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Retention of the native chondrocyte pericellular matrix results in significantly improved matrix production
Matrix Biology 21 (2002) 349–359
Retention of the native chondrocyte pericellular matrix results in significantly improved matrix production Christopher M. Larsona,1, Scott S. Kelleya, A. Denene Blackwooda, Albert J. Banesa, Greta M. Leea,b,* a
Department of Orthopedics, University of North Carolina School of Medicine, Chapel Hill, NC, USA Thurston Arthritis Research Center, University of North Carolina School of Medicine, Chapel Hill, NC, USA
b
Received 4 September 2001; received in revised form 6 February 2002; accepted 25 March 2002
Abstract The interaction of the cell with its surrounding extracellular matrix (ECM) has a major effect on cell metabolism. We have previously shown that chondrons, chondrocytes with their in vivo-formed pericellular matrix, can be enzymatically isolated from articular cartilage. To study the effect of the native chondrocyte pericellular matrix on ECM production and assembly, chondrons were compared with chondrocytes isolated without any pericellular matrix. Immediately after isolation from human cartilage, chondrons and chondrocytes were centrifuged into pellets and cultured. Chondron pellets had a greater increase in weight over 8 weeks, were more hyaline appearing, and had more type II collagen deposition and assembly than chondrocyte pellets. Minimal type I procollagen immunofluorescence was detected for both chondron and chondrocyte pellets. Chondron pellets had a 10-fold increase in proteoglycan content compared with a six-fold increase for chondrocyte pellets over 8 weeks (P-0.0001). There was no significant cell division for either chondron or chondrocyte pellets. The majority of cells within both chondron and chondrocyte pellets maintained their polygonal or rounded shape except for a thin, superficial edging of flattened cells. This edging was similar to a perichondrium with abundant type I collagen and fibronectin, and decreased type II collagen and proteoglycan content compared with the remainder of the pellet. This study demonstrates that the native pericellular matrix promotes matrix production and assembly in vitro. Further, the continued matrix production and assembly throughout the 8-week culture period make chondron pellet cultures valuable as a hyaline-like cartilage model in vitro. 䊚 2002 Elsevier Science B.V. and International Society of Matrix Biology. All rights reserved. Keywords: Chondrons; Chondrocytes; Cartilage; Cultures; Pericellular matrix
1. Introduction Removal of the pericellular matrix during chondrocyte isolation may have a significant effect on subsequent matrix production and assembly in culture. Cell–extracellular matrix interactions affect matrix metabolism, growth factor regulation, and gene expression (Adams and Watt, 1993; Klagsbrun and Baird, 1991; Hausser et al., 1994; Boudreau et al., 1995). The pericellular matrix may be an integral part of this regulation as it resides between the cell and the extracellular matrix. The *Corresponding author. Tel.: q1-919-966-0544; fax: q1-919-9661739. E-mail address: [email protected] (G.M. Lee). 1 Present address: Minneapolis Sports Medicine Center, Eden Prairie, MN, USA.
presence of a pericellular matrix on chondrocytes cultured in alginate eliminates the stimulation of matrix synthesis by TGF-b (van Osch et al., 1998). The presence of the native pericellular matrix also appears to promote the assembly of collagen fibrils (Lee and Caterson, 1995). Thus, the native pericellular matrix may be important for matrix production and assembly (Kelley et al., 1996). The pericellular matrix can be retained by isolating chondrons which consist of the chondrocyte and its native pericellular matrix (Poole et al., 1991). Chondrons can be enzymatically isolated by digestion with a mild protease, dispase, combined with collagenase (Lee and Caterson, 1995; Lee et al., 1997). Chondrons can be isolated because collagenase does not digest type VI collagen (Kielty et al., 1993), a major component of the
0945-053X/02/$ - see front matter 䊚 2002 Elsevier Science B.V. and International Society of Matrix Biology. All rights reserved. PII: S 0 9 4 5 - 0 5 3 X Ž 0 2 . 0 0 0 2 6 - 4
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Fig. 1. Photographs of whole chondrocyte (A,C) and chondron (B,D) pellets after 8 weeks in culture. (A,B) Pellet cultures photographed lying flat. (C,D) Pellet cultures photographed on their side. Ruler shows millimeters.
pericellular matrix. The pericellular matrix of enzymatically isolated chondrons retains types II and VI collagen, and aggrecan (Lee et al., 1997). It has been well established that chondrocytes grown in monolayer cultures lose their polygonal morphology as they undergo phenotypic change (Von der Mark et al., 1977). Chondrocytes that have lost their chondrocyte phenotype produce type I collagen instead of type II collagen and have decreased proteoglycan synthesis (Stewart et al., 2000; Von der Mark et al., 1977; Manning and Bonner, 1967). This is characteristic of fibrocartilage rather than hyaline cartilage. Our recent observations have shown that chondrons also lose their chondrocyte phenotype within 1 week in monolayer culture (unpublished). High density pellet culture systems, however, have been shown to maintain the chondrocyte phenotype (Stewart et al., 2000; Kato et al., 1988a, 1993; Johnstone et al., 1998; Ballock and Reddi, 1994; Manning and Bonner, 1967; Solursh, 1991). Manning and Bonner (1967) used chondrocyte pellet cultures obtained from adult human articular cartilage and observed maintenance of the chondrocyte phenotype and increased matrix synthesis compared with monolayer cultures over 2 weeks. Johnstone et al. (1998), achieved chondrogenesis of mesenchymal cells in pellet culture, but observed extensive type X collagen labeling, in addition to type II collagen. Stewart et al. (2000) evaluated several cartilage culture systems using an equine model and found that pellet and aggregate cultures supported matrix protein gene expression profiles more reflective of in vivo levels when compared with monolayer and explant cultures. Many studies utilizing chondrocyte pellet culture have used growth plate cartilage obtained from fetal, neonatal or immature animals and have observed chondrocyte hypertrophy and a tendency of the initial hyaline-like tissue to mineralize (Kato et al., 1988a, 1993; Ballock and Reddi, 1994; Solursh, 1991). More recent studies have used chondrocyte pellet cultures for in vitro assays (Croucher et al., 2000; Rebuck et al., 1999; Xu et al., 1996).
We hypothesized that, in addition to maintaining the chondrocyte phenotype, pellet culture would allow the chondron to retain its native pericellular matrix and that the presence of the native pericellular matrix would affect matrix production and assembly. We compared chondrons with chondrocytes obtained from adult human articular cartilage maintained for up to 8 weeks in pellet culture. For control of differences between individuals, chondrons and chondrocytes were isolated from each cartilage specimen. The pellets were evaluated with respect to size, DNA content, and matrix production including collagen types I, II and VI. 2. Experimental procedures 2.1. Cell culture Cartilage was obtained from osteoarthritic human knees at the time of joint replacement surgery as surgical waste (seven individuals, mean age 68 years, range 54– 81 years) with approval from our Institutional Review Board, Office of Human Research Studies. Within 1 h of removal from the knee, the cartilage was dissected from the whole joint except the patella and obvious osteophytic regions. Cartilage from each joint was handled separately. For comparison of chondrocytes to chondrons, cartilage from a joint was pooled and minced prior to dividing for enzymatic digestion. Chondrocytes were isolated by digestion with 1320 PUKyml pronase (Boehringer-Mannheim and Calbiochem) for 1 h followed by 0.4% collagenase (CLS-2, Worthington) for 3 h as previously described (Kuettner et al., 1982). Chondrons were isolated by digestion with 0.3% dispase (a neutral protease classified as an amino-endo peptidase produced by Bacillus polymyxa) (GIBCO) plus 0.2% collagenase (CLS-2, Worthington) in phosphate-buffered saline (PBS) for 5 h as previously described (Lee et al., 1997). After filtering through a 70 mm nylon mesh cell strainer (Falcon) and centrifuging at 400=g for 6 min, the cells were counted using a hemocytometer. Six hundred thousand cells in 2 ml aliquots of OptiMEM1 with GlutaMax1 (GIBCO, cat噛 51985-034) with additives (see below) were placed in 15 ml sterile, conical polypropylene culture tubes, and the cells were centrifuged at 400=g for 6 min. Cultures were maintained in Opti-MEM1 with penicillinystreptomycin, 2% FBS, 2.7 mM CaCl2, and 25 mgyml Phospitan C (L ascorbic acid-2-monophosphate, provided by Showa Denko America, Inc., Tokyo, Japan). The cell cultures were incubated in a humidified incubator at 378 C, 5% CO2. The medium was changed at 2-day intervals for all cultures. The pellets were harvested on day 0, and periodically through 8 weeks. 2.2. Immunofluorescence One-, 3- and 8-week pellet cultures were embedded in OCT embedding medium and frozen at y20 8C. A
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single pellet was used for each time point; duplicate sections were used for each antibody. The time course for three different knee specimens was examined. Twenty micron sections were cut using a cryostat (Frigocut 2800, Reichert-Jung). The sections were fixed in 3.7% formaldehyde, rinsed in PBS and blocked with 1% BSAyPBS. The following primary antibodies were used: C4F6 to type II collagen (provided by C. Chichester; Srinivas et al., 1993), Hfn7.1 to fibronectin (prepared by R.J. Klebe and obtained from the Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa City, IA; Schoen et al., 1982), H4C4 to CD44 (prepared by J.E.K. Hildreth and J.T. August, DSHB; Belitsos et al., 1990), 5D4 to keratan sulfate (provided by B. Caterson; Caterson et al., 1983), a rabbit polyclonal to type VI collagen with no cross-reactivity to fibronectin (GIBCO; Engvall et al., 1986), and Sp1.D8 to type I procollagen (prepared by H. Furthmayr, DSHB; Foellmer et al., 1983). The secondary antibodies were Cy3 labeled donkey anti-mouse, LRSC labeled donkey anti-mouse, and FITC labeled donkey anti-rabbit (Jackson Immunoresearch Laboratories, Inc., West Grove, PA). Nuclei were stained with Hoechst 33342 (10 mgy ml, Molecular Probes) and coverslips were mounted with polyvinyl alcohol mounting medium (Osborn and Weber, 1982) containing 1% n-propylgallate (Giloh and Sedat, 1982). 2.3. DNA analysis
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point for a given specimen. The pellets were not pooled at each time point. For each experiment, pellets were individually measured for proteoglycan and DNA content, and a mean of all measures at a given time point was used for statistical analysis. Chondron and chondrocyte pellets from the same specimen were used for comparative analysis of proteoglycan and DNA content. The experiments were repeated with cartilage from two different knee specimens, and comparisons were done between each of these by normalizing microgram proteoglycan to microgram DNA. 2.5. Competitive ELISA for type II collagen Pellets were frozen in liquid nitrogen and then pulverized using a seed crusher in 96-well format (HSC200, Perkin Elmer Wallac Inc.). The pulverized pellets were extracted with 4 M GuHCl overnight, rinsed with PBS then digested three times using freshly prepared 1 mgyml pepsin in 0.5 M acetic acid at 4 8C for 24 h for a total digestion time of 72 h. The digests were combined. The ELISA assay was performed essentially as previously described (Srinivas et al., 1993) using the C4F6 antibody to type II collagen at 1:8000 to coat the plates and horseradish peroxidase conjugated secondary antibody (Jackson Immmunoresearch Laboratories, Inc.) at approximately 0.1 mgyml with TMB Microwell Peroxidase Substrate System (KPL, Gaithersburg, MD). The ELISA was done on both the GuHCl extract and the pepsin digest.
For fluorometric DNA assays, the samples were digested with papain (Sigma Chemical Co., St. Louis, MO) at 60 8C overnight. Calf thymus DNA standards were prepared (0–50 mgyml DNA). Samples and standards were diluted in 10 mM Tris, 1 mM Na4EDTA, 0.1 mM NaCl (pH 7.3) and 33342 Hoechst (10 mgyml, Molecular Probes, Eugene, OR) was added at a 1:10 000 dilution. DNA content was then determined with a fluorometer using A365 nm excitation and A458 nm emission. DNA analyses were done on the same pellets as the sulfated glycosaminoglycan analyses.
One-way analysis of variance (ANOVA) was used to evaluate significant differences in PG and DNA content with time in culture for chondron and chondrocyte pellets. Statistical significance was determined within groups using multiple t-tests by the Bonferroni method and between chondron and chondrocyte pellets using two factor ANOVA with replication. The level of significance was set at P-0.05.
2.4. Measure of proteoglycan (PG) content
3. Results
Determination of sulfated-GAG was done on the papain digests using the dimethylmethylene blue (DMMB) dye from Serva Feinbiochemica (Heidelberg, Germany) in a binding assay as previously described (Chandrasekhar et al., 1987) using chondroitin sulfate C (shark cartilage extract, Sigma, St. Louis, MO) as the standard. The plates were read at an absorbance of 525 nm on a microtiter plate reader (Titertek Multiskan, MCCy340, ICN, Costa Mesa, CA) and the standards were plotted on a linear regression curve (Delta Soft 3, Biometallics). All analyses were carried out using quadruplicate chondron and chondrocyte pellets for each time
3.1. Gross pellets
2.6. Statistical analysis
The chondron pellets were more uniform, hyaline appearing and cohesive than the chondrocyte pellets which were very friable and difficult to handle for the first few weeks in culture. By 8 weeks, chondron pellets were well formed and similar to articular cartilage in vivo with respect to palpation and texture. The chondron pellets were noticeably larger than the chondrocyte pellets (Fig. 1). Over the 8 weeks in culture, the wet weight per pellet increased more for chondron (6.2"1.8 mg, mean"S.D.) than for chondrocyte pellets (3.1"0.9
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Fig. 2. Comparison of type II collagen immunofluorescence in chondron (A,C) and chondrocyte (B,D) pellets after 3 weeks (A,B) and 7 weeks (C,D) of culture. Nuclei (A9–D9) in viable chondrocytes were stained with Hoechst 33342. The insets show collagen fibrils at six-fold greater magnification. The photomicrographs in this figure and subsequent figures are of 20-mm-thick cryostat sections and were obtained as video images using an intensified video camera with gain and offset held constant. Bar, 50 mm.
mg) (P-0.001). This increase when normalized to DNA was 0.7 mgymg DNA and 0.2 mgymg DNA for chondrons and chondrocytes, respectively. 3.2. Immunofluorescence microscopy All chondron and chondrocyte pellet sections were evaluated using the same gain and offset camera settings so that the relative immunofluorescence between sections was directly comparable for a given antibody. Hoechst labeling of viable nuclei revealed good viability for both chondron and chondrocyte pellets and greater cell density for the chondrocyte pellets compared with chondron pellets at all times in culture. The most dramatic difference between chondron and chondrocyte pellets was seen with type II collagen immunofluoresence. Both chondron and chondrocyte pellets had an obvious increase in type II collagen immunofluorescence, including fibril formation, with time in culture but chondrons had dramatically more type II collagen immunofluoresence throughout the culture period (Fig. 2). There was no obvious difference in type I procollagen immunofluorescence between chondron and chondrocyte pellet cultures. With the exception of a thin edging of immunofluorescence to
type I procollagen that developed circumferentially about the outer surface of the pellets (see below), there was minimal immunofluorescence throughout the pellets (Fig. 3). The cells within chondron and chondrocyte pellets retained their polygonal cell shape as revealed by immunolabeling for CD44, a hyaluronan receptor on the chondrocyte cell membrane (Fig. 4). An exception was the thin superficial edging around the pellets where the cells were elongated. The polygonal shape and type II collagen production indicate that cells within both chondron and chondrocyte pellets maintained their chondrocyte phenotype. Compared with chondrocyte pellets, chondron pellets initially had much greater immunofluorescence for type VI collagen owing to retention of the native pericellular matrix. This disparity markedly decreased with time in culture, apparently due to type VI collagen synthesis increasing much more in chondrocyte than chondron pellets. In fact, there was only a slight increase in type VI collagen immunofluoresence for chondron pellets with time in culture (Fig. 5). The chondron morphology is still evident after 3 weeks in culture (Fig. 5C). The superficial layer or rim of flattened, fibroblastlike cells developed circumferentially about both chon-
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Fig. 4. After 3 weeks in culture, the chondrocytes had a polygonal shape as revealed by immunofluorescence for CD44, a hyaluronan receptor on the plasma membrane. (A) Three-week chondron pellet culture. (B) Three-week chondrocyte pellet culture. Bar, 20 mm.
Fig. 3. Type I procollagen labeling of chondron (A,C) and chondrocyte (B,D) pellet cultures at 3 weeks (A,B) and 8 weeks (C,D) shows labeling primarily in the thin rim area surrounding the pellet. The chondrocyte pellet section in (B) is narrower than the other sections. This is because the pellets were sectioned perpendicular to their long axis or flat surface and at 3 weeks the chondrocyte pellets were much thinner especially in the center of the pellet. The procollagen labeling is intracellular. Bar, 50 mm.
When examined over time, there was a significant increase in the amount of PG per microgram DNA for both chondron and chondrocyte pellets from day 0 to 8 weeks in culture (P-0.00001). When compared with chondrocyte pellets, chondron pellets had a significantly greater increase in total PG (5.2 mg PGymg DNA for chondrons, and 1.8 mg PGymg DNA for chondrocytes) (P-0.0001) from day 0 to 8 weeks in culture. There
dron and chondrocyte pellet cultures. This layer was not evident at 1 week in culture but became evident at 2–3 weeks and increased in cell number with time in culture (Fig. 6). Similar findings were noted previously with chondrocytes in pellet, micromass, and collagen disk cultures (Hauselmann et al., 1994; Johnstone et al., 1998; Nixon et al., 1993; Xu et al., 1996). In the present study, this rim had increased cell density and increased immunofluorescence to fibronectin and type I procollagen relative to the interior of the pellet (Figs. 3, 4 and 6). There was minimal to no immunofluorescence for type II collagen and keratan sulfate in this rim (Fig. 7). Table 1 summarizes the observed changes in immunofluorescence for the various extracellular matrix molecules for chondron and chondrocyte pellets and their rim with time in culture. 3.3. Proteoglycan analysis To account for any disparities in the cell number for chondron and chondrocyte pellet cultures, the PG data are presented as micrograms of PG per micrograms of DNA for pellets at each time point in culture (Fig. 8a).
Fig. 5. Immunolabeling for type VI collagen remained fairly constant for the chondron pellets (A, 3 week; C, 8 week), but was dramatically increased for chondrocyte pellets (B,D) from 3 weeks (B) to 8 weeks (D) of culture. Bar, 20 mm.
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Fig. 6. Immunolabeling for fibronectin reveals the development of a rim of flattened cells (arrows) surrounding the pellets. This figure is of chondron pellets at (A) 1 week, (B) 2 weeks, (C) 3 weeks and (D) 8 weeks of culture. The reduced immunofluorescence in the center of the pellet with time in culture may be due to either less fibronectin in this area or to epitope masking. The monoclonal, Hfn 7.1, is highly specific to human fibronectin and appears to bind to the cell-binding region (Schoen et al., 1982). Bar, 50 mm.
was a 10-fold increase in PG content for chondron pellets compared with a six-fold increase for chondrocyte pellets over 8 weeks in culture (P-0.001). Chondron and chondrocyte pellets released similar amounts of proteoglycan into the culture media over time (Fig. 8b). Comparison of proteoglycan in the pellets with the cumulative amount of proteoglycan released into the media shows that during the first week of culture there was more proteoglycan released than retained by both chondrons and chondrocytes. By day 21, the chondrons had retained considerably more proteoglycan in the pellets than was released into the media while the chondrocytes had a similar amount of proteoglycan in the pellets and media. 3.4. DNA analysis There was no significant difference in DNA content at day 0 between chondron (6.8 mg) and chondrocyte
pellets (6.2 mg) (Ps0.61) (Fig. 9). Both chondron and chondrocyte pellets had a decrease (15–37%) in DNA content from day 0 to 1 weeks, but this was only statistically significant for chondrocytes (P-0.01). Throughout this experiment we noted a thin lining of cells in the culture tubes formed by cells that had migrated from the pellets. This appeared to occur more frequently with chondrocyte than chondron pellet cultures and was evident after several days in culture. Cell adherence to the tube sidewall occurred despite using polypropylene tubes treated with 0.6 mgyml poly-Hema (Sigma). From 1 to 8 weeks in culture there was a minimal increase in DNA content for both chondron (13%) and chondrocyte (21%) pellets, but this was not statistically significant. 3.5. Type II collagen analysis Competitive ELISA of the GuHCl extracts and pepsin digests showed an increase in type II collagen with time
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Fig. 7. The superficial rim of the pellets did not label with antibodies to type II collagen (A) or keratan sulfate (C). These are sections of a chondron pellet after 8 weeks of culture. The corresponding nuclei of the chondrocytes are shown in (B) and (D). Note the increased cell density in the outer area. The edge of the sections is shown by white lines in (A,C). Bars (B) 20 mm and (D) 10 mm.
in culture (Fig. 10). The chondron pellets had a significantly greater amount of type II collagen than the chondrocyte pellets at all time points. This was most dramatic after 7 weeks in culture. 4. Discussion The present study demonstrated that chondron pellets had a significantly greater increase in size, proteoglycan
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content, and type II collagen content than chondrocyte pellets. The results of the DNA and sulfated glycosaminoglycan content analysis indicate that this greater increase in size for chondron pellets reflects increased matrix deposition rather than cell division. Matrix synthesis was not monitored in this study so we cannot say at this time whether the increased matrix deposition is due to differences in rates of synthesis or to greater retention and assembly. In preliminary work, we have found that, when maintained in alginate bead culture, chondrocytes synthesized more proteoglycans during the first week of culture, but chondrons synthesized more after the second week of culture (Kelley et al., 1996). Osteoarthritic human cartilage was used as a source of chondrocytes and chondrons primarily because normal human cartilage specimens were not as readily available. Chondrons isolated from osteoarthritic human cartilage are larger than those isolated from normal human cartilage (Lee et al., 2000). The increase in size is due to a larger pericellular matrix rather than to larger chondrocytes (Lee et al., 2000). However, the presence may be more important than the size of the pericellular matrix. Preliminary studies indicate that when normal cartilage is used, chondron pellets produce more matrix than chondrocyte pellets (Lee, unpublished). In addition, chondrons isolated from juvenile porcine cartilage have only a thin rim of pericellular matrix and yet produce more ECM in pellet culture than chondrocytes isolated from the same cartilage (R. Graff, personal communication). Some osteoarthritic joints have osteophytes on the edges of the cartilage. Although we avoided using obvious osteophytic regions for cell isolation, there may have been areas with subtle osteophytic changes that were included with the specimens. This, together with evidence that type I collagen production may be slightly increased in osteophytic regions of osteoarthritic cartilage (Mankin et al., 1994), may account for the isolated cells with type I collagen immunofluoresence noted infrequently within pellet cultures in the present study. Stewart et al. (2000) evaluated expression of types I and II collagen, aggrecan, and fibronectin in various
Table 1 Observed changes in immunofluorescence for the various extracellular matrix molecules for chondron and chondrocyte pellets and their rim with time in culture Chondrocyte
Type I collagen Type II collagen Type VI collagen Keratan sulfate Fibronectin (Hfn) CD44 (cell shape)
Chondron
Rim chondron and chondrocyte
3 weeks
8 weeks
3 weeks
8 weeks
3 weeks
8 weeks
q q F0 q q qq Fq Round
q qq Fq qq qq qq Fq Round
q qq Fq qq qq qq Fq Round
q qqqFq qq qqq qqFq Round
qq qyy F0 qyy qyy qq Fq Flattened
qqq q F0 qq q qqq Fq Flattened
Immunofluorescence was graded as qyysminimal to no immunofluorescence (IF), qsIF present in isolated areas, qqsmoderate IF throughout, and qqqsbright IF throughout. Fibrils were noted as being present (Fq) or absent (F0).
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Fig. 9. DNA content with time in culture for chondron and chondrocyte pellet cultures. DNA content was determined using Hoechst fluorometric analysis as described in the methods and is plotted as percent DNA content of day 0 for each time point in culture. This represents two separate experiments. Each experiment used 32 pellets (16 chondron and 16 chondrocyte pellets) from a single specimen. Each bar represents the mean"S.D. for eight pellets (four from each specimen). *P-0.05 vs. day 0 (one way ANOVA).
Fig. 8. Proteoglycan content and release with time in culture for chondron and chondrocyte pellets. Proteoglycan was measured as sulfated glycosaminoglycans by DMMB assay as described in the methods. (a) Values were plotted as micrograms sulfated glycosaminoglycans (PG) per microgram DNA for each time point in culture. The PG in pellets were averaged from two separate experiments. Each experiment used 32 pellets (16 chondron and 16 chondrocyte pellets) from a single specimen. Each bar represents the mean"S.D. for eight pellets (four from each specimen). *P-0.05 vs. day 0 (one way ANOVA). (b) PG released into the media averaged from seven separate experiments. Each experiment used one specimen with three pellets each for chondrons and chondrocytes. (c) Comparison of proteoglycan retained in the pellet with proteoglycan released into the media. The values for the media are cumulative. The data are from a representative specimen with triplicate pellets for each time point.
culture systems using equine chondrocytes. Monolayer, aggregate, and explant cultures were found to have no more than 50% of the in vivo levels of mRNA for type II collagen. In addition, explant cultures had only 50% of the in vivo levels of aggrecan mRNA. Pellet cultures, however, most closely resembled in vivo levels, having 80% and 150% of the in vivo levels of type II collagen mRNA and aggrecan mRNA, respectively. This study also found that serum supplementation significantly down-regulated the expression of type II collagen and aggrecan in monolayer culture; an effect that was not present in the three dimensional culture models. These
findings further support the value of pellet culture as a model system for studies of chondrocyte metabolism in vitro. Our studies indicate that chondron pellets may be superior to chondrocyte pellets for studies of matrix metabolism. Chondrocytes cultured in alginate beads for up to 8 months and agarose gels for 1 month maintain their chondrocyte phenotype with the continued ability to synthesize type II collagen and aggrecan (Hauselmann et al., 1994; Aydelotte et al., 1986). However, these studies did not evaluate the total increase in matrix content throughout the culture period. Other studies using chondrocytes incorporated into collagen gels, disks, and collagen-coated filter inserts have shown that proteoglycan synthesis and content increase for 1–3 weeks in culture and then stabilize thereafter for up to 7 weeks in culture (Boyle, et al., 1995; Nixon et al., 1993; Sams et al., 1995). Another study looking at chondrocytes centrifuged into collagen disks showed
Fig. 10. Comparison of type II collagen content for chondron and chondrocyte pellets over time in culture as measured by competitive ELISA. The micrograms collagen in the GuHCl extracts and pepsin digests were combined. The data are from triplicate pellets from one specimen. The experiment was repeated several times with similar results.
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that the total proteoglycan content increased by 78% from 1 to 3 weeks as judged by DMMB analysis (Nixon et al., 1993). The present study revealed that the total proteoglycan content per unit DNA in pellets increased by six- to 10-fold over 8 weeks in culture. This increase in proteoglycans was evident by immunofluorescence throughout the pellet cultures with the exception of the superficial rim. Increased immunofluorescence for type II collagen also indicates that type II collagen deposition and assembly continued with the pellet cultures throughout the eight weeks in culture. This is further supported by the increase in type II collagen in 7-week pellet cultures as determined by competitive ELISA. Other studies using a pellet culture system have not used chondrons (Manning and Bonner, 1967; Kato et al., 1988a, 1993; Xu et al., 1996; Johnstone et al., 1998; Ballock and Reddi, 1994). In our study matrix production was significantly different between chondron and chondrocyte pellets throughout the 8 weeks in culture. Chondron pellets had a significantly greater increase in proteoglycan content as measured by DMMB assay and increased type II collagen immunofluoresence compared with chondrocyte pellet cultures. The difference between a chondron and a chondrocyte is the presence of the in vivo-formed pericellular matrix in the chondron. Therefore, the results of this study support previous work (Lee and Caterson, 1995; Kelley et al., 1996) indicating that the in vivo-formed pericellular matrix promotes matrix deposition and assembly. The mechanism by which the in vivo-formed pericellular matrix results in increased matrix production and assembly is not known at this time. Kuijer et al. (1988), found that proteoglycan aggregates accelerated type II collagen fibrillogenesis. It may be that proteoglycan retention in the pericellular matrix after isolation and the increased proteoglycan content of the chondron pellets with time in culture result in accelerated type II collagen assembly compared with chondrocyte pellets. Retention of type II collagen in the in vivo-formed pericellular matrix of the chondron may also result in earlier production and assembly of fibrils compared with chondrocytes. The chondrons were isolated with a mixture of two enzymes, dispase and collagenase. A mild protease with limited activity against extracellular matrix proteins, dispase cleaves fibronectin (Stenn et al., 1989). A previous report indicated that fibronectin was lost from enzymatically isolated chondrons but that intracellular fibronectin was evident within 1 h after isolation and abundant in the pericellular matrix by 3 days of culture (Lee et al., 1997). This was based on immunolabeling with the monoclonal antibody to fibronectin, HFN 7.1 (Schoen et al., 1982). A polyclonal antibody to fibronectin (from Sigma Chemical Co.) used in Western blot analysis of freshly isolated chondrons revealed intact fibronectin but no low molecular weight degradation products (Lee, unpublished); these were probably
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washed away when the enzyme was removed. Fibronectin fragments initially have a similar effect as IL-1b on chondrocyte metabolism, but the inhibition of proteoglycan synthesis is transient (Homandberg and Wen, 1998). We found that treatment with 29 kDa fibronectin fragments for 3–5 h transiently increased NO synthesis but did not have a significant effect on proteoglycan content or pellet weight (Lee et al., unpublished). Thus, the generation of fibronectin fragments during enzymatic isolation does not appear to play a major role in subsequent matrix synthesis especially considering that retention of the pericellular matrix has a net positive effect. Previous studies using articular cartilage chondrocytes in a pellet culture system did not evaluate DNA content or rates of cell division (Manning and Bonner, 1967; Xu et al., 1996). We found that maintaining the chondrocyte phenotype in a pellet culture system resulted in minimal to no significant cell division for both chondrons and chondrocytes. This finding is supported by others who have found that chondrocytes cultured in three-dimensional culture systems have markedly decreased rates of cell division compared with chondrocytes in monolayer culture (Guo et al., 1989; Kato et al., 1988b; Kimura et al., 1984). Although a need for cell amplification has been emphasized for cartilage engineering, it may also be desirable to use a transplant with a similar cell to matrix ratio as articular cartilage. Evaluation of the spatial arrangement of nuclei stained with Hoechst 33342 revealed that the separation between cells increased with time in both chondrocyte and chondron pellets as more matrix was produced. The chondron pellets had a greater cell separation. When sections of the pellet cultures were compared with articular cartilage sections, the cell densities for both chondron and chondrocyte pellets appeared much higher, although the chondron pellets more closely approximated the cell density of cartilage especially after 8 weeks in culture. We looked at percent DNA content relative to proteoglycan content (wmg DNAy(mg DNA plus mg of PG)x=100) of three specimens of OA knee articular cartilage and three specimens from normal femoral head in addition to the chondron and chondrocyte pellets. The mean relative percent DNA content was 1.5% for cartilage from the normal femoral heads, and 1.2% for the OA knee cartilage. Chondron and chondrocyte pellets had a much higher mean relative percent DNA content: initially, 64% and 72%, respectively, at day 0 but decreased to 15% and 29%, respectively, after 8 weeks in culture. As matrix production and cell viability was maintained throughout the 8 week culture period while cell division was minimal, it may be that, with time, the chondron pellet cell density would more closely approximate that of cartilage in vivo.
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At 2–3 weeks in culture, we noted the development of a superficial rim of flattened cells that increased in cell thickness with time in culture. This rim of cells has been noted previously in chondrocyte and mesenchymal cell pellet cultures, chondrocyte collagen disk, alginate bead, and micromass cultures, but has not been well defined (Hauselmann et al., 1994; Johnstone et al., 1998; Nixon et al., 1993; Xu et al., 1996). Some investigators have suggested that it was reminiscent of the in vivo superficial layer of articular cartilage (Hauselmann et al., 1994; Xu et al., 1996), whereas others have likened it to a perichondrium. Although the present study has shown that this rim has increased fibronectin and decreased proteoglycan content in common with the superficial layer of articular cartilage, the findings of abundant type I collagen and little or no type II collagen in this rim clearly demonstrate that it is more like a perichondrium and is fibrocartilagenous in nature. 5. Conclusion The present study demonstrates that the in vivoformed pericellular matrix is important for matrix production and assembly, resulting in a three-fold increase in matrix production and significantly greater type II collagen content for chondrons compared with chondrocytes. This study also indicates that chondron pellet culture systems, with more type II collagen throughout and continued matrix deposition in long-term culture, may be useful as a hyaline-like cartilage model in vitro and may have potential applications for cartilage engineering. The demonstrated ability of osteoarthritic chondrons to deposit and assemble significant amounts of extracellular matrix indicate that they are capable of building new cartilage matrix when placed in an appropriate, nurturing environment. This may have applications for cartilage engineering for patients with osteoarthritis. Acknowledgments We thank Eleanor Hilliard, Greg Faris, and Marianne Tioran for technical assistance. This work was funded by a Thurston Arthritis Research Center Grant to SSK; an Alumni Endowment Fund grant, UNC School of Medicine, to CML; an Arthritis Foundation Investigator Award to GML and by the National Institutes of Health (AR 43883 to GML; AR38121 to AJB). References Adams, J.C., Watt, F.M., 1993. Regulation of development and differentiation by the extracellular matrix. Development 117, 1183–1198. Aydelotte, M.B., Schleyerbach, R., Zeck, B.J., Kuettner, K.E., 1986. Articular chondrocytes cultured in agarose gel for study of chon-
drocytic chondrolysis. In: Kuettner, K.E., Schleyerbach, R., Hascall, V.C. (Eds.), Articular Cartilage Biochemistry. Raven Press, New York, pp. 235–256. Ballock, R.T., Reddi, A.H., 1994. Thyroxine is the serum factor that regulates morphogenesis of columnar cartilage from isolated chondrocytes in chemically defined medium. J. Cell Biol. 126, 1311–1318. Belitsos, P.C., Hildreth, J.E., August, J.T., 1990. Homotypic cell aggregation induced by anti-CD44 (Pgp-1) monoclonal antibodies and related to CD44 (Pgp-1) expression. J. Immunol. 144, 1661–1670. Boudreau, N., Myers, C., Bissell, M.J., 1995. From laminin to lamin: regulation of tissue-specific gene expression by the ECM. Trends Cell Biol. 5, 1–4. Boyle, J., Luan, B., Cruz, T.F., Kandel, R.A., 1995. Characterization of proteoglycan accumulation during formation of cartilagenous tissue in vitro. Osteoarth. Cart. 3, 117–125. Caterson, B., Christner, J.E., Baker, J.R., 1983. Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. Monoclonal antibodies to cartilage proteoglycan. J. Biol. Chem. 258, 8848–8854. Chandrasekhar, S., Esterman, M.A., Hoffman, H.A., 1987. Microdetermination of proteoglycans and glycosaminoglycans in the presence of guanidine hydrochloride. Anal. Biochem. 161, 103–108. Croucher, L.J., Crawford, A., Hatton, P.V., Russell, R.G., Buttle, D.J., 2000. Extracellular ATP and UTP stimulate cartilage proteoglycan and collagen accumulation in bovine articular chondrocyte pellet cultures. Biochim. Biophys. Acta 1502, 297–306. Foellmer, H.G., Kawahara, K., Madri, J.A., Furthmayr, H., Timpl, R., Tuderman, L., 1983. A monoclonal antibody specific for the amino terminal cleavage site of procollagen type I. Eur. J. Biochem. 134, 183–189. Engvall, E., Hessle, H., Klier, G., 1986. Molecular assembly, secretion, and matrix deposition of type VI collagen. J. Cell Biol. 102, 703–710. Giloh, H., Sedat, J.W., 1982. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217, 1252–1255. Guo, J., Jourdian, G.W., MacCallum, D.K., 1989. Culture and growth characteristics of chondrocytes encapsulated in alginate beads. Connect. Tissue Res. 19, 277–297. Hauselmann, H.J., Fernandes, R.J., Mok, S.S., et al., 1994. Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads. J. Cell Sci. 107, 17–27. Hausser, H., Groning, A., Hasilik, A., Schonherr, E., Kresse, H., 1994. Selective inactivity of TGF-bydecorin complexes. FEBS Lett. 353, 243–245. Homandberg, G.A., Wen, C., 1998. Exposure of cartilage to a fibronectin fragment amplifies catabolic processes while also enhancing anabolic processes to limit damage. J. Orthop. Res. 16, 237–246. Johnstone, B., Hering, T.M., Caplan, A.I., Godberg, V.M., Yoo, J.U., 1998. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell Res. 238, 265–272. Kato, Y., Iwamoto, M., Koike, T., Suzuki, F., Takano, Y., 1988. Terminal differentiation calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor b and serum factors. Proc. Natl. Acad. Sci. USA 85, 9552–9556. Kato, Y., Iwamoto, M., Nakashima, K., Sato, K., Yan, W., Koike, T., 1988. Rabbit articular chondrocytes in soft agar are useful to test the effect of non-steroidal anti-inflammatory drugs on chondrocyte replication. Scand. J. Rheum. Suppl. 77, 3–6. Kato, Y., Nakashima, K., Iwamoto, M., et al., 1993. Effects of interleukin-1 on synthesis of alkaline phosphatase, type X collagen,
C.M. Larson et al. / Matrix Biology 21 (2002) 349–359 and 1,25-dihydroxyvitamin D3 receptor, and matrix calcification in rabbit chondrocyte cultures. J. Clin. Invest. 92, 2323–2330. Kelley, S.S., Blackwood, A.D., Caterson, B., Lee, G.M., 1996. Retention of the in vivo-formed pericellular matrix affects proteoglycan synthesis in vitro. Trans. Orthop. Res. Soc. 21, 768. Kielty, C.M., Lees, M., Shuttleworth, C.A., Wolley, D., 1993. Catabolism of intact type VI collagen microfibrils: susceptibility to degradation by serine proteases. Biochem. Biophys. Res. Commun. 191, 1230–1236. Kimura, T., Yasui, N., Ohsawa, S., Ono, K., 1984. Chondrocytes embedded in collagen gels maintain cartilage phenotype during long-term cultures. Clin. Orthop. Rel. Res. 186, 231–239. Klagsbrun, M., Baird, A., 1991. A dual receptor system is required for basic fibroblast growth factor activity. Cell 67, 229–231. Kuettner, K.E., Pauli, B.U., Gall, G., Memoli, V.A., Schenk, R.K., 1982. Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology. J. Cell. Biol. 93, 743–750. Kuijer, R., von de Stadt, R.J., de Koning, M.H., van Kampen, G.P., van der Karst, J.K., 1988. Influence of cartilage proteoglycans on type II collagen fibrillogenesis. Connect. Tissue Res. 17, 83–97. Lee, G.M., Caterson, B., 1995. When cultured in alginate beads, chondrons produce a more fibrillar matrix than isolated chondrocytes. Trans. Orthop. Res. Soc. 20, 394. Lee, G.M., Poole, C.A., Kelley, S.S., Chang, J., Caterson, B., 1997. Isolated chondrons: a viable alternative for studies of chondrocyte metabolism in vitro. Osteoarth. Cart. 5, 261–274. Lee, G.M., Paul, T.A., Slabaugh, M., Kelley, S.S., 2000. The incidence of enlarged chondrons in normal and osteoarthritic human cartilage and their relative matrix density. Osteoarth. Cart. 8, 44–52. Mankin, H.J., Mow, V.C., Buckwalter, J.A., Ianotti, J.P., Ratcliffe, A., 1994. Form and function of articular cartilage. In: Simon, S.R. (Ed.), Orthopaedic Basic Science. American Academy of Orthopaedic Surgeons, Rosemont, pp. 1–41. Manning, W.K., Bonner, W.M., 1967. Isolation and culture of chondrocytes from adult human articular cartilage. Arthritis Rheum. 10, 235–239. Nixon, A.J., Sams, A.E., Lust, G., Grande, D., Mohammed, H.O., 1993. Temporal matrix synthesis and histologic features of a chondrocyte-laden porous collagen cartilage analogue. Am. J. Vet. Res. 54, 349–356.
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Osborn, M., Weber, K., 1982. Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulincontaining structures. Methods Cell Biol. 24, 97–132. Poole, C.A., Glant, T.T., Schofield, J.R., 1991. Chondrons from articular cartilage. Immunolocalization of proteoglycan epitopes in isolated tibial chondrons. J. Histochem. Cytochem. 39, 1175–1187. Rebuck, N., Croucher, L.J., Hollander, A.P., 1999. Distribution of two alternatively spliced variants of the type II collagen Npropeptide compared with C-propeptide in bovine chondrocyte pellet cultures. J. Cell Biochem. 75, 13–21. Sams, A.E., Minor, R.R., Wootton, J.A., Mohammed, H., Nixon, A.J., 1995. Local and remote matrix responses to chondrocyte-laden collagen scaffold implantation in extensive articular cartilage defects. Osteoarth. Cart. 3, 61–70. Schoen, R.C., Bentley, K.L., Klebe, R.J., 1982. Monoclonal antibody against human fibronectin which inhibits cell attachment. Hybridoma 1, 99–108. Solursh, M., 1991. Formation of cartilage tissue in vitro. J. Cell Biochem. 45, 258–260. Srinivas, G.R., Barrach, H.J., Chichester, C.O., 1993. Quantitative immunoassays for type II collagen and its cyanogen bromide peptides. J. Immunol. Methods 159, 53–62. Stenn, K.S., Link, R., Moellmann, G., Kuklinska, E., 1989. Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase. J. Invest. Dermatol. 93, 287–290. Stewart, M.C., Saunders, K.M., Burton-Wurster, N., Macleod, J.N., 2000. Phenotypic stability of articular chondrocytes in vitro: the effects of culture models, bone morphogenetic protein 2, and serum supplementation. J. Bone Miner. Res. 15, 166–174. van Osch, G.J., van der Veen, S.W., Buma, P., Verwoerd-Verhoef, H.L., 1998. Effect of transforming growth factor-b on proteoglycan synthesis by chondrocytes in relation to differentiation stage and the presence of pericellular matrix. Matrix Biol. 17, 413–424. Von der Mark, K., Gauss, V., von der Mark, H., Muller, P., 1977. Relationship between cell shape and type of collagen synthesized as chondrocytes lose their cartilage phenotype in culture. Nature 267, 532–533. Xu, C., Babatunde, O.O., Frazer, A., Kozaci, L.D., Russell, R.G.G., Hollander, A.P., 1996. Effects of growth factors and interleukin-1a on proteoglycan and type II collagen turnover in bovine nasal and articular chondrocyte pellet cultures. Endocrinology 137, 3557–3565.
Retention of the native chondrocyte pericellular matrix results in significantly improved matrix production Christopher M. Larsona,1, Scott S. Kelleya, A. Denene Blackwooda, Albert J. Banesa, Greta M. Leea,b,* a
Department of Orthopedics, University of North Carolina School of Medicine, Chapel Hill, NC, USA Thurston Arthritis Research Center, University of North Carolina School of Medicine, Chapel Hill, NC, USA
b
Received 4 September 2001; received in revised form 6 February 2002; accepted 25 March 2002
Abstract The interaction of the cell with its surrounding extracellular matrix (ECM) has a major effect on cell metabolism. We have previously shown that chondrons, chondrocytes with their in vivo-formed pericellular matrix, can be enzymatically isolated from articular cartilage. To study the effect of the native chondrocyte pericellular matrix on ECM production and assembly, chondrons were compared with chondrocytes isolated without any pericellular matrix. Immediately after isolation from human cartilage, chondrons and chondrocytes were centrifuged into pellets and cultured. Chondron pellets had a greater increase in weight over 8 weeks, were more hyaline appearing, and had more type II collagen deposition and assembly than chondrocyte pellets. Minimal type I procollagen immunofluorescence was detected for both chondron and chondrocyte pellets. Chondron pellets had a 10-fold increase in proteoglycan content compared with a six-fold increase for chondrocyte pellets over 8 weeks (P-0.0001). There was no significant cell division for either chondron or chondrocyte pellets. The majority of cells within both chondron and chondrocyte pellets maintained their polygonal or rounded shape except for a thin, superficial edging of flattened cells. This edging was similar to a perichondrium with abundant type I collagen and fibronectin, and decreased type II collagen and proteoglycan content compared with the remainder of the pellet. This study demonstrates that the native pericellular matrix promotes matrix production and assembly in vitro. Further, the continued matrix production and assembly throughout the 8-week culture period make chondron pellet cultures valuable as a hyaline-like cartilage model in vitro. 䊚 2002 Elsevier Science B.V. and International Society of Matrix Biology. All rights reserved. Keywords: Chondrons; Chondrocytes; Cartilage; Cultures; Pericellular matrix
1. Introduction Removal of the pericellular matrix during chondrocyte isolation may have a significant effect on subsequent matrix production and assembly in culture. Cell–extracellular matrix interactions affect matrix metabolism, growth factor regulation, and gene expression (Adams and Watt, 1993; Klagsbrun and Baird, 1991; Hausser et al., 1994; Boudreau et al., 1995). The pericellular matrix may be an integral part of this regulation as it resides between the cell and the extracellular matrix. The *Corresponding author. Tel.: q1-919-966-0544; fax: q1-919-9661739. E-mail address: [email protected] (G.M. Lee). 1 Present address: Minneapolis Sports Medicine Center, Eden Prairie, MN, USA.
presence of a pericellular matrix on chondrocytes cultured in alginate eliminates the stimulation of matrix synthesis by TGF-b (van Osch et al., 1998). The presence of the native pericellular matrix also appears to promote the assembly of collagen fibrils (Lee and Caterson, 1995). Thus, the native pericellular matrix may be important for matrix production and assembly (Kelley et al., 1996). The pericellular matrix can be retained by isolating chondrons which consist of the chondrocyte and its native pericellular matrix (Poole et al., 1991). Chondrons can be enzymatically isolated by digestion with a mild protease, dispase, combined with collagenase (Lee and Caterson, 1995; Lee et al., 1997). Chondrons can be isolated because collagenase does not digest type VI collagen (Kielty et al., 1993), a major component of the
0945-053X/02/$ - see front matter 䊚 2002 Elsevier Science B.V. and International Society of Matrix Biology. All rights reserved. PII: S 0 9 4 5 - 0 5 3 X Ž 0 2 . 0 0 0 2 6 - 4
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Fig. 1. Photographs of whole chondrocyte (A,C) and chondron (B,D) pellets after 8 weeks in culture. (A,B) Pellet cultures photographed lying flat. (C,D) Pellet cultures photographed on their side. Ruler shows millimeters.
pericellular matrix. The pericellular matrix of enzymatically isolated chondrons retains types II and VI collagen, and aggrecan (Lee et al., 1997). It has been well established that chondrocytes grown in monolayer cultures lose their polygonal morphology as they undergo phenotypic change (Von der Mark et al., 1977). Chondrocytes that have lost their chondrocyte phenotype produce type I collagen instead of type II collagen and have decreased proteoglycan synthesis (Stewart et al., 2000; Von der Mark et al., 1977; Manning and Bonner, 1967). This is characteristic of fibrocartilage rather than hyaline cartilage. Our recent observations have shown that chondrons also lose their chondrocyte phenotype within 1 week in monolayer culture (unpublished). High density pellet culture systems, however, have been shown to maintain the chondrocyte phenotype (Stewart et al., 2000; Kato et al., 1988a, 1993; Johnstone et al., 1998; Ballock and Reddi, 1994; Manning and Bonner, 1967; Solursh, 1991). Manning and Bonner (1967) used chondrocyte pellet cultures obtained from adult human articular cartilage and observed maintenance of the chondrocyte phenotype and increased matrix synthesis compared with monolayer cultures over 2 weeks. Johnstone et al. (1998), achieved chondrogenesis of mesenchymal cells in pellet culture, but observed extensive type X collagen labeling, in addition to type II collagen. Stewart et al. (2000) evaluated several cartilage culture systems using an equine model and found that pellet and aggregate cultures supported matrix protein gene expression profiles more reflective of in vivo levels when compared with monolayer and explant cultures. Many studies utilizing chondrocyte pellet culture have used growth plate cartilage obtained from fetal, neonatal or immature animals and have observed chondrocyte hypertrophy and a tendency of the initial hyaline-like tissue to mineralize (Kato et al., 1988a, 1993; Ballock and Reddi, 1994; Solursh, 1991). More recent studies have used chondrocyte pellet cultures for in vitro assays (Croucher et al., 2000; Rebuck et al., 1999; Xu et al., 1996).
We hypothesized that, in addition to maintaining the chondrocyte phenotype, pellet culture would allow the chondron to retain its native pericellular matrix and that the presence of the native pericellular matrix would affect matrix production and assembly. We compared chondrons with chondrocytes obtained from adult human articular cartilage maintained for up to 8 weeks in pellet culture. For control of differences between individuals, chondrons and chondrocytes were isolated from each cartilage specimen. The pellets were evaluated with respect to size, DNA content, and matrix production including collagen types I, II and VI. 2. Experimental procedures 2.1. Cell culture Cartilage was obtained from osteoarthritic human knees at the time of joint replacement surgery as surgical waste (seven individuals, mean age 68 years, range 54– 81 years) with approval from our Institutional Review Board, Office of Human Research Studies. Within 1 h of removal from the knee, the cartilage was dissected from the whole joint except the patella and obvious osteophytic regions. Cartilage from each joint was handled separately. For comparison of chondrocytes to chondrons, cartilage from a joint was pooled and minced prior to dividing for enzymatic digestion. Chondrocytes were isolated by digestion with 1320 PUKyml pronase (Boehringer-Mannheim and Calbiochem) for 1 h followed by 0.4% collagenase (CLS-2, Worthington) for 3 h as previously described (Kuettner et al., 1982). Chondrons were isolated by digestion with 0.3% dispase (a neutral protease classified as an amino-endo peptidase produced by Bacillus polymyxa) (GIBCO) plus 0.2% collagenase (CLS-2, Worthington) in phosphate-buffered saline (PBS) for 5 h as previously described (Lee et al., 1997). After filtering through a 70 mm nylon mesh cell strainer (Falcon) and centrifuging at 400=g for 6 min, the cells were counted using a hemocytometer. Six hundred thousand cells in 2 ml aliquots of OptiMEM1 with GlutaMax1 (GIBCO, cat噛 51985-034) with additives (see below) were placed in 15 ml sterile, conical polypropylene culture tubes, and the cells were centrifuged at 400=g for 6 min. Cultures were maintained in Opti-MEM1 with penicillinystreptomycin, 2% FBS, 2.7 mM CaCl2, and 25 mgyml Phospitan C (L ascorbic acid-2-monophosphate, provided by Showa Denko America, Inc., Tokyo, Japan). The cell cultures were incubated in a humidified incubator at 378 C, 5% CO2. The medium was changed at 2-day intervals for all cultures. The pellets were harvested on day 0, and periodically through 8 weeks. 2.2. Immunofluorescence One-, 3- and 8-week pellet cultures were embedded in OCT embedding medium and frozen at y20 8C. A
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single pellet was used for each time point; duplicate sections were used for each antibody. The time course for three different knee specimens was examined. Twenty micron sections were cut using a cryostat (Frigocut 2800, Reichert-Jung). The sections were fixed in 3.7% formaldehyde, rinsed in PBS and blocked with 1% BSAyPBS. The following primary antibodies were used: C4F6 to type II collagen (provided by C. Chichester; Srinivas et al., 1993), Hfn7.1 to fibronectin (prepared by R.J. Klebe and obtained from the Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa City, IA; Schoen et al., 1982), H4C4 to CD44 (prepared by J.E.K. Hildreth and J.T. August, DSHB; Belitsos et al., 1990), 5D4 to keratan sulfate (provided by B. Caterson; Caterson et al., 1983), a rabbit polyclonal to type VI collagen with no cross-reactivity to fibronectin (GIBCO; Engvall et al., 1986), and Sp1.D8 to type I procollagen (prepared by H. Furthmayr, DSHB; Foellmer et al., 1983). The secondary antibodies were Cy3 labeled donkey anti-mouse, LRSC labeled donkey anti-mouse, and FITC labeled donkey anti-rabbit (Jackson Immunoresearch Laboratories, Inc., West Grove, PA). Nuclei were stained with Hoechst 33342 (10 mgy ml, Molecular Probes) and coverslips were mounted with polyvinyl alcohol mounting medium (Osborn and Weber, 1982) containing 1% n-propylgallate (Giloh and Sedat, 1982). 2.3. DNA analysis
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point for a given specimen. The pellets were not pooled at each time point. For each experiment, pellets were individually measured for proteoglycan and DNA content, and a mean of all measures at a given time point was used for statistical analysis. Chondron and chondrocyte pellets from the same specimen were used for comparative analysis of proteoglycan and DNA content. The experiments were repeated with cartilage from two different knee specimens, and comparisons were done between each of these by normalizing microgram proteoglycan to microgram DNA. 2.5. Competitive ELISA for type II collagen Pellets were frozen in liquid nitrogen and then pulverized using a seed crusher in 96-well format (HSC200, Perkin Elmer Wallac Inc.). The pulverized pellets were extracted with 4 M GuHCl overnight, rinsed with PBS then digested three times using freshly prepared 1 mgyml pepsin in 0.5 M acetic acid at 4 8C for 24 h for a total digestion time of 72 h. The digests were combined. The ELISA assay was performed essentially as previously described (Srinivas et al., 1993) using the C4F6 antibody to type II collagen at 1:8000 to coat the plates and horseradish peroxidase conjugated secondary antibody (Jackson Immmunoresearch Laboratories, Inc.) at approximately 0.1 mgyml with TMB Microwell Peroxidase Substrate System (KPL, Gaithersburg, MD). The ELISA was done on both the GuHCl extract and the pepsin digest.
For fluorometric DNA assays, the samples were digested with papain (Sigma Chemical Co., St. Louis, MO) at 60 8C overnight. Calf thymus DNA standards were prepared (0–50 mgyml DNA). Samples and standards were diluted in 10 mM Tris, 1 mM Na4EDTA, 0.1 mM NaCl (pH 7.3) and 33342 Hoechst (10 mgyml, Molecular Probes, Eugene, OR) was added at a 1:10 000 dilution. DNA content was then determined with a fluorometer using A365 nm excitation and A458 nm emission. DNA analyses were done on the same pellets as the sulfated glycosaminoglycan analyses.
One-way analysis of variance (ANOVA) was used to evaluate significant differences in PG and DNA content with time in culture for chondron and chondrocyte pellets. Statistical significance was determined within groups using multiple t-tests by the Bonferroni method and between chondron and chondrocyte pellets using two factor ANOVA with replication. The level of significance was set at P-0.05.
2.4. Measure of proteoglycan (PG) content
3. Results
Determination of sulfated-GAG was done on the papain digests using the dimethylmethylene blue (DMMB) dye from Serva Feinbiochemica (Heidelberg, Germany) in a binding assay as previously described (Chandrasekhar et al., 1987) using chondroitin sulfate C (shark cartilage extract, Sigma, St. Louis, MO) as the standard. The plates were read at an absorbance of 525 nm on a microtiter plate reader (Titertek Multiskan, MCCy340, ICN, Costa Mesa, CA) and the standards were plotted on a linear regression curve (Delta Soft 3, Biometallics). All analyses were carried out using quadruplicate chondron and chondrocyte pellets for each time
3.1. Gross pellets
2.6. Statistical analysis
The chondron pellets were more uniform, hyaline appearing and cohesive than the chondrocyte pellets which were very friable and difficult to handle for the first few weeks in culture. By 8 weeks, chondron pellets were well formed and similar to articular cartilage in vivo with respect to palpation and texture. The chondron pellets were noticeably larger than the chondrocyte pellets (Fig. 1). Over the 8 weeks in culture, the wet weight per pellet increased more for chondron (6.2"1.8 mg, mean"S.D.) than for chondrocyte pellets (3.1"0.9
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Fig. 2. Comparison of type II collagen immunofluorescence in chondron (A,C) and chondrocyte (B,D) pellets after 3 weeks (A,B) and 7 weeks (C,D) of culture. Nuclei (A9–D9) in viable chondrocytes were stained with Hoechst 33342. The insets show collagen fibrils at six-fold greater magnification. The photomicrographs in this figure and subsequent figures are of 20-mm-thick cryostat sections and were obtained as video images using an intensified video camera with gain and offset held constant. Bar, 50 mm.
mg) (P-0.001). This increase when normalized to DNA was 0.7 mgymg DNA and 0.2 mgymg DNA for chondrons and chondrocytes, respectively. 3.2. Immunofluorescence microscopy All chondron and chondrocyte pellet sections were evaluated using the same gain and offset camera settings so that the relative immunofluorescence between sections was directly comparable for a given antibody. Hoechst labeling of viable nuclei revealed good viability for both chondron and chondrocyte pellets and greater cell density for the chondrocyte pellets compared with chondron pellets at all times in culture. The most dramatic difference between chondron and chondrocyte pellets was seen with type II collagen immunofluoresence. Both chondron and chondrocyte pellets had an obvious increase in type II collagen immunofluorescence, including fibril formation, with time in culture but chondrons had dramatically more type II collagen immunofluoresence throughout the culture period (Fig. 2). There was no obvious difference in type I procollagen immunofluorescence between chondron and chondrocyte pellet cultures. With the exception of a thin edging of immunofluorescence to
type I procollagen that developed circumferentially about the outer surface of the pellets (see below), there was minimal immunofluorescence throughout the pellets (Fig. 3). The cells within chondron and chondrocyte pellets retained their polygonal cell shape as revealed by immunolabeling for CD44, a hyaluronan receptor on the chondrocyte cell membrane (Fig. 4). An exception was the thin superficial edging around the pellets where the cells were elongated. The polygonal shape and type II collagen production indicate that cells within both chondron and chondrocyte pellets maintained their chondrocyte phenotype. Compared with chondrocyte pellets, chondron pellets initially had much greater immunofluorescence for type VI collagen owing to retention of the native pericellular matrix. This disparity markedly decreased with time in culture, apparently due to type VI collagen synthesis increasing much more in chondrocyte than chondron pellets. In fact, there was only a slight increase in type VI collagen immunofluoresence for chondron pellets with time in culture (Fig. 5). The chondron morphology is still evident after 3 weeks in culture (Fig. 5C). The superficial layer or rim of flattened, fibroblastlike cells developed circumferentially about both chon-
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Fig. 4. After 3 weeks in culture, the chondrocytes had a polygonal shape as revealed by immunofluorescence for CD44, a hyaluronan receptor on the plasma membrane. (A) Three-week chondron pellet culture. (B) Three-week chondrocyte pellet culture. Bar, 20 mm.
Fig. 3. Type I procollagen labeling of chondron (A,C) and chondrocyte (B,D) pellet cultures at 3 weeks (A,B) and 8 weeks (C,D) shows labeling primarily in the thin rim area surrounding the pellet. The chondrocyte pellet section in (B) is narrower than the other sections. This is because the pellets were sectioned perpendicular to their long axis or flat surface and at 3 weeks the chondrocyte pellets were much thinner especially in the center of the pellet. The procollagen labeling is intracellular. Bar, 50 mm.
When examined over time, there was a significant increase in the amount of PG per microgram DNA for both chondron and chondrocyte pellets from day 0 to 8 weeks in culture (P-0.00001). When compared with chondrocyte pellets, chondron pellets had a significantly greater increase in total PG (5.2 mg PGymg DNA for chondrons, and 1.8 mg PGymg DNA for chondrocytes) (P-0.0001) from day 0 to 8 weeks in culture. There
dron and chondrocyte pellet cultures. This layer was not evident at 1 week in culture but became evident at 2–3 weeks and increased in cell number with time in culture (Fig. 6). Similar findings were noted previously with chondrocytes in pellet, micromass, and collagen disk cultures (Hauselmann et al., 1994; Johnstone et al., 1998; Nixon et al., 1993; Xu et al., 1996). In the present study, this rim had increased cell density and increased immunofluorescence to fibronectin and type I procollagen relative to the interior of the pellet (Figs. 3, 4 and 6). There was minimal to no immunofluorescence for type II collagen and keratan sulfate in this rim (Fig. 7). Table 1 summarizes the observed changes in immunofluorescence for the various extracellular matrix molecules for chondron and chondrocyte pellets and their rim with time in culture. 3.3. Proteoglycan analysis To account for any disparities in the cell number for chondron and chondrocyte pellet cultures, the PG data are presented as micrograms of PG per micrograms of DNA for pellets at each time point in culture (Fig. 8a).
Fig. 5. Immunolabeling for type VI collagen remained fairly constant for the chondron pellets (A, 3 week; C, 8 week), but was dramatically increased for chondrocyte pellets (B,D) from 3 weeks (B) to 8 weeks (D) of culture. Bar, 20 mm.
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Fig. 6. Immunolabeling for fibronectin reveals the development of a rim of flattened cells (arrows) surrounding the pellets. This figure is of chondron pellets at (A) 1 week, (B) 2 weeks, (C) 3 weeks and (D) 8 weeks of culture. The reduced immunofluorescence in the center of the pellet with time in culture may be due to either less fibronectin in this area or to epitope masking. The monoclonal, Hfn 7.1, is highly specific to human fibronectin and appears to bind to the cell-binding region (Schoen et al., 1982). Bar, 50 mm.
was a 10-fold increase in PG content for chondron pellets compared with a six-fold increase for chondrocyte pellets over 8 weeks in culture (P-0.001). Chondron and chondrocyte pellets released similar amounts of proteoglycan into the culture media over time (Fig. 8b). Comparison of proteoglycan in the pellets with the cumulative amount of proteoglycan released into the media shows that during the first week of culture there was more proteoglycan released than retained by both chondrons and chondrocytes. By day 21, the chondrons had retained considerably more proteoglycan in the pellets than was released into the media while the chondrocytes had a similar amount of proteoglycan in the pellets and media. 3.4. DNA analysis There was no significant difference in DNA content at day 0 between chondron (6.8 mg) and chondrocyte
pellets (6.2 mg) (Ps0.61) (Fig. 9). Both chondron and chondrocyte pellets had a decrease (15–37%) in DNA content from day 0 to 1 weeks, but this was only statistically significant for chondrocytes (P-0.01). Throughout this experiment we noted a thin lining of cells in the culture tubes formed by cells that had migrated from the pellets. This appeared to occur more frequently with chondrocyte than chondron pellet cultures and was evident after several days in culture. Cell adherence to the tube sidewall occurred despite using polypropylene tubes treated with 0.6 mgyml poly-Hema (Sigma). From 1 to 8 weeks in culture there was a minimal increase in DNA content for both chondron (13%) and chondrocyte (21%) pellets, but this was not statistically significant. 3.5. Type II collagen analysis Competitive ELISA of the GuHCl extracts and pepsin digests showed an increase in type II collagen with time
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Fig. 7. The superficial rim of the pellets did not label with antibodies to type II collagen (A) or keratan sulfate (C). These are sections of a chondron pellet after 8 weeks of culture. The corresponding nuclei of the chondrocytes are shown in (B) and (D). Note the increased cell density in the outer area. The edge of the sections is shown by white lines in (A,C). Bars (B) 20 mm and (D) 10 mm.
in culture (Fig. 10). The chondron pellets had a significantly greater amount of type II collagen than the chondrocyte pellets at all time points. This was most dramatic after 7 weeks in culture. 4. Discussion The present study demonstrated that chondron pellets had a significantly greater increase in size, proteoglycan
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content, and type II collagen content than chondrocyte pellets. The results of the DNA and sulfated glycosaminoglycan content analysis indicate that this greater increase in size for chondron pellets reflects increased matrix deposition rather than cell division. Matrix synthesis was not monitored in this study so we cannot say at this time whether the increased matrix deposition is due to differences in rates of synthesis or to greater retention and assembly. In preliminary work, we have found that, when maintained in alginate bead culture, chondrocytes synthesized more proteoglycans during the first week of culture, but chondrons synthesized more after the second week of culture (Kelley et al., 1996). Osteoarthritic human cartilage was used as a source of chondrocytes and chondrons primarily because normal human cartilage specimens were not as readily available. Chondrons isolated from osteoarthritic human cartilage are larger than those isolated from normal human cartilage (Lee et al., 2000). The increase in size is due to a larger pericellular matrix rather than to larger chondrocytes (Lee et al., 2000). However, the presence may be more important than the size of the pericellular matrix. Preliminary studies indicate that when normal cartilage is used, chondron pellets produce more matrix than chondrocyte pellets (Lee, unpublished). In addition, chondrons isolated from juvenile porcine cartilage have only a thin rim of pericellular matrix and yet produce more ECM in pellet culture than chondrocytes isolated from the same cartilage (R. Graff, personal communication). Some osteoarthritic joints have osteophytes on the edges of the cartilage. Although we avoided using obvious osteophytic regions for cell isolation, there may have been areas with subtle osteophytic changes that were included with the specimens. This, together with evidence that type I collagen production may be slightly increased in osteophytic regions of osteoarthritic cartilage (Mankin et al., 1994), may account for the isolated cells with type I collagen immunofluoresence noted infrequently within pellet cultures in the present study. Stewart et al. (2000) evaluated expression of types I and II collagen, aggrecan, and fibronectin in various
Table 1 Observed changes in immunofluorescence for the various extracellular matrix molecules for chondron and chondrocyte pellets and their rim with time in culture Chondrocyte
Type I collagen Type II collagen Type VI collagen Keratan sulfate Fibronectin (Hfn) CD44 (cell shape)
Chondron
Rim chondron and chondrocyte
3 weeks
8 weeks
3 weeks
8 weeks
3 weeks
8 weeks
q q F0 q q qq Fq Round
q qq Fq qq qq qq Fq Round
q qq Fq qq qq qq Fq Round
q qqqFq qq qqq qqFq Round
qq qyy F0 qyy qyy qq Fq Flattened
qqq q F0 qq q qqq Fq Flattened
Immunofluorescence was graded as qyysminimal to no immunofluorescence (IF), qsIF present in isolated areas, qqsmoderate IF throughout, and qqqsbright IF throughout. Fibrils were noted as being present (Fq) or absent (F0).
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Fig. 9. DNA content with time in culture for chondron and chondrocyte pellet cultures. DNA content was determined using Hoechst fluorometric analysis as described in the methods and is plotted as percent DNA content of day 0 for each time point in culture. This represents two separate experiments. Each experiment used 32 pellets (16 chondron and 16 chondrocyte pellets) from a single specimen. Each bar represents the mean"S.D. for eight pellets (four from each specimen). *P-0.05 vs. day 0 (one way ANOVA).
Fig. 8. Proteoglycan content and release with time in culture for chondron and chondrocyte pellets. Proteoglycan was measured as sulfated glycosaminoglycans by DMMB assay as described in the methods. (a) Values were plotted as micrograms sulfated glycosaminoglycans (PG) per microgram DNA for each time point in culture. The PG in pellets were averaged from two separate experiments. Each experiment used 32 pellets (16 chondron and 16 chondrocyte pellets) from a single specimen. Each bar represents the mean"S.D. for eight pellets (four from each specimen). *P-0.05 vs. day 0 (one way ANOVA). (b) PG released into the media averaged from seven separate experiments. Each experiment used one specimen with three pellets each for chondrons and chondrocytes. (c) Comparison of proteoglycan retained in the pellet with proteoglycan released into the media. The values for the media are cumulative. The data are from a representative specimen with triplicate pellets for each time point.
culture systems using equine chondrocytes. Monolayer, aggregate, and explant cultures were found to have no more than 50% of the in vivo levels of mRNA for type II collagen. In addition, explant cultures had only 50% of the in vivo levels of aggrecan mRNA. Pellet cultures, however, most closely resembled in vivo levels, having 80% and 150% of the in vivo levels of type II collagen mRNA and aggrecan mRNA, respectively. This study also found that serum supplementation significantly down-regulated the expression of type II collagen and aggrecan in monolayer culture; an effect that was not present in the three dimensional culture models. These
findings further support the value of pellet culture as a model system for studies of chondrocyte metabolism in vitro. Our studies indicate that chondron pellets may be superior to chondrocyte pellets for studies of matrix metabolism. Chondrocytes cultured in alginate beads for up to 8 months and agarose gels for 1 month maintain their chondrocyte phenotype with the continued ability to synthesize type II collagen and aggrecan (Hauselmann et al., 1994; Aydelotte et al., 1986). However, these studies did not evaluate the total increase in matrix content throughout the culture period. Other studies using chondrocytes incorporated into collagen gels, disks, and collagen-coated filter inserts have shown that proteoglycan synthesis and content increase for 1–3 weeks in culture and then stabilize thereafter for up to 7 weeks in culture (Boyle, et al., 1995; Nixon et al., 1993; Sams et al., 1995). Another study looking at chondrocytes centrifuged into collagen disks showed
Fig. 10. Comparison of type II collagen content for chondron and chondrocyte pellets over time in culture as measured by competitive ELISA. The micrograms collagen in the GuHCl extracts and pepsin digests were combined. The data are from triplicate pellets from one specimen. The experiment was repeated several times with similar results.
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that the total proteoglycan content increased by 78% from 1 to 3 weeks as judged by DMMB analysis (Nixon et al., 1993). The present study revealed that the total proteoglycan content per unit DNA in pellets increased by six- to 10-fold over 8 weeks in culture. This increase in proteoglycans was evident by immunofluorescence throughout the pellet cultures with the exception of the superficial rim. Increased immunofluorescence for type II collagen also indicates that type II collagen deposition and assembly continued with the pellet cultures throughout the eight weeks in culture. This is further supported by the increase in type II collagen in 7-week pellet cultures as determined by competitive ELISA. Other studies using a pellet culture system have not used chondrons (Manning and Bonner, 1967; Kato et al., 1988a, 1993; Xu et al., 1996; Johnstone et al., 1998; Ballock and Reddi, 1994). In our study matrix production was significantly different between chondron and chondrocyte pellets throughout the 8 weeks in culture. Chondron pellets had a significantly greater increase in proteoglycan content as measured by DMMB assay and increased type II collagen immunofluoresence compared with chondrocyte pellet cultures. The difference between a chondron and a chondrocyte is the presence of the in vivo-formed pericellular matrix in the chondron. Therefore, the results of this study support previous work (Lee and Caterson, 1995; Kelley et al., 1996) indicating that the in vivo-formed pericellular matrix promotes matrix deposition and assembly. The mechanism by which the in vivo-formed pericellular matrix results in increased matrix production and assembly is not known at this time. Kuijer et al. (1988), found that proteoglycan aggregates accelerated type II collagen fibrillogenesis. It may be that proteoglycan retention in the pericellular matrix after isolation and the increased proteoglycan content of the chondron pellets with time in culture result in accelerated type II collagen assembly compared with chondrocyte pellets. Retention of type II collagen in the in vivo-formed pericellular matrix of the chondron may also result in earlier production and assembly of fibrils compared with chondrocytes. The chondrons were isolated with a mixture of two enzymes, dispase and collagenase. A mild protease with limited activity against extracellular matrix proteins, dispase cleaves fibronectin (Stenn et al., 1989). A previous report indicated that fibronectin was lost from enzymatically isolated chondrons but that intracellular fibronectin was evident within 1 h after isolation and abundant in the pericellular matrix by 3 days of culture (Lee et al., 1997). This was based on immunolabeling with the monoclonal antibody to fibronectin, HFN 7.1 (Schoen et al., 1982). A polyclonal antibody to fibronectin (from Sigma Chemical Co.) used in Western blot analysis of freshly isolated chondrons revealed intact fibronectin but no low molecular weight degradation products (Lee, unpublished); these were probably
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washed away when the enzyme was removed. Fibronectin fragments initially have a similar effect as IL-1b on chondrocyte metabolism, but the inhibition of proteoglycan synthesis is transient (Homandberg and Wen, 1998). We found that treatment with 29 kDa fibronectin fragments for 3–5 h transiently increased NO synthesis but did not have a significant effect on proteoglycan content or pellet weight (Lee et al., unpublished). Thus, the generation of fibronectin fragments during enzymatic isolation does not appear to play a major role in subsequent matrix synthesis especially considering that retention of the pericellular matrix has a net positive effect. Previous studies using articular cartilage chondrocytes in a pellet culture system did not evaluate DNA content or rates of cell division (Manning and Bonner, 1967; Xu et al., 1996). We found that maintaining the chondrocyte phenotype in a pellet culture system resulted in minimal to no significant cell division for both chondrons and chondrocytes. This finding is supported by others who have found that chondrocytes cultured in three-dimensional culture systems have markedly decreased rates of cell division compared with chondrocytes in monolayer culture (Guo et al., 1989; Kato et al., 1988b; Kimura et al., 1984). Although a need for cell amplification has been emphasized for cartilage engineering, it may also be desirable to use a transplant with a similar cell to matrix ratio as articular cartilage. Evaluation of the spatial arrangement of nuclei stained with Hoechst 33342 revealed that the separation between cells increased with time in both chondrocyte and chondron pellets as more matrix was produced. The chondron pellets had a greater cell separation. When sections of the pellet cultures were compared with articular cartilage sections, the cell densities for both chondron and chondrocyte pellets appeared much higher, although the chondron pellets more closely approximated the cell density of cartilage especially after 8 weeks in culture. We looked at percent DNA content relative to proteoglycan content (wmg DNAy(mg DNA plus mg of PG)x=100) of three specimens of OA knee articular cartilage and three specimens from normal femoral head in addition to the chondron and chondrocyte pellets. The mean relative percent DNA content was 1.5% for cartilage from the normal femoral heads, and 1.2% for the OA knee cartilage. Chondron and chondrocyte pellets had a much higher mean relative percent DNA content: initially, 64% and 72%, respectively, at day 0 but decreased to 15% and 29%, respectively, after 8 weeks in culture. As matrix production and cell viability was maintained throughout the 8 week culture period while cell division was minimal, it may be that, with time, the chondron pellet cell density would more closely approximate that of cartilage in vivo.
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At 2–3 weeks in culture, we noted the development of a superficial rim of flattened cells that increased in cell thickness with time in culture. This rim of cells has been noted previously in chondrocyte and mesenchymal cell pellet cultures, chondrocyte collagen disk, alginate bead, and micromass cultures, but has not been well defined (Hauselmann et al., 1994; Johnstone et al., 1998; Nixon et al., 1993; Xu et al., 1996). Some investigators have suggested that it was reminiscent of the in vivo superficial layer of articular cartilage (Hauselmann et al., 1994; Xu et al., 1996), whereas others have likened it to a perichondrium. Although the present study has shown that this rim has increased fibronectin and decreased proteoglycan content in common with the superficial layer of articular cartilage, the findings of abundant type I collagen and little or no type II collagen in this rim clearly demonstrate that it is more like a perichondrium and is fibrocartilagenous in nature. 5. Conclusion The present study demonstrates that the in vivoformed pericellular matrix is important for matrix production and assembly, resulting in a three-fold increase in matrix production and significantly greater type II collagen content for chondrons compared with chondrocytes. This study also indicates that chondron pellet culture systems, with more type II collagen throughout and continued matrix deposition in long-term culture, may be useful as a hyaline-like cartilage model in vitro and may have potential applications for cartilage engineering. The demonstrated ability of osteoarthritic chondrons to deposit and assemble significant amounts of extracellular matrix indicate that they are capable of building new cartilage matrix when placed in an appropriate, nurturing environment. This may have applications for cartilage engineering for patients with osteoarthritis. Acknowledgments We thank Eleanor Hilliard, Greg Faris, and Marianne Tioran for technical assistance. This work was funded by a Thurston Arthritis Research Center Grant to SSK; an Alumni Endowment Fund grant, UNC School of Medicine, to CML; an Arthritis Foundation Investigator Award to GML and by the National Institutes of Health (AR 43883 to GML; AR38121 to AJB). References Adams, J.C., Watt, F.M., 1993. Regulation of development and differentiation by the extracellular matrix. Development 117, 1183–1198. Aydelotte, M.B., Schleyerbach, R., Zeck, B.J., Kuettner, K.E., 1986. Articular chondrocytes cultured in agarose gel for study of chon-
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