ARTICLE

Immunochemical and Mechanical Characterization of Cartilage Subtypes in Rabbit

Correspondence to: James E. Dennis, Skeletal Research Center, Dept. of Biology, Case Western Reserve University, 2080 Adelbert Road, Cleveland, OH 44106. E-mail: jed4@po.cwru.edu

	Summary

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Cartilage is categorized into three general subgroups, hyaline, elastic, and fibrocartilage, based primarily on morphologic criteria and secondarily on collagen (Types I and II) and elastin content. To more precisely define the different cartilage subtypes, rabbit cartilage isolated from joint, nose, auricle, epiglottis, and meniscus was characterized by immunohistochemical (IHC) localization of elastin and of collagen Types I, II, V, VI, and X, by biochemical analysis of total glycosaminoglycan (GAG) content, and by biomechanical indentation assay. Toluidine blue staining and safranin-O staining were used for morphological assessment of the cartilage subtypes. IHC staining of the cartilage samples showed a characteristic pattern of staining for the collagen antibodies that varied in both location and intensity. Auricular cartilage is discriminated from other subtypes by interterritorial elastin staining and no staining for Type VI collagen. Epiglottal cartilage is characterized by positive elastin staining and intense staining for Type VI collagen. The unique pattern for nasal cartilage is intense staining for Type V collagen and collagen X, whereas articular cartilage is negative for elastin (interterritorially) and only weakly positive for collagen Types V and VI. Meniscal cartilage shows the greatest intensity of staining for Type I collagen, weak staining for collagens V and VI, and no staining with antibody to collagen Type X. Matching cartilage samples were categorized by total GAG content, which showed increasing total GAG content from elastic cartilage (auricle, epiglottis) to fibrocartilage (meniscus) to hyaline cartilage (nose, knee joint). Analysis of aggregate modulus showed nasal and auricular cartilage to have the greatest stiffness, epiglottal and meniscal tissue the lowest, and articular cartilage intermediate. This study illustrates the differences and identifies unique characteristics of the different cartilage subtypes in rabbits. The results provide a baseline of data for generating and evaluating engineered repair cartilage tissue synthesized in vitro or for post-implantation analysis. (J Histochem Cytochem 50:1049–1058, 2002)

Key Words: cartilage subtypes, collagen, glycosaminoglycan, biomechanics

	Introduction

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CARTILAGE is an avascular tissue that consists of chondrocytes and an extensive extracellular matrix (ECM) that is produced and maintained by chondrocytes. All cartilage matrices are composed predominantly of aggrecan and Type II collagen. Type II collagen is primarily responsible for tensile strength, while aggrecan molecules, when entrapped within the Type II collagen lattice, provide compressive strength. The highly sulfated glycosaminoglycan (GAG) side chains of chondroitin sulfate and keratan sulfate enable the matrix to structure large amounts of water and thereby create a large osmotic pressure (Ehrlich et al. 1998 ). Long chains of hyaluronan are present throughout the matrix and serve to specifically bind aggrecan and link protein to retain the aggrecan molecules within the cartilage ECM (Caplan 1984 ). Cartilage is further classified as hyaline cartilage, elastic cartilage, or fibrocartilage, based on the presence of collagen and elastic fibers and on tissue morphology (Ross et al. 1995 ). The goal of this study was to further define the subtypes of cartilage based on the molecular composition and structure of the cartilage matrix, glycosaminoglycan (GAG) content, and mechanical properties.

Type I collagen is the prominent structural element in all coarse fibrous connective tissue, including fibrocartilage (Eyre and Wu 1983 ), skin, bone, and tendon. Type II collagen, which represents approximately 85–90% of the total tissue collagen of hyaline cartilage (Burgeson et al. 1982 ), is also present in different amounts in elastic cartilage and fibrocartilage. The presence of Type X collagen is associated mainly with hypertrophic cartilage, either in vitro (Capasso et al. 1982 ; Gibson et al. 1982 ) or in vivo in growth plate (Nerlich et al. 1992 ) or sternal cartilage destined to become mineralized (Gibson et al. 1984 ). Type X collagen has also been identified in the fibrocartilage at the bone insertion site of the Achilles tendon (Fukuta et al. 1998 ). Type V (Evans et al. 1983 ; Eyre et al. 1987 ; Bland and Ashhurst 1996 ) and Type VI (Sakai et al. 1986 ; Keene et al. 1988 ; Poole et al. 1988 ) collagen have been reported to be present in different degrees in the three cartilage subtypes.

Cartilage tissues located at different anatomic sites have different physiological requirements and are subjected to site-specific mechanical stresses. Either in response to these physiological and mechanical conditions or as a consequence of developmental events, cartilage from different anatomic sites varies in total aggrecan and collagen content and also varies in the amount and in the localization pattern of minor collagens. Although reports have described the presence of collagen Types V, VI, and X at various anatomic sites and in different species, no study has comprehensively characterized the distribution of these collagens at different anatomic sites from a single species. In this study, histological, immunohistochemical (IHC), and biomechanical methods were used to obtain a bank of information on the distribution of collagens, the GAG contents, and the biomechanical properties of native rabbit cartilage from different sources. These results provide a standard profile for native cartilage against which engineered replacement tissues can be evaluated.

	Materials and Methods

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Cartilage Harvest
Native cartilage was obtained from six 4-month-old male New Zealand White rabbits. The different subtypes of cartilage, such as hyaline cartilage (nasal septum, articular), elastic cartilage (auricle, epiglottis), and fibrocartilage (meniscus), were surgically removed and immediately fixed in 10% neutral buffered formalin. Articular cartilage was obtained from the medial portion of the distal femoral condyle. Auricular cartilage samples were obtained from the central region of the ear cartilage. Epliglottal cartilage was obtained by cutting the epiglottis off at the height of the vallecula, and cross-sections were obtained from an anterior to posterior direction. For nasal cartilage, a 10-mm sample of septal cartilage was taken starting at approximately 5 mm from the nasal tip Samples destined for mechanical testing were placed in saline plus protease inhibitors (0.1 M sodium chloride, 10 mM ethylenediaminetetraacetic acid [EDTA], 5 mM benzamidine-HCl, 10 mM N-ethylmaleimide, 0.1 M 6-aminocaproic acid, and 1 mM phenylmethylsufonyl fluoride).

Histology and IHC
The samples were dehydrated, embedded in paraffin, and 5-µm sections were cut. For histochemical staining of GAGs, representative sections from all cartilage subtypes were stained with 0.2% Toluidine blue (Sigma T-3260; Sigma Chemicals, St Louis, MO) for 5 min, and neighboring sections were stained for 6 min in 0.1% safranin-O containing 1% acetic acid and counter-stained for 2 min in 1% Fast Green containing 7% acetic acid.

For IHC analysis, sections of all cartilage subtypes were rehydrated, incubated in 1 mg/ml pronase (Sigma P-5147) in PBS (10 mM sodium phosphate, 0.15 M sodium chloride, pH 7.4) for 15 minutes at room temperature (RT), and stained with antibodies to Type I, II, V, VI, or X collagen or to elastin. The sources and dilutions for the primary antibodies are listed in Table 1. Secondary antibody was either biotinylated goat anti-mouse (Cappel, Organon Technica; West Chester, PA) diluted 1:100 in 1% BSA (bovine serum albumin) in PBS or horseradish peroxidase-conjugated goat-anti-rabbit IgG, diluted 1:250 in 1% BSA/PBS. Samples incubated in biotinylated secondary antibody were then incubated in streptavidin–peroxidase, diluted 1:300 in 1% BSA/PBS. Slides were washed in PBS and contrasted with solution from the Vector VIP Substrate Kit (Vector Labs; Burlingame, CA) according to the manufacturer's instructions, washed, and counterstained with Fast Green. The slides were observed on an Olympus BH-2 fluorescence microscope.

Preliminary studies were conducted to determine the optimal pretreatment conditions for immunostaining with the different antibodies. In addition to pronase, testing was performed using chondroitinase ABC and hyluronidase, alone or in combination, or in combination with pronase digestion, to determine the optimal pretreatment protocol. Sections were digested for up to 1hr with 0.1 U/ml chondroitinase ABC, 2.5% testicular hyluronidase, with and without digestion in 1 mg/ml pronase. The sections were then stained with each of the antibodies used in this study and compared for staining intensity. To confirm the effectiveness of chondroitinase ABC digestion, a matching slide was stained with the antibody 3B3, whose antigenic site on sulfated proteoglycans (PGs) is revealed only after chondroitinase digestion. No significant increase in staining intensity was observed with hyluronidase and/or chondroitinase digestion in any of the samples, and hyluronidase and chondroitinase digestion alone, without pronase digestion, always resulted in only minimal or no positive staining. Interestingly, matching studies in human tissues showed significant differences in staining intensity when pronase is used in combination with hyluronidase or chondroitinase digestion (unpublished observations).

Safranin O Assay
Before measurement of total GAG content, the samples of cartilage subtypes were cut with a punch (5.0 mm in diameter) and enzymatically digested. A matching punch sample was taken from an adjacent region and prepared for histology to determine the exact thickness of the sample. The samples were digested with a solution containing 20 mM sodium phosphate buffer, pH 6.8, 1 mM EDTA, 2 mM dithiothreitol, and 300 µg/ml papain. Samples were incubated for 60 min at 60C. Digestion was stopped by the addition of 110 mM iodoacetic acid, followed by incubation for 5 min at RT. The samples were centrifuged at 1200 rpm for 5 min, and then aliquots were removed for the Safranin O assay.

The total GAG content was assayed colorimetrically by a Safranin O assay as previously described (Carrino et al. 1991 ). All standard curves were fitted by linear regression through the linear portion of the data. The sizes of sample aliquots were adjusted so that the absorbance values were within the linear portion of the standard curves. The final results are expressed as micrograms of GAG per cubic centimeter of tissue [determined from the measured thickness of the sample times 6.25 x 10^-2 (0.25-cm radius squared) times ].

Biomechanical Testing
To determine the indentation creep behavior of cartilage subtypes, a biphasic indentation assay was performed with a plane-ended circular porous microindentor (0.91 mm in diameter), as previously described (Jurvelin et al. 1986 , Jurvelin et al. 1990 ; Mow et al. 1989 ; Athanasiou et al. 1991 ; Palmer et al. 1995 ). Before testing, each cartilage subtype sample was placed for 1 hr at RT in saline solution (0.15 M NaCl) containing enzyme inhibitors (10 mM EDTA, 5 mM benzamidine, 10 mM N-ethylmaleimide, 6-aminocaproic acid, and 1 mM PMSF). The cartilage specimen was fixed to the center of a cylindrical container with a cyanoacrylate adhesive. Each specimen was visually aligned perpendicular to the indentor tip. After alignment of the specimen, the test chamber was filled with saline solution and a tare load of 0.015 N was applied to ensure full contact between the cartilage sample and the indentor tip. A porous indentor was used to maintain a zero pressure boundary condition on the surface (a requirement for later processing of data to obtain material properties). The purpose of the tare load is to endure good contact between the indentor tip and the sample. Under the tare load, the surface deformed at a decreasing rate and came to equilibrium in 900–1500 sec. A constant test load of 0.029 N was then applied, and the sample displacement was again allowed to come to equilibrium. Values of the applied load were chosen to limit the deformation of the sample to approximately 20% of its thickness, another requirement for subsequent processing to determine material properties: aggregate modulus, Poisson's ratio, and permeability. Total time to creep equilibrium varied from 2000 to 4000 sec. The criterion for equilibrium was no more than a 1-µm change in displacement in a 10-min-period. The exact cartilage thickness of each sample was determined by fixing and paraffin-embedding the tissue, and measuring the thickness in cross-sections using a light microscope. By curve-fitting the experimental creep curves with the master solution via a nonlinear regression procedure, the intrinsic material properties (aggregate modulus, Poisson's ratio, and permeability) of the different cartilage subtypes were calculated (Mak et al. 1987 ; Mow et al. 1989 ). Aggregate modulus is a measure of the stiffness of the material at equilibrium, Poisson's ratio is a measure of compressibility, and permeability is a measure of the resistance to fluid flow through the matrix.

Significance of differences in mechanical properties between different cartilage subtypes was analyzed by ANOVA and pairwise comparisons were tested by the Student–Newman–Keuls post-hoc method. p<0.05 was defined as a significant difference between the properties of cartilage subtypes.

	Results

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Histological Observations
The morphology of the different cartilage subtypes is shown in Fig 1. Nasal and auricular cartilage are quite similar when examined after Toluidine blue or Saffranin-O staining [ Fig 1A and Fig 1F (nasal) and Fig 1C and Fig 1H (auricle)]. In contrast, epiglottis (Fig 1B) and meniscus (Fig 1E) show more metachromatic staining in the Toluidine blue-stained preparations. Safranin-O staining shows the highest intensity in auricular (Fig 1F) and nasal cartilage (Fig 1H), both of which are intensely blue in the Toluidine blue-stained sections instead of the metachromatic pink staining usually correlated with high GAG content. The lack of pink metachromasia in regions that stain intensely with Safranan-O shows that metachromasia is not a reliable indicator of high GAG concentration or density within the tissue. Another prominent feature in the Toluidine blue-stained sections is the fibrillar nature of the matrix in the meniscus (Fig 1E).

View larger version (120K): [in this window] [in a new window]   Figure 1. Samples of cartilage subtypes from auricular (A,F,K), epiglottal (B,G,L), nasal (C,H,M), articular (D,I,N), and meniscal (E,J,O) cartilage are shown after staining with Toluidine blue (A–E) or Safranin-O (F–J) and after immunostaining with antibody to elastin (K–O). Bars = 100 mm.

Immunohistochemistry
Immunochemical staining with an antibody to elastin (Fig 1K–1O) revealed significant differences between the cartilage subtypes. Only auricular (Fig 1K) and epiglottal cartilage (Fig 1L) showed detectable staining for elastin within the interterritorial matrix. The epiglottis exhibited the most intense staining within focal areas of the matrix. Nasal cartilage showed only minimal pericellular staining (Fig 1M), while meniscus showed only cellular staining (Fig 1O). ("Cellular" refers to staining that appears to be cytoplasmic, but is interpreted to arise from matrix material that collapses into chondrocytes during the fixation process. This fixation artifact can be avoided but at the expense of antigenicity to the antibodies used in this study.) Articular cartilage was mostly negative except for rare cells that were intensely stained (Fig 1N). It was a recurring observation that immunostaining patterns varied among cartilage subtypes with respect to matrix, pericellular, and cellular localization. For this reason, the observations for all of the immunostaining patterns were categorized for their intensity and cellular and matrix localization; these data are summarized in Table 2. Samples of cartilage subtypes from three different rabbits were examined to determine staining patterns.

Immunostaining for collagens showed specific staining patterns for each of the cartilage subtypes. Articular cartilage was easily differentiated from the other cartilage subtypes collagens because it stained weakly for collagen Type I, although adjacent osseous tissue stained intensely (Fig 2D). In contrast, each of the other cartilage subtypes showed at least some staining for Type I collagen. Only meniscus showed any Type I collagen staining in the matrix (Fig 2E). Nasal, epiglottal, and auricular cartilage showed either cellular or pericellular staining (Fig 2A–2C). As expected, all cartilage subtypes were positive for Type II collagen (Fig 2F–2J) and differed from each other primarily in the distribution of collagen within the matrix. Staining with antibody to Type V collagen gave unexpected results. Auricular and nasal cartilage were intensely stained for this minor collagen (Fig 2K and Fig 2M), and meniscus showed focally intense staining (Fig 2O). Epiglottal and articular cartilage were only moderately positive for Type V collagen (Fig 2L and Fig 2N). Type VI collagen immunostaining (Fig 3A–3E) further revealed differences in the cartilage subtypes, in that auricular cartilage was completely negative (Fig 3A) and epiglottis was highly positive in the pericellular and cellular regions (Fig 3B). Articular cartilage showed negative or very weak diffuse staining (Fig 3D), and nasal cartilage and meniscal cartilage were stained at intermediate intensity (Fig 3C and Fig 3E).

View larger version (155K): [in this window] [in a new window]   Figure 2. Samples of cartilage subtypes from auricular (A,F,K), epiglottal (B,G,L), nasal (C,H,M), articular (D,I,N), and meniscal (E,J,O) cartilage are shown after immunohistochemical staining with antibodies to Type I collagen (A–E), Type II collagen (F–J), and Type V collagen (K–O). Bars = 100 mm.

View larger version (148K): [in this window] [in a new window]   Figure 3. Samples of cartilage subtypes from auricular (A,F,K), epiglottal (B,G,L), nasal (C,H,M), articular (D,I,N), and meniscal (E,J,O) cartilage are shown after immunohistochemical staining with antibodies to Type VI collagen (A–E), Type X collagen (F–J), and no first antibody controls (K–O). Bars = 100 mm.

Type X collagen staining (Fig 3F–3J) for epiglottis and meniscus was completely negative (Fig 3G and Fig 3J), and articular cartilage (Fig 3I) showed staining only in the matrix near the tidemark. Interestingly, both auricular (Fig 3F) and nasal cartilage (Fig 3H) are positive for Type X collagen in the pericellular zone. Together, these immunolocalization data provide sufficient information to clearly distinguish each of the cartilage subtypes by a number of staining characteristics, as summarized in Table 2.

Safranin-O Assays
Quantification of GAG by Safranin-O assay of tissue digests (Fig 4) revealed significant differences in total GAG content between elastic cartilage subtypes (auricle and epiglottis) and hyaline cartilage subtypes (nose and knee joint). Fibrocartilage (meniscus) contained significantly less GAG than hyaline cartilage and slightly higher GAG than elastic cartilage (Fig 4). There were no significant differences within the hyaline and elastic cartilage subtypes. Total GAG content did not correlate with the results of biomechanical testing.

View larger version (15K): [in this window] [in a new window]   Figure 4. Total GAG content was determined by Safranin-O assay for each of the different cartilage subtypes. Assays were run in duplicate on samples obtained from six different rabbits.

Indentation Assay
Indentation assays were used to generate material properties (thickness, permeability, Poisson's ratio, and aggregate modulus) of the different cartilage subtypes; these biomechanical measurements are summarized in Table 3. Although the auricle and epiglottis are both elastic cartilage, biomechanical measurements showed significant differences in stiffness (aggregate modulus). There were significant differences in stiffness between elastic cartilage of epiglottis and hyaline cartilage of nose, and nasal cartilage was also significantly higher in stiffness compared to meniscal fibrocartilage. Auricular elastic cartilage had an aggregate modulus similar to that of nasal hyaline cartilage. No significant differences were found between the elastic epiglottis, hyaline articular, and fibrous meniscal cartilage, which indicates that aggregate modulus did not correlate with the cartilage subtype. Because of the large standard deviation in the permeability measurements, no significant differences were detected between the cartilage subtypes. There were no significant differences in the Poisson's ratio (a measure of compressibility) across the cartilage subtypes tested. It was not technically feasible to obtain valid Poisson's ratio measurements on meniscus samples because of the physical constraints (size and availability of a large enough flat surface) of the material.

	Discussion

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The rabbit is a well-accepted model for histological, biochemical, and biomechanical analyses of in vitro and in vivo cartilage and bone repair (Floman et al. 1980 ; Setton et al. 1993 ; Sah et al. 1997 ; LeRoux et al. 2000 ). The results of repair and in vitro studies are usually correlated with native cartilage tissue and are based on morphological observations, biomechanical testing, and biochemical analysis. Although subtle matrix and biomechanical differences between the cartilage subtypes have been recognized, no comprehensive database is available on the characterization and distinction among cartilage subtypes from rabbit or any other animal model. In this study we describe, for the first time, the characteristic differences between hyaline cartilage, elastic cartilage, and fibrocartilage in the rabbit by histology, by IHC for collagen Types I, II, V, VI, X, and for elastin, by biochemical analysis of total GAG content, and by biomechanical testing. This report shows that the matrix of cartilage subtypes is unique with respect to the presence and distribution of specific matrix molecules, and that as few as two markers (e.g., collagen Types VI and X) are sufficient to distinguish among these five cartilage subtypes.

The observation of collagen Type X expression in the deep zone of articular cartilage near the tidemark was somewhat unexpected, because Type X collagen is generally associated with the developing mid-diaphysis, growth plate, and mineralizing regions (von der Mark et al. 1976 ; Gibson et al. 1982 ; Schmid et al. 1990 ; Iyama et al. 1991 ). However, others have shown Type X collagen expression in articular chondrocytes (Eerola et al. 1998 ), and there is a report of transient Type X collagen expression at the articular surface in the marsupial Monodelphis domestica (Morrison et al. 1996 ). Another site of Type X collagen localization proximal to a mineralizing front is in the calcifying region of the insertion site of bovine Achilles tendon (Fukuta et al. 1998 ). Type X collagen has also been detected in the intervertebral disc (Aigner et al. 1998 ), and its increased expression may be associated with pathological conditions such as disc degeneration (Roberts et al. 1998 ). In a study in the developing rat, it was reported that Type X collagen could be found in tracheal cartilage in the peripheral regions that do not mineralize (Sasano et al. 1998 ). Our results indicate that auricular and nasal cartilage are additional cartilage subtypes that synthesize a Type X collagen matrix that persists within the matrix without mineralizing.

Some observations of Type I collagen immunostaining were also somewhat surprising. In the present study, positive staining for collagen Type I was detected in the matrix and pericellular regions of meniscal fibrocartilage, whereas nasal hyaline cartilage showed pericellular staining and epiglottical elastic cartilage showed only cellular staining. Articular cartilage differed from the other cartilage subtypes in having only a trace amount of staining for collagen Type I. This observation of trace amounts of Type I collagen is consistent with studies showing the expression of collagen Type I in chicken articular cartilage and embryonic bovine epiphyseal cartilage (Seyer et al. 1974 ) and with other studies showing Type I collagen in rat (Nakamura et al. 1997 ) and porcine (Wardale and Duance 1993 ) articular cartilage.

Although Type V collagen is accepted as a minor collagen present in bone, skin and other connective tissues in which collagen Type I prevails (Rhodes and Miller 1978 ), its presence in cartilage has been controversial. Immunofluorescence studies have demonstrated collagen Type V in fully differentiated hyaline cartilage in the pericellular area adjacent to the surface of chondrocytes (Gay et al. 1981 ). After some controversial IHC findings of the collagen Type V localization in cartilage (Burgeson et al. 1982 ; von der Mark and Ocalan 1982 ), collagen Types V and VI were conclusively identified in bovine articular cartilage (Eyre et al. 1987 ). Our observations indicate a slight extracellular staining for collagen Type V in all cartilage subtypes, which is consistent with the IHC observations of Furuto et al. 1991 in articular cartilage of rats.

Several IHC studies have reported the localization of collagen Type VI in different tissues, such as skin, kidney (McComb et al. 1987 ), and muscle (Linsenmayer et al. 1986 ). In cartilage, collagen Type VI was found in elastic cartilage (Sakai et al. 1986 ) and articular hyaline cartilage throughout the matrix and concentrated in areas surrounding chondrocytes (Keene et al. 1988 ; Hagiwara et al. 1993 ). In the rabbit specimens analyzed here, collagen Type VI was the most highly positive in the cellular and pericellular regions of all cartilage subtypes, with the exception of auricular elastic cartilage, which exhibited no positive staining. Although epiglottis and auricle are both representatives of elastic cartilage, they appear to differ in collagen Type VI deposition. From a technical standpoint, it is possible that the penetration of antibody to collagen Type VI may depend on the permeability of soft epiglottal cartilage in contrast to firm auricular cartilage, although the same enzymatic pretreatment was used throughout these experiments and, in principle, minimized this problem.

The biomechanical properties of the different cartilage subtypes were analyzed by a biphasic indentation assay. Our results for Poisson's ratio, aggregate modulus, and permeability in articular cartilage were slightly lower than previously reported by Athanasiou et al. 1991 . This small difference could be related to age, because we tested 4-month-old rabbits and Athanasiou et al. tested skeletally mature rabbits that were older (9–12 months old). Differences in biomechanical properties of articular cartilage as a function of species, a particular joint, or location within the joint have been reported previously (Athanasiou et al. 1994 ; Palmer et al. 1995 ; Fortin et al. 2000 ; Jurvelin et al. 2000 ). As previously described by Hunziker 1999 , there is a high variation of mechanical properties between different animal models and limited transferability to human cartilage tissue.

The study reported here is the first in which biomechanical properties have been comparatively tested for different cartilage subtypes in the rabbit. These data will be useful for comparison in studies where chondrocytes are transplanted to another site, where the question arises as to whether transplanted chondrocytes produce a tissue that assumes the mechanical properties matching the new physical environment or produce a tissue having the mechanical properties of the tissue of origin. This is an important issue for tissue engineering of cartilage replacement through the substitution of different cartilage subtypes.

Received for publication November 14, 2001; accepted February 20, 2002.

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