons, respectively. The quantities of ΔDi0S were very similar for chondrons and chondrocytes but levels of ΔDi4S and ΔDi6S were higher in the chondron preparations. Although chondrons had the higher concentrations, the values were lower than previously reported for bovine-derived cells (Larson et al. 2002). The difference could be explained by the fact that Larson et al. quantitated total sulphated GAGs using the widely used dimethylmethylene blue (DMMB) assay (Farndale et al. 1986). DMMB is unable to differentiate between GAG types and can be inaccurate due to interference from polyanions from other sources.

There were more striking differences between the quantities of ΔDi4S and ΔDi6S, as assessed by FACE. The differences for ΔDi6S detected by FACE mirrored the immunofluorescence data obtained for chondroitin-6 sulphate. The CS digestion products identified in this study corresponded to those found in previously published studies (Calabro et al. 2000b; Sauerland et al. 2003). In our study, the ratios of CS disaccharides (ΔDi6S:ΔDi4S:ΔDi0S) in enzymatically digested chondrocytes and chondrons were both comparable to the ratio reported for cartilage explants (Sauerland et al. 2003). No non-reducing termini were detectable. The non-reducing termini are normally used to determine the average CS chain length.

For KS digestion, the proportion of each sugar following enzyme digestion coupled with FACE separation was comparable to previously published work (Whitham et al. 1999; Plaas et al. 2001). For both chondrocytes and chondrons, four internal sugars were detected; galβ1, 4glcNAc6S, gal6Sβ1, 4glcNAc6S, glcNAcβ1, 3gal, and glcNAc6Sβ1, 3gal. All four internal sugars were present at significantly higher concentrations in the chondron preparations. A previous study (Lee et al. 2000) detected KS on the plasma membrane of chondrons. In our study, the plasma membranes surrounding the chondrocytes may have contained remnants of KS that had been detected by FACE but not immunolocalization. We did not detect any galβ1, 4glcNAc6Sβ1, 3galβ1, 4glcNAc6S or galβ1, 4glcNAc6Sβ1, 3gal6Sβ1, 4glcNAc6S, which are reported to be present at very low concentrations in bovine cartilage (Poon et al. 2005). Their levels may have been too low for detection by FACE.

HA is known to be an integral component of both the PCM and the extracellular matrix enabling aggrecan interactions and cartilage homeostasis (Knudson & Knudson, 2001). HA has been reported to be present in the PCM surrounding chondrons isolated from human, chick and bovine cartilage tissues (Mason, 1981; Poole et al. 1991). In our study, HA was not detected by immunofluorescence despite attempting different enzyme treatments to unmask epitopes (data not shown). We were able to detect HA by enzyme digestion and FACE separation although it was at the lower limits of FACE detection. It is possible that HA was not retained during the enzymatic preparation of our bovine chondrons and chondrocytes. It has previously been reported that the hyaluronan receptor, CD44, can be partially lost from freshly isolated chondrons (Lee et al. 1997).

In this study, FACE separation revealed new information about the differences in GAGs from isolated chondrocyte and chondron preparations. Further investigation of these differences in cell preparations from different zones of articular cartilage could provide useful information for the development of tissue engineered cartilage. Many evolving tissue engineering strategies for cartilage repair rely upon either passage expanded chondrocytes or chemically manipulated progenitor cells. These cells are expected to differentiate towards a chondrogenic phenotype and are therefore required to undergo rapid changes in their phenotypes. Some studies have demonstrated that chondrons have been shown to produce more matrix over time than chondrocytes (Larson et al. 2002; Graff et al. 2003). Graff et al. (2003) maintained porcine chondrons in pellet culture for up to 12 weeks and demonstrated that the retention of the in vivo PCM during chondrocyte isolation promoted the formation of a mechanically functional neocartilage construct. Larson et al. (2002) utilized enzymatically isolated chondrons in pellet culture to demonstrate that chondrons increased matrix production and assembly during an 8-week culture period. There may be a population of chondrons which would be better suited for tissue engineering strategies. Consequently, it would be useful to study the effect of parameters such as a different cartilage source, age, or time in culture upon the GAG profiles of the PCM of chondrons from different zones.

Acknowledgments

This work was funded by the Engineering & Physical Sciences Research Council (EP/C511727/1). We thank Professor Sally Roberts, Director of Spinal Research, The Robert Jones & Agnes Hunt Orthopaedic Hospital, Oswestry, UK for the bovine intervertebral disc sections.

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