Chondrocytes Attach to Hyaline Or Calcified Cartilage and Bone1

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

International Cartilage Repair Society

Chondrocytes attach to hyaline or calciﬁed cartilage and bone1 Hangjun Wang M.D. and Rita A. Kandel M.D.* Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, University of Toronto, Toronto, Canada

Summary

Objective: The initial attachment of transplanted chondrocytes to the surface of a cartilage defect is crucial for the success of chondrocyte transplantation. The purpose of this study was to investigate the early interaction of chondrocytes with the deep or calcified zones of cartilage or the subchondral bone, joint surfaces to which transplanted chondrocytes might have to attach in vivo. Design: Freshly isolated (primary) or passaged (P1) chondrocytes were seeded on the top of bone plugs having either a surface composed of mid-deep zone hyaline cartilage or calcified cartilage or bone only. The percent of cells that attached, the role of integrins in cell attachment, and gene expression after placement of the cells on the different surfaces were determined. Results: Both primary and passaged chondrocytes attached efficiently to all three surfaces (over 88% of seeded cells). The chondrocytes showed a punctate distribution of 1-integrin and vinculin, which in areas co-localized with actin, suggesting that the cells formed focal adhesions. The primary chondrocytes had a different shape, appearance of focal contacts, and actin distribution when compared to passaged cells and these did not appear to be influenced by the type of surface to which the cells attached. Blocking either 1-integrin or v5 integrin partially inhibited (between 27 to 48% and 26 to 37% respectively) attachment of both primary and passaged chondrocytes to all surfaces. Blocking v3 had no effect on adhesion. There was expression of type II collagen and aggrecan core protein mRNA by 2 h. The different surfaces did not appear to affect the expression of these genes up to 24 h although gene levels were lower in passaged cells. Conclusions: Chondrocytes, either freshly isolated or passaged, have the potential to adhere to the different joint surfaces that could be exposed in a cartilage defect. Understanding how chondrocytes adhere and interact with damaged joint surfaces may help identify methods to enhance the retention of transplanted cells in the defect site and cartilage tissue formation. © 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Chondrocytes, Attachment, Focal adhesions, Joint surfaces.

Introduction 1, v1, and v5 integrins were responsible for chondrocyte adhesion to articular cartilage17. Their role in attach- Articular cartilage when damaged has limited, if any, ability ment to calcified cartilage or bone is not known. for repair especially if the defect is confined to cartilage1,2. Integrins are ubiquitous heterodimeric transmembrane A variety of reasons for this have been postulated such as proteins composed of one chain and one chain that the inability of chondrocytes to migrate into the site of injury, participate in adhesion to a variety of extracellular matrix the avascular nature of cartilage, and the absence of repair components11,15,16. Although at least 18 chains and eight elements such as the ingrowth of mesenchymal cells and a chains have been identified in different cell types, fibrin clot scaffold into which the cells can migrate1. Several chondrocytes have been shown to express only a restricted surgical approaches have been developed to treat cartilage number of integrins such as 11, 21, 31, 51, 61, defects one of which is autogenous chondrocyte transplan- v 3, and v 5. Integrins combine with other proteins such tion3,4. This treatment consists of placing chondrocytes into as vinculin, which together form the focal adhesions that a cartilage defect and oversewing a periosteal flap to help 11,18–20 retain the cells5,6. The success of this procedure depends mediate cell adhesion . Formation of these structures activates signaling pathways that ultimately regulate in part on the initial attachment of the transplanted chondro- 14 cytes to the cartilage. Chondrocytes can adhere to both gene expression and could influence whether these cells devitalized and live cartilage7–10, but whether they can will maintain their phenotype. attach to calcified cartilage or bone has not been estab- Although in vitro experimental studies have demon- lished. Chondrocytes express integrins, annexin V, and strated chondrocyte attachment to cartilage, animal studies CD44 all of which could mediate attachment to these have suggested that the transplanted cells may not always surfaces10–17. A recent study by Kurtis et al suggested that be retained in the defect suggesting that attachment may be impaired21–23. Clearly the placement of the cells on the 1 Funding Support: This work was supported by Genzyme Bio- cartilage is important as is the duration of seeding time24, surgery, Boston, USA and CIHR. however it is possible that the state of chondrocyte differ- *Address correspondence and reprint requests to: Rita Kandel, entiation (use of passaged cells) or the type of articular MD, Dept. of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Ste 600, Toronto, Ontario, surface onto which the cells are implanted may also influ- Canada M5G 1X5. Tel: +1 416 586-8516; Fax: +1 416 586-8628; ence cell attachment. Depending on the depth of the E-mail: [email protected] cartilage defect the cells may be placed on hyaline carti- Received 6 November 2002; revision accepted 24 August 2003. lage, calcified cartilage or even bone if the calcified zone

56 Osteoarthritis and Cartilage Vol. 12, No. 1 57 has been penetrated3,25,26. The purpose of this study was CELL CULTURE to determine whether different joint surfaces, such as Articular cartilage was harvested from metacarpal-carpal hyaline cartilage (the mid-deep zone which might be the bovine joints and the cartilage from several animals (be- portion of cartilage that remains in a damaged joint), tween two to four calves) pooled together in order to be calcified cartilage or bone influences chondrocyte attach- able to obtain sufficient cells for each experiment. Chondro- ment. We also examined if integrins were involved in cytes were isolated by sequential enzyme digestion as attachment to the different surfaces, whether focal adhe- described previously27. The cells were allowed to recover sions were formed and the effect of the surfaces on actin in medium with serum for 20 min. They were then pelleted, organization in the cells as these can regulate gene expres- washed three times in Ham’s F12 (serum-free) and then sion. The expression of type II collagen and aggrecan core placed on the top surface of the plugs at a density of protein was also examined. Both freshly isolated (primary 16×106 cells/cm2 in serum-free Ham’s F12. This cell den- cells) and passaged chondrocytes were used in this study sity was selected as we observed in previous studies to determine whether this will influence cell attachment. (unpublished data) that cells under these conditions would form cartilage tissue on the plug surface. The cells were cultured at 37°C under standard cell culture conditions for various times up to 24 h. To obtain passaged cells, freshly isolated chondrocytes Materials and methods were plated at low cell density (3×103/cm2) in flasks and cultured in Ham’s F12 supplemented with 5% FBS. The MATERIALS media was changed every other day. When the cells reached confluence (around day 14), cells were harvested Fetal bovine serum (FBS) and protease were using trypsin (0.05%) and allowed to recover for up to 3 h. purchased from Sigma Chemical Co (St. Louis, MO, USA). The cells were washed and re-suspended in serum-free Collagenase was from Boehringer Mannhein GmbH Ham’s F12 media and placed on the top of the plugs as (Indianapolis IN). Tygon tubing was from Norton Perform- described above. ance Plastic Corporation (Akron, Ohio). The antibodies In the experiments evaluating the role of integrins on cell used for immunostaining were obtained from the following attachment, the cells were plated at a lower density sources: rat monoclonal anti-human 1 integrin (AIIB2, (0.8×106 cells/cm2). This was done to ensure that cell Hybridoma Bank, IW); mouse monoclonal anti-human attachment was being examined. vinculin, control mouse and rat IgG1, FITC-conjugated goat anti-rat IgG antibody or goat anti-mouse IgG antibody (Sigma, St. Louise, MI). The antibodies used for the block- ATTACHMENT ASSAY ing experiments were obtained from: 1 integrin (4B4) and mouse IgG1 (Coulter Immunology, Hialeah, FL); v5 Before placing the cells on the plug surface, the chondro- (LM609) and v5 (P1F6) (Chemicon, Temecula, CA). cytes were incubated with SYTO-13 (5 µM in PBS) for Alexa Fluor 568-phalloidin and Syto-13 were from 30 min at 37°C to label the cells. The efficiency of labeling Molecular Probes (Eugene OR). The RNeasy kit was from was confirmed by fluorescent microscopy prior to plug Qiagen Inc (Valencia CA). ™First-strand Synthesis System seeding. If at least 98% of cells were fluorescent the cells for RT-PCR and the primers for type II collagen and were used in the experiment. The cells were labeled with aggrecan genes were purchased from GIBCO BRL this fluorescent dye to ensure that only the cells that had (Gaithersburg MD, U.S.A). Perkin Elmer DNA thermal been placed on the plugs were counted. After 24 h the cycler 480 was used for the PCR. plugs were gently washed (Ham’s F12) to remove the unattached cells. The attached cells were harvested using collagenase (0.1%, 20 min) and counted using a hemo- GENERATING BONE PLUGS WITH DIFFERENT SURFACES cytometer and fluorescent microscopy. This method of retrieving cells was developed as we found that trypsin was Plugs of cartilage and subchondral bone were hand- ineffective in releasing all the cells consistently from the drilled from bovine metacarpal–carpal joints under sterile plug surface. To evaluate the efficiency of cell release, the conditions. The osteochondral plugs were 4 mm plug surface was examined by fluorescent microscopy for diameter×6 mm long. To generate plugs with a mid-deep the presence of residual fluorescent cells after the colla- zone cartilage surface, the upper third of the cartilage was genase digestion. Unlabeled cells, when present amongst removed using a scalpel. To generate plugs with a calcified the harvested cells, comprised much less than 1% of the cartilage surface only, the entire cartilage was removed total cells. As an additional control, plugs that did not with a single cut whereas to get plugs without a cartilage receive any chondrocytes were incubated in SYTO-13 for surface the full thickness cartilage including the calcified 30 min and then underwent identical collagenase treatment zone was removed to expose the subchondral bone. The to determine whether any of the endogenous cells would plugs were washed vigorously with Ham’s F12 and main- take up the dye. tained in media for up to 24 h prior to cell seeding. The plugs were placed in tubing to create a ‘well effect’ in order to prevent the cells and media from spilling over the edge. STAINING AND MICROSCOPY Random plugs were submitted for histology to assess the surfaces that had been generated. These plugs were fixed After 24 h the cells attached to the plugs were fixed in 4% in 10% buffered formalin immediately upon removal of the paraformaldehyde and permeablized with 0.2% Triton surface, decalcified in 10% formic acid, and paraffin em- X-100. The multi-layer of cells was gently scraped off with bedded. Sections were cut (5 µm), stained with toluidine the edge of a scalpel blade leaving behind the cells that blue, and examined by light microscopy to confirm the were attached to the surface. This latter treatment had no extent of cartilage removed. effect on the cells that remain attached and was done to 58 H. Wang and R. A. Kandel: Chondrocyte attachment facilitate evaluation of the cell-surface interface and elimi- STATISTICAL ANALYSIS nate non-specific sticking of the antibody. The samples Results are presented as the mean±the standard devia- were blocked with 3% goat serum and then incubated with tion (S.D.). Two-way analysis of variance (ANOVA) was used antibody reactive with 1 integrin (rat monoclonal, 1:5 to determine the statistical significance. Significance was dilution) or vinculin (mouse monoclonal, 1:200 dilution) at assigned at P<0.05. room temperature for 1 h. After washing (PBS containing 0.2% Triton X-100), fluorescein isothiocyanate (FITC)- conjugated goat anti-rat IgG (1:300 dilution) or goat anti- mouse IgG (1:400 dilution), depending on the primary Results antibody, was applied for 1 h at room temperature. The HISTOLOGY OF THE SURFACES OF THE BONE PLUGS negative control consisted of replacing the primary antibody with either rat or mouse IgG1 (depending on the Histological sections of the plugs were stained with primary antibody). toluidine blue, which stains cartilage preferentially, and Actin microfilaments were visualized by staining the cells examined after processing by light microscopy to confirm with Alexa Fluor 568-phalloidin (1:20 dilution in PBS) for the extent of the cartilage removed. As shown in Fig. 1,a 20 min at room temperature. For double stained exper- layer of mid-deep zone hyaline cartilage remained on the iments, the cells were incubated with phalloidin sequen- plugs with a cartilage surface, the calcified zone of cartilage tially after antibody staining. The cells were washed with was seen on the surfaces of the calcified cartilage-bone PBS containing 0.2% Triton X-100. The plug surface was plugs and subchondral bone was seen on the surface of mounted in Immuno Floure mounting medium and the cells the bone plugs only. The cores of some trabeculae con- were visualized either using confocal (Zeiss LSM 510) or tained calcified cartilage but these were surrounded by deconvolution microscopy (Olympus 1X70). By scanning in bone and were not exposed to the cells. Occasional plugs step sections (Z series) it was possible to ensure that only with calcified cartilage or bone surfaces were incompletely the cells attached to the surface were examined. covered by the desired surface. When this occurred it was only present focally and usually affected less than 5% of the surface available for cell attachment.

EFFECT OF INTEGRIN BLOCKING ON CELL ATTACHMENT ATTACHMENT ASSAY To examine whether integrins were involved in chondrocyte attachment, the cells were pre-incubated with antibody The percent of chondrocytes that adhered to the different that reacts with either 1, v3orv5 integrins. These plug surfaces was determined by counting the numbers of antibodies prevent ligand binding and block cell attach- fluorescently-labeled cells removed from the plug surface ment15,17. The freshly isolated or passaged cells were 24 h after seeding and dividing that number by the initial incubated with the following integrin blocking antibodies: 1 number of cells placed onto the plug. As shown in Table I, (4B4, 20 ug/ml), av3 (LM609, 20 ug/ml) or av5 (P1F6, similar percentages of freshly isolated cells attached to the 20 ug/ml) in Ham’s F-12 containing 0.5% BSA, 20 mM different surfaces. This ranged from 88.8±3 to 90.8±4%. HEPES and SYTO-13 at 4°C for 1 h with agitation. The cell Similar percentages of passaged cells attached to the suspension was then seeded on the different surfaces in different surfaces. Although passaged chondrocytes the presence of the appropriate antibody. After 24 h the showed a trend to a higher percent of cell attachment when attached cells were harvested using collagenase and the compared to the freshly isolated cells, the differences were number of fluorescent cells counted using a hemocytom- not significant. No fluorescently-labeled cells were obtained eter and fluorescent microscopy. Negative controls, which from the control plugs that had not received cells but had was done with each experiment, consisted of replacing been incubated with the SYTO-13 dye. the antibody with IgG1 (20 µg/ml, same isotype for all antibodies). DISTRIBUTION OF ACTIN, 1-INTEGRIN, AND VINCULIN IN CHONDROCYTES ATTACHED TO THE PLUGS To examine whether chondrocytes express 1 integrin RT-PCR and formed focal adhesions when attached to hyaline or Total RNA was extracted from the cells attached to the calcified cartilage and bone surfaces the adherent cells plugs 2, 6, and 24 h after being placed on the plug surface, were stained for 1 integrin, actin and vinculin, components using the Qiagen RNeasy kit, to determine expression of focal adhesions. The freshly isolated chondrocytes that levels of aggrecan core protein and type II collagen RNA. had been stained for actin and examined by confocal One microgram of total RNA underwent reverse- microscopy appeared round or polygonal in shape and transcription polymerase chain reaction (RT-PCR) using demonstrated a ring-like subcortical distribution of actin. By primers previously described28. The PCR was done in an deconvolution microscopy the cells, in some of the planes automated thermal cycler for 26 cycles as follows: dena- of imaging, showed a punctate actin distribution in keeping turation at 95°C for 30 s, annealing at 55°C for 60 s and with a network of actin filaments. The ring-like actin organi- elongation for 75 s at 72°C with a terminal cycle of 72°C for zation was present in the cells attached to all three surfaces 5 min. An equal volume of the PCR product from each [Fig. 2 (A–C)]. This differed from the passaged chondro- sample was loaded on a 1.5% agarose gel and was cytes where the cells were an admixture of round and visualized by ethidium bromide staining. Photographs were spread cells and the actin filaments were visible both as obtained and levels of gene expression were semi- fibrils at the periphery of the cells and as clusters randomly quantitated using a densitometer. The results were normal- distributed in the cytoplasm. Occasional spread cells had ized for the number of cells analysed. Each time point was stress fibers. Similar actin organization was seen in the done in triplicate. Three independent experiments were passaged cells attached to the different surfaces [Fig. 2 performed and the results were combined. (D–F)]. Osteoarthritis and Cartilage Vol. 12, No. 1 59

Fig. 1. Appearance of subchondral bone plug surfaces. Photomicrographs of histological sections from representative bone plugs immediately after removing the cartilage as described in the Methods. The sections showed (A) the mid-deep zone cartilage (↓) overlying the subchondral bone, (B) calciﬁed cartilage (↓) overlying the subchondral bone and (C) bone (↓) only, no cartilage. In (B) and (C), cartilage cores are present in some of the bone trabeculae. (Toluidine blue, original magniﬁcation ×40.)

Table I INTEGRINS AND CHONDROCYTE ATTACHMENT Chondrocyte attachment to different joint surfaces To investigate the role of integrins in the attachment of Chondrocyte type Plug surface % attachment chondrocytes to the plug surfaces, the cells were exposed Primary* Hyaline cartilage 89.5±3 to an antibody that was reactive with either 1 integrin or Calcified cartilage 90.8±4 v3orv5 and then placed on the plug surface in the Bone (no cartilage) 88.8±3 presence of the same antibody. As summarized in Fig. 5, Passaged* Hyaline cartilage 95.4±2 adhesion of either freshly isolated (A) or passaged Calcified cartilage 94.8±2 chondrocytes (B) to the three surfaces was partially inhib- Bone (no cartilage) 94.4±4 ited in the presence of either 1orv5 blocking antibody. The percent inhibition ranged from 27 to 34% for 1 and 27 *Freshly isolated (primary) or passaged (P1) chondrocytes were to 29% for v5 for the primary cells. There appeared to be seeded on the surfaces of bone plugs with either mid-deep zone greater inhibition of attachment of passaged cells ranging hyaline cartilage, or calcified cartilage or no cartilage (bone). After 24 h the cells that attached were counted as described under the from 41 to 48% for 1 and 26 to 37% for v5 when Materials and methods. The percent of cells attached was calcu- compared to primary chondrocytes. However these differ- lated. Each condition was done in triplicate and the experiment ences were not significant. Treatment of the cells with was repeated five times. The results were pooled and expressed antibody that blocks v3 had no effect on adhesion. as the mean±SD (P>0.05).

EFFECT OF THE DIFFERENT SURFACES ON CHONDROCYTE GENE EXPRESSION Deconvolution microscopy showed that both primary and To determine whether placing the cells on the different passaged chondrocytes expressed 1 integrin and vinculin surfaces inﬂuenced gene expression, the levels of aggre- in a punctate distribution associated with the cell mem- can core protein and type II collagen mRNA were evaluated brane which in areas co-localized with actin (Fig. 3 and Fig. semi-quantitatively by RT-PCR. PCR performed at different 4). These were also present at the bottom of the cells cycle numbers showed that the PCR products were in the where they attached to the plug surface [Fig. 3 (B and D) linear range between 25 to 35 cycles. All of the analyses and Fig. 4)]. This suggested that focal adhesions were were done at 26 cycles. Freshly isolated and passaged present and were seen in the cells attached to all three chondrocytes express type II collagen and aggrecan core different surfaces. Interestingly in the passaged cells the protein mRNA by 2 h after seeding. The levels of expres- spread cells also had elongated focal adhesions, a feature sion are higher in the primary cells than the passaged cells not seen in the primary cells. at all time points. No differences in expression of these two 60 H. Wang and R. A. Kandel: Chondrocyte attachment

$ % &

' ( )

Fig. 2. Visualization of actin cytoskeleton. Confocal images of freshly isolated chondrocytes (A–C) or passaged chondrocytes (D–F) attached to hyaline cartilage (A,D), or calcified cartilage (B,E) or bone (C,F) and stained for actin using Alexa Fluor 568-phalloidin. The freshly isolated cells have a ring-like distribution of actin whereas in the passaged cells that were spread the actin was present at the periphery and as clusters in the cytoplasm (arrow indicates round cells; arrowhead indicates polygonal shaped cell). genes were seen in chondrocytes 2, 6 or 24 h after attach- the three surfaces during the first 24 h. Furthermore both ment to hyaline cartilage, calcified layer, or bone surfaces primary cells and passaged cells were able to adhere to (Fig. 6). these surfaces. It is possible that there was an overestima- To ensure that we were examining RNA extracted from tion of cell attachment to the bone because there might chondrocytes that had been placed on the plug, plugs that have been a greater surface area available for attachment. had not been seeded with chondrocytes were subjected to These plugs had focal areas where the surface was not RNA extraction and RT-PCR. No expression of aggrecan continuous because of the porous nature of bone [see edge core protein or type II collagen RNA was detected indicat- of Fig. 1 (C)]. This could have allowed some flow through of ing that any gene expression observed was from the cells cells into the pores of the bone. Also it is possible that the placed on the plugs. percentages of cell attachment to the different surfaces were similar because some of the calcified and bone plugs were incompletely covered with the desired surface. How- Discussion ever this was considered unlikely as most of the plugs that were examined appeared to have the appropriate surface. This study demonstrated that freshly isolated and pas- When remnants of cartilage remained on the plug surface it saged chondrocytes can adhere to hyaline or calcified represented only a small part of the available surface, as cartilage or bone. 1 and v5 integrins appears to be determined by the random histological evaluation of the involved in the attachment to these surfaces. The cells that plugs. At least 15 plugs in five independent experiments attach can form focal adhesions. The chondrocytes ex- were assessed for each experimental condition to minimize press type II collagen and aggrecan core protein during the the effect of this factor. The observation that chondrocytes first 24 h after the placement of the cells on the different can attach to cartilage is in keeping with results from other surfaces. The expression of these genes is detected as studies showing that passaged articular chondrocytes can early as 2 h and does not appear to be influenced by the attach to fresh, frozen and lyophilized articular cartilage surface to which the cells are attached. in vitro7–10. However, in none of those studies did they The type of surface did not appear to influence cell examine whether chondrocytes could attach to the calcified attachment as similar percentages of cells were retained on zone of cartilage or bone. Osteoarthritis and Cartilage Vol. 12, No. 1 61

$ %

& '

Fig. 3. Vinculin and actin localization. Freshly isolated (A,B) or passaged (C,D) chondrocytes attached to mid-deep hyaline cartilage were stained for actin and vinculin and imaged using deconvolution microscopy. Actin ﬁlaments were stained with phalloidin and appear red, the immunostained vinculin appears green, and co-localization of the two is yellow (→). (A) and (C) are images from the z series representing the middle of the cells whereas (B) and (D) are images from the bottom of the cells (adjacent to the plug surface). The bar is 5 µm.

Under the conditions used in this study integrins ap- variety of proteins, such as collagens, ﬁbronectin, and peared to be involved in chondrocyte attachment to joint osteopontin11,15,16. These macromolecules are present in surfaces. 1 and v5 contributed to chondrocyte adhesion the surfaces examined so although different the appropri- whereas v3 did not. This is in keeping with studies by ate ligands for integrin mediated attachment are present in Kurtis et al. who also demonstrated the involvement of all of them. similar integrins in the attachment of passaged human Further studies are required to determine the exact roles chondrocytes to cartilage slices17. Although the plug sur- of 1 and v5 in cell adhesion and whether they interact faces differ in composition, the chondrocytes appeared to during this process. For example it has been shown that use similar integrins to attach to them all. The exact nature blocking 51 prevented chondrocyte spreading on ﬁbro- of the ligand(s) involved in these interactions are not nectin29 and perhaps this is the mechanism by which 1 known. Integrins have been shown to recognize a wide affected attachment. Nevertheless preventing v5or1 62 H. Wang and R. A. Kandel: Chondrocyte attachment

Fig. 5. Effect of integrins on cell attachment to different surfaces. Either primary (freshly isolated) (A) or passaged cells (B) were placed on plugs with either mid-deep zone hyaline cartilage, calcified cartilage or bone surface in the presence or absence of antibodies that block 1orv3orv5 integrin as described under the Materials and methods. As a control the cells were incubated with IgG1, which is the same isotype as all the antibodies. Twenty-four hours later the cells were harvested. The results from at least two independent experiments were pooled and expressed as the mean±SD. The percent of cells attached in the presence of 1 (diagonal slashed bar), or v3 (dotted bar) or Fig. 4. 1 integrin and actin localization. Freshly isolated (A) or v5 (unfilled bar) or in the absence (in the presence of IgG1) passaged chondrocytes (B) attached to mid-deep hyaline cartilage (solid bar) of blocking antibody is shown. *P<0.05. were doubly stained for actin (red) and 1 integrin (green) and were visualized using deconvolution microscopy. Co-localization of actin and 1 integrin are yellow (↓). The images were selected from the bottom of the z series and show the cells adjacent to the The chondrocytes, both the freshly isolated and pas- plug surface. The bar is 5 µm. saged cells, formed focal adhesions after attachment to the different surfaces. These contacts were visualized by both confocal and deconvolution microscopy after staining for 1 integrin, vinculin and actin. This was not unexpected as integrin-ligand interactions did not completely prevent chondrocytes in native cartilage have focal adhesions33–35. chondrocyte attachment. There are several possible expla- Others have also reported their presence in chondrocytes nations for this. Firstly, chondrocyte adhesion could be attached to materials36. The adhesions present in the influenced by a variety of technical factors related to the primary cells appeared smaller than those seen in pas- assay itself, including the amount of time allowed for saged chondrocytes. Reddi et al. also observed small focal attachment, the amount of antibody used relative to the cell adhesions in chondrocytes in suspension (agarose coated number or the sensitivity and specificity of the adhesion dishes) culture as compared to cells grown in monolayer37. assay9,15. Secondly, the culture conditions utilized in the The significance of these smaller adhesions and their study may influence attachment. Thirdly, other types of relationship to focal contacts, which have been described in integrins, not evaluated in this study, may also contribute to other cell types, is not clear38. Nevertheless these contacts attachment. Alternatively other chondrocyte receptors, are functional as inhibition of 1 integrin binding decreased such as CD44 and annexin V, which bind to matrix mol- cell attachment to the surface. ecules may be involved13,14,30,31. This is considered less Chondrocytes placed on the three surfaces expressed likely as previous work showed that blocking CD44 or type II collagen and aggrecan core protein within 2 h. The annexin V had no detectable effect on bovine chondrocyte type of surface did not seem to influence the gene expres- adhesion to hyaline cartilage10. However this does not rule sion as similar levels were seen in chondrocytes up to 24 h out a role for these receptors in mediating attachment to after attachment to hyaline or calcified cartilage or bone. calcified cartilage or bone. Finally it is possible that the Primary chondrocytes had higher levels of expression extracellular matrix proteins may bind to more than one when compared to passaged cells and this did not appear type of adhesion molecule32. to be influenced by the type of surface on to which they are Osteoarthritis and Cartilage Vol. 12, No. 1 63

$ % FHOOV FHOOV 2' 2' $JJUHFDQ ([SUHVVLRQ

7\SH,, &ROODJHQ ([SUHVVLRQ 7LPH KUV 7LPH KUV

& '

FHOOV FHOOV 2' 2' $JJUHFDQ ([SUHVVLRQ 7\SH ,, &ROODJHQ ([SUHVVLRQ 7LPH KUV 7LPH KUV

Fig. 6. Type II collagen and aggrecan core protein expression by chondrocytes on different surfaces. Either freshly isolated (A,B) or passaged cells (C,D) were placed on plugs with either mid-deep zone hyaline cartilage (black), calcified cartilage (grey), or bone (white) surface. After various times total RNA was extracted and underwent reverse transcription followed by PCR. The products were run on an agarose gel, semi-quantified using densitometry and the results normalized for cell number (OD/104 cells). Three independent experiments were performed and the results pooled and expressed as the mean±SD. (A,C): type II collagen; (B,D): aggrecan core protein. placed. This suggests that factors other than the type of Acknowledgements surface to which the cells attach will effect the gene expression of these two molecules. For example the or- We would like to thank Hanje Chen for his help with tissue ganization of the actin cytoskeleton may regulate gene culture, Veronica Ciolfi for advice regarding confocal expression39. Brown and Benya showed that dedifferenti- microscopy and immunostaining, and Isabelle Schell for ated chondrocytes can be induced to re-express type II her secretarial assistance. The 1 integrin antibody (SP2/0) collagen when incubated with cytochalasin under con- developed by Caroline Damsky was obtained from the ditions that modify actin filament organization40. Interest- Developmental Studies Hybridoma Bank under the aus- ingly we observed that actin mRNA levels increased in both pices of the NICHD and maintained by the University of primary and passaged cells over time (data not shown), the Iowa, Department of Biological Sciences, Iowa City, IA significance of which is not known but may indicate an 52242. ongoing role for actin in chondrocytes. In summary, this study suggests that chondrocytes, either freshly isolated or cells that have been passaged once, have the potential to adhere to the different joint References surfaces that could be exposed in a cartilage defect. Furthermore the cells that attach to these surfaces do so in 1. Hunziker EB. Articular cartilage repair: are the intrinsic part through 1 and v5 integrins. The cells express type biological constraints undermining this process II collagen and aggrecan RNA within 2 h of placement of insuperable? Osteoarthritis Cartilage 1999;7:15–28. the cells on the surfaces. Actin organization in the cells and 2. Buckwalter JA, Rosenberg LC, Hunziker EB. Articular gene expression of type II collagen and aggrecan core cartilage: composition, structure, response to injury, protein appeared to be influenced more by the state of and methods of facilitating repair. In: Ewing JW, Ed. differentiation of the cells (passaged vs freshly isolated) Articular Cartilage and Knee Joint Function: Basic than the surface on to which they were placed within the Science and Arthroscopy. New York: Raven Press, first 24 h. Further experimentation is required to delineate Ltd 1990. the entire mechanism of attachment and whether the 3. Frenkel SR, Di Cesare P. Degradation and Repair of chondrocytes behave similarly when implanted in vivo. Articular Cartilage. Frontiers in Bioscience. 1999; Understanding how chondrocytes adhere and interact with 4:671–85. damaged joint surfaces may help identify methods to 4. Gilbert JE. Current treatment options for the restora- enhance the retention of transplanted cells in the defect site tion of articular cartilage. Am J Knee Surg 1998; and accumulation of matrix molecules. 11:42–6. 64 H. Wang and R. A. Kandel: Chondrocyte attachment

5. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson 24. Schinagl RM, Kurtis MS, Ellis KD, Chien S, Sah RL. O, Peterson L. Treatment of deep cartilage defects Effect of seeding duration on the strength of chondro- in the knee with autologous chondrocyte trans- cyte adhesion to articular cartilage. J Orthop Res plantation. N Engl J Med 1994;331:889–95. 1999;17:121–9. 6. Brittberg M. Autologous chondrocyte transplantation. 25. Shortkroff S, Barone L, Hsu H-P, Wrenn C, Gagne T, Clin Orthop Rel Res 1999;367S:S147–55. Chi T, et al. Healing of chondral and osteochondral 7. Lee MC, Sung KLP, Kurtis MS, Akeson WH, Sah RL. defects in a canine model; the role of cultured Adhesive force of chondrocytes to cartilage. Clin chondrocytes in regeneration of articular cartilage. Orthop Rel Res 2000;370:286–94. Biomaterials 1996;17:147–54. 8. Peretti GM, Randolph MA, Caruso EM, Rossetti F, 26. Breinan HA, Minas T, Hsu H-P, Nehrer S, Sledge CB, Zaleske DJ. Bonding of cartilage matrices with cul- Spector M. Effect of cultured autologous chondro- tured chondrocytes: an experimental model. J Orthop cytes on repair of chondral defects in a canine model. Res 1998;16:89–95. J Bone Joint Surg 1997;79:1439–51. 9. Lee MC, Sung KL, Kurtis MS, Akeson WH, Sah RL. 27. Boyle J, Bo L, Cruz TF, Kandel RA. Characterization of Adhesive force of chondrocytes to cartilage. Effects proteoglycan accumulation during formation of carti- of chondroitinase ABC. Clin Orthop 2000; lagenous tissue in vitro. Osteoarthritis Cartilage 370:286–94. 1995;3:117–25. 10. Kurtis MS, Tu BP, Gaya OA, Mollenhauer J, Knudson 28. Sun YL, Kandel R. Deep zone articular chondrocytes W, Loeser RF, et al. Mechanisms of chondrocyte in vitro express genes that show speciﬁc changes adhesion to cartilage: role of ß1-integrins, CD44, and with mineralization. J Bone Miner Res. 1997; annexin V. J Orthop Res 2001;19:1122–30. 12:1124–32. 11. Eble JA. The molecular basis of integrin-extracellular 29. Enomoto-Iwamoto M, et al. Involvement of 51 integrin matrix interactions. Osteoarthritis Cartilage 2001; in matrix interactions and proliferation of 9(A):S131–40. chondrocytes. 12. Loeser RF, Sadiev S, Tan L, Goldring MB. Integrin 30. Mollenhauer J, Mok MT, King KB, Gupta M, expression by primary and immortalized human Chubinskaya S, Koepp H, et al. Expression of an- chondrocytes: evidence of a differential role for a11 chorin CII (cartilage annexin V) in human young, and a21 integrins in mediating chondrocyte adhe- normal adult, and osteoarthritic cartilage. J Histo- sion to types II and VI collagen. Osteoarthritis chem Cytochem 1999;47:209–20. Cartilage 2000;8:96–105. 31. Reid DL, Aydelotte MB, Mollenhauer J. Cell attach- 13. Ishida O, Tanaka Y, Morimoto I, Takigawa M, Eto S. ment, collagen binding, and receptor analysis on Chondrocytes are regulated by cellular adhesion bovine articular chondrocytes. J Orthop Res 2000; through CD44 and hyaluronic acid pathway. J Bone 18:364–73. Miner Res 1997;12:1657–63. 32. Giancotti FG, Ruoslalhti E. Integrin signaling. Science 1999;285:1028–32. 14. Knudson W, Loeser RF. CD 44 and integrin matrix 33. Durrant LA, Archer CW, Benjamin M, Ralphs JR. receptors participate in cartilage homeostasis. Cell Organization of the chondrocyte cytoskeleton and its Mol Life Sci 2002;59:36–44. response to changing mechanical conditions in organ 15. Loeser RF. Chondrocyte integrin expression and culture. J Anat 1999;194:343–53. function. Biorheology 2000;37:109–16. 34. Langelier E, Suetterlin R, Hoemann CD, Aebi U, 16. Loeser RF. Integrins and cell signalling in Buschmann MD. The chondrocyte cytoskeleton in chondrocytes. Biorheology 2002;39:119–24. mature articular cartilage: structure and distribution of 17. Kurtis MS, Tannin A, Bugbee WD, Loesser RF, Sah actin, tubulin and vimentin ﬁlaments. J Histochem RL. Integrin-mediated adhesion of human articular Cytochem 2000;48:1307–20. chondrocytes to cartilage. Arth Rheum 2003; 35. Vinall RL, Lo SH, Reddi AH. Regulation of articular 48:110–8. chondrocyte phenotype by bone morphogenetic pro- 18. Petit V, Thiery J-P. Focal adhesions: structure and tein 7, interleukin 1, and cellular context is dependent dynamics. Biol Cell 2000;92:477–94. on the cytoskeleton. Exp Cell Res 2002;272:32–44. 19. Zamir E, Geiger B. Molecular complexity and dynamics 36. Loty C, Forest N, Boulekbache H, Kokubo T, Sautier of cell-matrix adhesions. J Cell Sci 2001; JM. Behaviour of fetal rat chondrocytes cultured on a 114:3583–90. bioactive glass-ceramic. J Biomed Mater Res 1997; 20. Adams JC. Cell-matrix contact structures. Cell Mol Life 37:137–49. Sci 2001;58:371–92. 37. Vinall RL, Reddi AH. The effect of BMP on the expres- 21. Grande DA, Pitman MI, Peterson L, Menche D, Klein sion of cytoskeleton proteins and its potential M. The repair of experimentally produced defects in relevance. JBJS 2001;83-AS1:63–9. rabbit articular cartilage by autologous chondrocyte 38. Renaudin A, Lehmann A, Girault J-A, McKerracher A. transplantation. J Orthop Res 1989;7:208–18. Organization of point contacts in neuronal growth 22. Breinan HA, Minas T, Barone L, Tubo R, Hsu H-P, cones. J Neuroscience Res 1999;55:458–71. Shortkroff S, et al. Histological evaluation of the 39. Mallein-Gerin F, Garrone R, van der Rest M. Proteo- course of healing of canine articular cartilage de- glycan and collagen synthesis are correlated with fects treated with cultured autologous chondrocytes. actin organization in dedifferentiating chondrocytes. Tissue Eng 1998;4:1011–4. Eur J Cell Biol 1991;56:364–73. 23. Sohn DH, Lottman LM, Lum LY, Kim SG, Pedowitz RA, 40. Brown PD, Benya PD. Alterations in chondrocyte cyto- Coutts RD, et al. Effect of gravity on localization of skeletal architecture during phenotypic modulation by chondrocytes implanted in cartilage defects. Clin retinoic acid and dihydrocytochalasin B-induced Orthop 2002;394:254–62. reexpression. J Cell Biol 1988;0:171–9.