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Tissue engineering of stratified articular cartilage from chondrocyte subpopulations
OsteoArthritis and Cartilage (2003) 11, 595–602 © 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S1063-4584(03)00090-6
International Cartilage Repair Society
Tissue engineering of stratified articular cartilage from chondrocyte subpopulations T. J. Klein M.S.†, B. L. Schumacher B.S.†, T. A. Schmidt M.S.†, K. W. Li Ph.D.†, M. S. Voegtline Ph.D.†, K. Masuda M.D.‡§, E. J.-M. A. Thonar Ph.D.‡§i and R. L. Sah M.D., Sc.D.†¶††* †Department of Bioengineering, University of California-San Diego, La Jolla, CA, USA ‡Department of Biochemistry, Rush Medical College, Chicago, IL, USA §Department of Orthopedic Surgery, University of California-San Diego, La Jolla, CA, USA iDepartment of Internal Medicine, Rush Medical College, Chicago, IL, USA ¶Department of Orthopedic Surgery, Rush Medical College, Chicago, IL, USA ††Whitaker Institute of Biomedical Engineering, University of California-San Diego, La Jolla, CA, USA Summary Objective: To test if subpopulations of chondrocytes from different cartilage zones could be used to engineer cartilage constructs with features of normal stratification. Design: Chondrocytes from the superficial and middle zones of immature bovine cartilage were cultured in alginate, released, and seeded either separately or sequentially to form cartilage constructs. Constructs were cultured for 1 or 2 weeks and were assessed for growth, compressive properties, and deposition, and localization of matrix molecules and superficial zone protein (SZP). Results: The cartilaginous constructs formed from superficial zone chondrocytes exhibited less matrix growth and lower compressive properties than constructs from middle zone chondrocytes, with the stratified superficial-middle constructs exhibiting intermediate properties. Expression of SZP was highest at the construct surfaces, with the localization of SZP in superficial-middle constructs being concentrated at the superficial surface. Conclusions: Manipulation of subpopulations of chondrocytes can be useful in engineering cartilage tissue with a biomimetic approach, and in fabricating constructs that exhibit stratified features of normal articular cartilage. © 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Cartilage, Tissue engineering, Superficial zone protein.
and allow more rapid patient remobilization4,5. A common design goal for such constructs is to form tissue with properties similar to those of immature articular cartilage, which has a capacity to repair6. The alginate-recoveredchondrocyte method recapitulates certain features of immature cartilage, including a high cell density in a tissue free of exogenous scaffolds, by first culturing cells in a three-dimensional gel to generate a pericellular matrix, then releasing these cells and their newly formed matrix from the gel, and seeding them on cell culture inserts at a high density7. The cell phenotype and matrix properties of articular cartilage normally vary with depth from the articular surface. This stratification confers specialized functional properties to the superficial, middle, and deep zones of cartilage8. The superficial zone has relatively low sulfatedglycosaminoglycan (GAG) and aggrecan content8 with a correspondingly low compressive modulus9, allowing the superficial zone to conform to the opposing tissue surface and thereby distribute stress under compressive load. In contrast, collagen content is similar among the zones8. Differences in the phenotype of chondrocytes, the cells within articular cartilage, appear to be responsible primarily for these variations. The chondrocytes of the superficial
Introduction Articular cartilage normally functions as the low-friction, wear-resistant, load-bearing tissue at the ends of bones in synovial joints. Unfortunately, articular cartilage becomes degenerate frequently and increasingly with age, and has minimal capacity for self-repair1. To address this problem, tissue engineering approaches to repair cartilage defects are under active investigation. Clinical therapies currently range from transplanting osteochondral allografts with mature articular cartilage2 to isolating autogeneic chondrocytes from a biopsy of cartilage, proliferating these cells in culture, and then injecting them into the defect site under a periosteal patch3. Recent efforts have focused on fabricating cartilaginous tissue in vitro with the view that such constructs could circumvent the lack of donor supply for allografts, be implanted with a simpler surgical procedure, This work was supported by grants from NASA, NIH, and NSF. *Address correspondence and reprint requests to: Robert L. Sah, Department of Bioengineering, University of California-San Diego, 9500 Gilman Dr., Mail Code 0412, La Jolla, CA, USA. Tel: 858-534-0821; Fax: 858-534-6896; E-mail: [email protected] Received 5 November 2002; revision accepted 15 April 2003.
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Fig. 1. Schematic of methods used to engineer cartilage tissue using cells isolated separately from superficial and middle zones of articular cartilage. Homogeneous constructs were generated from chondrocytes of the superficial (S) or middle (M) zones. Stratified constructs (S/M) were formed by sequentially seeding different chondrocyte subpopulations, with time allowed between seedings for the initial layer to coalesce. Superficial (white) and middle (gray) zone chondrocytes and the tissues derived from them are shaded differently to emphasize their location in culture and in the engineered tissue.
zone secrete GAG at a relatively low rate10–12. Conversely, chondrocytes of the superficial zone secrete superficial zone protein (SZP) at a high rate, and biosynthesis of SZP by articular chondrocytes has been used to define this population of cells and also demarcate the superficial zone of cartilage13–15. SZP appears to have important mechanical properties, being identical or closely related to a molecule termed lubricin16 and imparting lubrication properties to the articular surface17–19. The superficial zone of cartilage also appears to be typically the region damaged first, during age-associated weakening and the early stages of osteoarthritis6. Despite the marked zonal variations in cartilage biology and function, and the importance of the superficial zone, few attempts have been made previously to fabricate articular cartilage tissue with stratification. Previous studies have focused on the differences between chondrocytes in the deeper regions of cartilage20. The objectives of this study were to determine if subpopulations of chondrocytes from the superficial or middle zones (1) would differentially express SZP, collagen, GAG, and compressive properties in engineered cartilage tissue, and (2) could be used to fabricate cartilage constructs with normal physiological stratification, including differential expression of SZP.
Materials and methods CARTILAGE HARVEST, CHONDROCYTE ISOLATION AND CULTURE, AND CONSTRUCT FORMATION
Osteochondral blocks were harvested from the patellofemoral groove of bovine calf knees using a scalpel. The blocks were from six animals, 1–3 weeks old, with the blocks from each animal treated separately. Blocks were then mounted on a sledge microtome and sectioned in 0.2 mm slices from the articular surface. Slices were pooled from the superficial (0–0.2 mm) and middle (0.4–1.6 mm) zones. Superficial and middle zone chondrocyte subpopulations were isolated by sequential digestion for 1 h with 0.2% pronase (Sigma–Aldrich, St. Louis, MO) and 12 h with 0.025% collagenase P (Roche Diagnostics, Indianapolis, IN)21. Chondrocytes were washed and resus-
pended in 1.2% alginate (Keltone LV, Kelco, Chicago, IL) in 150 mM NaCl at 4 million cells/ml, and then expressed from a 22-gage needle into a 102 mM CaCl2 solution to polymerize the alginate as small beads (∼2.7 mm in diameter) thus entrapping the chondrocytes22. The alginate beads were then transferred to T-175 flasks containing DMEM/ F12 with additives (10% fetal bovine serum, 25 µg/ml ascorbic acid, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml Fungizone, 0.1 mM MEM non-essential amino acids, 0.4 mM L-proline, 2 mM L-glutamine) and cultured for 7 days with daily medium changes (1 ml per million cells). Cells and their associated matrix (CM) were released from alginate using 55 mM sodium citrate in 150 mM NaCl. Selected cells were labeled by incubating for 2 h in medium containing 10 µM CellTracker™ Orange CMTMR (middle) or Green CMFDA (superficial) (Molecular Probes, Eugene, OR). CM were then seeded onto 6.5 or 12 mm diameter cell inserts with a 0.4 µm pore size polyester membrane (Corning Inc., Corning, NY) to achieve a final density of 5 million cells per cm2 membrane area. Three types of cartilage constructs were formed: homogeneous superficial (S), homogeneous middle (M), and stratified with superficial layered atop middle (S/M) (Fig. 1). S and M constructs were formed by seeding CM at one time, while S/M constructs were formed by seeding middle CM (2.5 million/cm2) followed 18 h later by superficial CM. All groups were incubated overnight, to allow the CM to coalesce, prior to flooding the tissue culture well with medium. Constructs were cultured for 1 or 2 weeks with daily changes of medium (DMEM with additives). At the end of culture, constructs were removed from the membrane, measured for thickness with a current sensing micrometer, and weighed wet. To assess the stability of cell stratification, some samples were sectioned after 1 week, and fluorescence and phase contrast photomicrographs of sections were overlayed using Adobe Photoshop (Adobe Systems, San Jose, CA).
BIOCHEMICAL ANALYSIS
Certain constructs (N⫽6–19 constructs per type per time point) were used for biochemical analyses. A single 3 mm
Osteoarthritis and Cartilage Vol. 11, No. 8 diameter disk, centered 1.5 mm from the center of the construct, was punched from each construct. The disks were then solubilized with proteinase K (Roche Diagnostics), and analyzed for DNA23, for collagen as hydroxyproline24 assuming 7.25 g collagen/g hydroxyproline25, and for GAG26. MECHANICAL TESTING
Other constructs (N⫽6–10 constructs per type per time point) were punched to a diameter of 4.8 mm, then transferred to a radially confining chamber filled with phosphate buffered saline (PBS) and subjected to compression tests essentially as described previously27. To increase the sensitivity of the system, a load cell with 1 mN resolution (Model 31/1435-03, Sensotec, Columbus, OH) was used. This allowed determination of the equilibrium confined compressive modulus, HA0, at a resolution of 0.1 kPa for the sample geometry and test protocol used. IMMUNOHISTOCHEMISTRY
A third set of constructs were treated for 16 h with 0.1 µM monensin (Sigma–Aldrich) in medium to prevent secretion of SZP from the Golgi apparatus14, frozen in O.C.T. compound (Fisher Scientific Co., Tustin, CA), and cut normal to the construct surface into 40 µm thick sections. Sections were probed for SZP and Col II using the Vectastain ABC Elite Kit (Vector Laboratories, Burlingame, CA) as per manufacturer’s instructions, employing an anti-bovine SZP monoclonal antibody (mAb), 3-A-4 (a generous gift from Dr Bruce Caterson, Wales, UK)14, a mouse mAb anti-Col II antibody cocktail (Chondrex, Redmond, WA), or mouse IgG (Vector Labs) as control. Full-thickness bovine articular cartilage treated with monensin served as a positive (in the superficial zone) and negative control (in the deeper zones) for SZP. Bovine cartilage and tendon served as positive and negative controls for Col II, respectively. Sections were counterstained with Methyl Green (Vector Labs) for 2–5 min at room temperature and mounted in Crystal Mount (Biomeda, Foster City, CA). Serial sections were stained with 0.1% Alcian Blue in 0.4 M MgCl2, 0.025 M sodium acetate, pH 5.6 to stain GAG11,28. Positive reactivity with SZP and Col II, and GAG staining were documented by photomicroscopy using brightfield illumination. SZP positive and negative cells were marked using Adobe Photoshop and analyzed to determine the percentage of positive cells as a function of depth from the top and bottom surfaces, using NIH Image 1.62. ELISA FOR SZP
Portions of medium from days 12 to 14 of culture (9–13 constructs per type) were analyzed for SZP by ELISA. Medium was allowed to bind to the wells of 96-well plates overnight at 4°C in PBS containing 0.1 M guanidine-HCl, pH 7.4. Conditioned medium from cartilage explants, which was standardized against purified SZP13,14, was also assayed in twofold dilutions to generate a sigmoidal standard curve. Plates were washed extensively with PBS containing 0.1% Tween-20 (PBS-T) between all steps. Non-specific binding was blocked with 5% non-fat milk in PBS for 1 h. Plates were then incubated for 1 h with a 1:1000 dilution of primary mAb 3-A-4 in PBS with 0.1% bovine serum albumin (Sigma–Aldrich), followed by an additional 1 h incubation with a 1:1000 dilution of a horseradish peroxidase conjugated goat-anti-mouse IgG (Pierce,
597 Rockford, IL) secondary antibody. ABTS substrate (Roche Diagnostics, Indianapolis, IN) was applied for 30 min and the reaction was stopped by the addition of 1% SDS in water. Optical density measurements were made at 405 nm on an EMAX microplate reader (Molecular Devices, Menlo Park, CA). Optical densities at three dilutions were used to calculate sample SZP concentration using the standard curve. WESTERN BLOT FOR SZP
Portions of medium from days 12 to 14 of culture were pooled from five constructs per experimental condition and analyzed for SZP by Western Blot. Pooled medium and purified SZP13,14 were digested with chondroitinase ABC (Seikagaku America, Rockville, MD) in sample buffer (0.1 M Tris and 0.05 M sodium acetate, pH 8.0) for 2 h at 37°C. Samples were mixed with an equal volume of 2× Laemmli sample buffer and boiled for 3 min immediately prior to electrophoresis on a 5% SDS-polyacrylamide gel. Separated proteins were transferred to an Immobilon-P membrane (Millipore, Burlington, MA) overnight at 4°C with 1.4 mA/cm2 in a transfer buffer containing 12 mM Tris, 0.3 mM EDTA, and 6 mM sodium acetate, pH 7.429. The membrane was then removed and blocked for 30 min with 5% non-fat dry milk in PBS-T. After rinsing, the membrane was incubated for 1 h with mAb 3-A-4 diluted 1:200 in PBS-T, followed by incubation with a secondary antibody, as above, for 2 h. The proteins were visualized with hyperfilm—ECL (Amersham Pharmacia, Piscataway, NJ). The approximate molecular weight of the SZP bands was determined using rainbow molecular weight standards (Amersham Pharmacia). STATISTICAL ANALYSIS
Data are expressed as mean±S.E.M. The effects of construct type and culture duration were assessed by two-way ANOVA (α⫽0.05) and appropriate Tukey post-hoc tests, with animal treated as a random variable, using Systat 5.2.1 (Systat Software, Inc., Richmond, CA).
Results CONSTRUCT PHYSICAL PROPERTIES
Cohesive cartilage constructs that could withstand manual manipulation after removal from the membrane were fabricated from chondrocytes, isolated separately from superficial and middle zones of bovine calf cartilage, using the alginate-recovered-chondrocyte method7 (Fig. 1). The cells isolated from the superficial and middle zones were relatively enriched and devoid, respectively, in the proportion of cells expressing SZP (data not shown). In S/M constructs, the tissue layers with cells of distinct origin were created and maintained, as demonstrated by localization of fluorescent-labeled superficial and middle zone cells (data not shown). Construct thickness varied with construct type (P<0.001) and increased with culture duration (P<0.001). After 1 and 2 weeks of culture, M constructs were significantly thicker than S/M constructs, which were in turn thicker than S constructs [Fig. 2(A)]. The wet weight of constructs varied in a manner consistent with thickness data [Fig. 2(B)]. Cell proliferation was also evident, as DNA content increased from 1 to 2 weeks (P<0.01), but
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Fig. 2. Structural, compositional, and functional properties of three types of cartilage constructs formed from superficial and/or middle zone chondrocytes after 1 and 2 weeks in culture. Wet weight (WW), DNA, GAG, and collagen (Col) were all normalized to the cross-sectional area of the construct. (F) The equilibrium modulus, HA0, was measured in confined compression. Data are expressed as mean±S.E.M. (P<0.001 (↔), P<0.01 (+), P<0.05 (♦)).
was similar for the three types of constructs (P⫽0.12) [Fig. 2(C)]. CARTILAGE MATRIX DEPOSITION AND LOCALIZATION, AND COMPRESSIVE PROPERTIES
Cartilage matrix constituents, GAG and collagen, were deposited at different rates by the various types of constructs. GAG content increased 51% from 1 to 2 weeks (P<0.001) and was significantly greater in M than in S and S/M constructs [each, P<0.001, Fig. 2(D)]. Collagen content tripled from 1 to 2 weeks (P<0.001) and was significantly greater in M than S constructs (P<0.001), and at an intermediate level in S/M constructs [P<0.001 vs S, P<0.05 vs M, Fig. 2(E)]. The equilibrium confined compressive modulus tended to increase with time (P⫽0.06), and was greater in M constructs than in S and in S/M constructs [P<0.01 and P<0.05, respectively, Fig. 2(F)]. Matrix constituents typical of normal articular cartilage were detected throughout the engineered constructs. Sulfated GAG was visualized throughout the thickness of all constructs with Alcian Blue dye, using conditions specific for chondroitin sulfate and keratan sulfate [Fig. 3(A, E, H)]. While GAG was present throughout the thickness of all sections, Alcian Blue staining of the S/M constructs was heavier in the bottom portion [Fig. 3(E)]. Collagen type II (Col II), a marker of hyaline cartilage, was also localized throughout the thickness of all types of constructs by immunohistochemistry [Fig. 3(B, F, I)]. SZP IMMUNOLOCALIZATION AND SECRETION
SZP, a molecule that is normally a product of chondrocytes in the superficial region of cartilage, was synthesized variably by S, S/M, and M constructs. In S and S/M constructs, SZP was immunolocalized within a high pro-
portion of the chondrocytes [Fig. 3(D, G)]. However, in M constructs SZP was localized within a low proportion of chondrocytes [Fig. 3(J)]. Cells near the top (free) and bottom (on the membrane) surfaces of S constructs stained more frequently than cells of the interior, while cells at the free surface stained more frequently than other cells in S/M constructs. After 2 weeks of culture, the percentage of cells positive for SZP averaged 52% in the top 0.2 mm, and then dropped to 36% in the next 0.2 mm, and only 13% in the remainder of the tissue [Fig. 4(A)]. In contrast, S constructs showed similarly high percentages of SZP-positive cells at both surfaces (48% in both the top 0.2 mm and the bottom 0.2 mm) and lower percentages in the interior (20% at 0.2–0.4 mm, 29% in the remainder of tissue). M constructs had a low proportion of SZP-positive cells throughout, ranging from 2% in the top 0.2 mm to 15% in the bottom 0.2 mm [Fig. 4(A)]. The extent of SZP secretion into the medium varied between the different constructs. S constructs secreted 21.5±4.0 µg/[cm2 day], a rate approximately 3.4-fold higher than both S/M and M constructs [P<0.001, Fig. 4(B)]. On Western blot, the SZP in media pooled from days 12 to 14 of culture had the same electrophoretic mobility as SZP secreted by chondrocytes in cartilage explants, with an approximate molecular weight of 345 kD (data not shown).
Discussion The above results indicate that chondrocyte subpopulations can play an important role in determining the structure, composition, metabolism, and mechanical function of tissue-engineered cartilage constructs, and that multiple chondrocyte populations may be used to form cartilage with stratification features that are typical of normal articular cartilage. A variety of cell sources could be used to impart
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Fig. 3. Localization of matrix and secreted molecules in S, S/M, and M cartilage constructs cultured for 2 weeks. Some tissue sections (A, E, H) were stained with Alcian Blue to detect glycosaminoglycan. Other sections were immunostained for (B, F, I) type II collagen or (D, G, J) SZP, and counterstained with Methyl Green. Positive immunoreactivity is indicated by a purple color. Counterstaining results in a blue–green color. For clarity, higher power images (D1, G1, J1) of the top and (D2, G2, J2) of the middle of each construct are shown. To show specificity of immunostaining, high power images (C1, C2) of a section treated with non-specific control IgG are shown. Bar, 100 µm.
Fig. 4. Depth-dependent SZP expression in each construct type after 2 weeks of culture. (A) Cells were counted in 0.2 mm bins, with cells between 0.4 mm from the top surface and 0.2 mm from the bottom surface combined ({) to allow for variations in construct thickness. (B) SZP measured in spent medium from days 12 to 14 of construct culture by indirect ELISA, normalized to construct area and culture duration (P<0.001 (↔)).
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on the engineered tissue the phenotypic zonal characteristics of articular cartilage, such as lubrication at the surface19, and higher resistance to compression in the deeper layers9. Like the distinctive superficial and middle zones of normal articular cartilage30, S constructs, formed from superficial zone chondrocytes, were different from M constructs, formed from middle zone chondrocytes, with the former having relatively low GAG content and compressive modulus (Fig. 2). Additionally, like articular cartilage with normal stratification14, S/M constructs, formed by layering chondrocytes from the superficial zone atop those from the middle zone, exhibited expression of SZP by cells within a few cell layers of the free surface of the tissue, whereas S and M constructs did not exhibit normal stratification (Figs. 3 and 4). The biomimetic approach of engineering cartilage tissue to have functional stratification may be useful with a variety of methods of forming cartilaginous tissue in vitro. The tissue engineering method used here, involving preincubation of chondrocytes within a three-dimensional gel, facilitates formation of cartilaginous matrix even of adult chondrocytes31. The method of seeding cells in biodegradable scaffolds32 could also be modified to form cartilage layers sequentially and with distinct cell populations. Important criteria for the cells of each layer of the construct may include SZP and collagen production in the superficial layer, and collagen expression in addition to a greater amount (relative to the superficial layer) of aggrecan production with little SZP expression in the deeper layers. Since there is a limited supply of primary chondrocytes from different zones, additional expansion of the few cells or an alternative cell source may be required for clinical application. Chondrocytes from the superficial zone can be expanded and maintain expression of SZP in monolayer culture33. Also, chondrocytes can be expanded and then induced to redifferentiate by three-dimensional culture34. Certain synovial cells may be useful as an alternative source of cells for the superficial layer of cartilage constructs, as they also express SZP14. Further, the individual layers may be composed of heterogeneous cell populations, rather than a single population. The degree of in vitro maturation of engineered cartilage typically varies with the duration of culture. The short-term in vitro studies, described here, have resulted in stratified tissue constructs that recapitulate certain features of immature, fetal-like cartilage tissue, including high cellularity and a low compressive modulus, while exhibiting SZP stratification akin to both immature and mature normal articular cartilage. While the compressive modulus of the constructs [4 kPa, Fig. 2(F)] fabricated in this study was ∼20 times lower than that of the top 1 mm of third trimester fetal bovine cartilage (80 kPa)35, the compressive modulus of other constructs become similar to that of even adult articular cartilage with extended culture duration, and stimulation with growth factors and bioreactor culture36–38. Both immature and mature cartilaginous tissue may be useful for repair of cartilage defects. The degree of construct maturation may affect the therapeutic efficacy of the engineered cartilage following transplantation. Immature cartilage, when injured, apparently has the intrinsic capacity to heal39, whereas adult cartilage does not40 and integrates poorly with biological grafts41. Engineered cartilage with immature mechanical properties also has a greater integration potential than engineered cartilage with more fully developed matrix properties42. In addition, immature cartilage constructs are likely to have a high growth potential and allow for enhanced filling of a defect
with irregularities. On the other hand, implants with fully functional articular cartilage, such as osteochondral allografts, may also be advantageous, and more effectively and quickly contribute to mechanical load bearing and a relatively short duration of post-operative rehabilitation. While formation of stratified cartilaginous tissue was achieved, refinement of the engineering methodology and biological understanding is likely to be useful for scientific and possible clinical applications. More complete characterization of the enriched subpopulations used in this study and mixed populations used in others could be achieved using zonal molecular markers such as SZP14 and clusterin43. Purification of cell subpopulations may allow generation of cartilage constructs with a greater degree of stratification and enable more detailed experimental studies. The mechanisms of regulation of chondrocyte phenotype and tissue growth during extended in vitro culture remain to be fully elucidated. Here, the number and proportion of cells in S constructs expressing SZP, as judged by immunohistochemistry of the parent population and the grown construct, declined with culture duration. This could be due to a relatively rapid proliferation of an initially small population of middle zone cells, which would tend to decrease the apparent population of SZP-positive cells in long term cultures. Alternately, the pattern of maintenance of SZP expression by cells near the surfaces of S constructs could suggest a phenotypic dependence on nutrient levels or chemical gradients in the tissue, possibly like those established in vivo due to the avascular nature of cartilage. Additionally, SZP expression could be regulated by cell–cell or cell–matrix interactions.
Acknowledgements This work was supported by grants from NASA, NIH, and NSF.
References 1. Mankin HJ. The response of articular cartilage to mechanical injury. J Bone Joint Surg Am 1982; 64:460–6. 2. Bugbee WD, Convery FR. Osteochondral allograft transplantation. Clin Sports Med 1999;18:67–75. 3. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994;331:889–95. 4. Sah RL, Chen AC, Chen SS, Li KW, DiMicco MA, Kurtis MS, et al. Articular cartilage repair. In: Koopman WJ, Ed. Arthritis and Allied Conditions. A Textbook of Rheumatology. Philadelphia: Lippincott, Williams and Wilkins 2001;2264–78. 5. Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage 2002; 10:432–63. 6. Sah RL. The biomechanical faces of articular cartilage. In: Kuettner KE, Hascall VC, Eds. The Many Faces of Osteoarthritis. New York: Raven Press 2002;409–22. 7. Masuda K, Sah RL, Hejna MJ, Thonar EJ. A novel two-step method for the formation of tissueengineered cartilage by mature bovine chondrocytes:
Osteoarthritis and Cartilage Vol. 11, No. 8
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
the alginate-recovered-chondrocyte (ARC) method. J Orthop Res 2003;21:139–48. Maroudas A. Physico-chemical properties of articular cartilage. In: Freeman MAR, Ed. Adult Articular Cartilage. Tunbridge Wells: Pitman Medical 1979;215–90. Schinagl RM, Gurskis D, Chen AC, Sah RL. Depthdependent confined compression modulus of fullthickness bovine articular cartilage. J Orthop Res 1997;15:499–506. Wong M, Wuethrich P, Eggli P, Hunziker E. Zonespecific cell biosynthetic activity in mature bovine articular cartilage: a new method using confocal microscopic stereology and quantitative autoradiography. J Orthop Res 1996;14:424–32. Aydelotte MB, Greenhill RR, Kuettner KE. Differences between sub-populations of cultured bovine articular chondrocytes. II. Proteoglycan metabolism. Connect Tissue Res 1988;18:223–34. Zanetti M, Ratcliffe A, Watt FM. Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate. J Cell Biol 1985; 101:53–9. Schumacher BL, Block JA, Schmid TM, Aydelotte MB, Kuettner KE. A novel proteoglycan synthesized and secreted by chondrocytes of the superficial zone of articular cartilage. Arch Biochem Biophys 1994; 311:144–52. Schumacher BL, Hughes CE, Kuettner KE, Caterson B, Aydelotte MB. Immunodetection and partial cDNA sequence of the proteoglycan, superficial zone protein, synthesized by cells lining synovial joints. J Orthop Res 1999;17:110–20. Flannery CR, Hughes CE, Schumacher BL, Tudor D, Aydelotte MB, Kuettner KE, et al. Articular cartilage superficial zone protein (SZP) is homologous to megakaryocyte stimulating factor precursor and Is a multifunctional proteoglycan with potential growthpromoting, cytoprotective, and lubricating properties in cartilage metabolism. Biochem Biophys Res Commun 1999;254:535–41. Swann DA, Silver FH, Slayter HS, Stafford W, Shore E. The molecular structure and lubricating activity of lubricin isolated from bovine and human synovial fluids. Biochem J 1985;225:195–201. Jay GD, Tantravahi U, Britt DE, Barrach HJ, Cha CJ. Homology of lubricin and superficial zone protein (SZP): products of megakaryocyte stimulating factor (MSF) gene expression by human synovial fibroblasts and articular chondrocytes localized to chromosome 1q25. J Orthop Res 2001;19:677–87. Jay GD, Haberstroh K, Cha CJ. Comparison of the boundary-lubricating ability of bovine synovial fluid, lubricin, and Healon. J Biomed Mater Res 1998; 40:414–8. Schmid TM, Su JL, Lindley KM, Soloveychik V, Madsen L, Block JA, et al. Superficial zone protein (SZP) is an abundant glycoprotein in human synovial fluid with lubricating properties. In: Kuettner KE, Hascall VC, Eds. The Many Faces of Osteoarthritis. New York: Raven Press 2002;159–61. Yu H, Grynpas M, Kandel RA. Composition of cartilagenous tissue with mineralized and non-mineralized zones formed in vitro. Biomaterials 1997; 18:1425–31. Mok SS, Masuda K, Ha¨uselmann HJ, Aydelotte MB, Thonar EJ. Aggrecan synthesized by mature bovine
601
22.
23. 24.
25.
26.
27.
28. 29. 30.
31.
32.
33.
34. 35.
36.
37.
chondrocytes suspended in alginate. Identification of two distinct metabolic matrix pools. J Biol Chem 1994;269:33021–7. Ha¨uselmann HJ, Aydelotte MB, Schumacher BL, Kuettner KE, Gitelis SH, Thonar EJ-MA. Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads. Matrix 1992;12:116–29. Kim YJ, Sah RLY, Doong JYH, Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 1988;174:168–76. Woessner JF. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys 1961; 93:440–7. Pal S, Tang L-H, Choi H, Habermann E, Rosenberg L, Roughley P, et al. Structural changes during development in bovine fetal epiphyseal cartilage. Collagen Rel Res 1981;1:151–76. Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 1986;883:173–7. Chen AC, Bae WC, Schinagl RM, Sah RL. Depth- and strain-dependent mechanical and electromechanical properties of full-thickness bovine articular cartilage in confined compression. J Biomech 2001;34:1–12. Scott JE, Dorling J. Differential staining of acid glycosaminoglycans (mucopolysaccharides) by alcian blue in salt solutions. Histochemie 1965;5:221–33. Schwartz NB. Synthesis and secretion of an altered chondroitin sulfate proteoglycan. J Biol Chem 1990; 254:2271–7. Hunziker EB, Quinn TM, Hauselmann H. Quantitative structural organization of normal adult human articular cartilage. Osteoarthritis Cartilage 2002; 10:564–72. Hauselmann HJ, Masuda K, Hunziker EB, Neidhart M, Mok SS, Michel BA, et al. Adult human chondrocytes cultured in alginate form a matrix similar to native human articular cartilage. Am J Physiol 1996; 271:C742–52. Freed LE, Marquis JC, Nohria A, Emmanual J, Mikos AG, Langer R. Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers. J Biomed Mater Res 1993;27:11–23. Schmidt TA, Schumacher BL, Klein TJ, Voegtline MS, Sah RL. Resurfacing articular cartilage with chondrocytes expressing superficial zone protein. Trans Orthop Res Soc 2003;28:137. Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 1982;30:215–24. Williamson AK, Chen AC, Sah RL. Compressive properties and function-composition relationships of developing bovine articular cartilage. J Orthop Res 2001;19:1113–21. Mauck RL, Soltz MA, Wang CC, Wong DD, Chao PH, Valhmu WB, et al. Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J Biomech Eng 2000;122:252–60. Martin I, Obradovic B, Treppo S, Grodzinsky AJ, Langer R, Freed LE, et al. Modulation of the mechanical properties of tissue engineered cartilage. Biorheology 2000;37:141–7.
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38. Vunjak-Novakovic G, Martin I, Obradovic B, Treppo S, Grodzinsky AJ, Langer R, et al. Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J Orthop Res 1999;17:130–9. 39. Namba RS, Meuli M, Sullivan KM, Le A, Adzick NS. Spontaneous repair of superficial defects in articular cartilage in a fetal lamb model. J Bone Joint Surg Am 1998;80:4–10. 40. Hunter W. On the structure and diseases of articulating cartilage. Philos Trans R Soc Lond 1743;42:514–21.
41. DiMicco MA, Waters SN, Akeson WH, Sah RL. Integrative articular cartilage repair: dependence on developmental stage and collagen metabolism. Osteoarthritis Cartilage 2002;10:218–25. 42. Obradovic B, Martin I, Padera RF, Treppo S, Freed LE, Vunjak-Novakovic G. Integration of engineered cartilage. J Orthop Res 2001;19:1089–97. 43. Khan IM, Salter DM, Bayliss MT, Thomson BM, Archer CW. Expression of clusterin in the superficial zone of bovine articular cartilage. Arthritis Rheum 2001; 44:1795–9.
International Cartilage Repair Society
Tissue engineering of stratified articular cartilage from chondrocyte subpopulations T. J. Klein M.S.†, B. L. Schumacher B.S.†, T. A. Schmidt M.S.†, K. W. Li Ph.D.†, M. S. Voegtline Ph.D.†, K. Masuda M.D.‡§, E. J.-M. A. Thonar Ph.D.‡§i and R. L. Sah M.D., Sc.D.†¶††* †Department of Bioengineering, University of California-San Diego, La Jolla, CA, USA ‡Department of Biochemistry, Rush Medical College, Chicago, IL, USA §Department of Orthopedic Surgery, University of California-San Diego, La Jolla, CA, USA iDepartment of Internal Medicine, Rush Medical College, Chicago, IL, USA ¶Department of Orthopedic Surgery, Rush Medical College, Chicago, IL, USA ††Whitaker Institute of Biomedical Engineering, University of California-San Diego, La Jolla, CA, USA Summary Objective: To test if subpopulations of chondrocytes from different cartilage zones could be used to engineer cartilage constructs with features of normal stratification. Design: Chondrocytes from the superficial and middle zones of immature bovine cartilage were cultured in alginate, released, and seeded either separately or sequentially to form cartilage constructs. Constructs were cultured for 1 or 2 weeks and were assessed for growth, compressive properties, and deposition, and localization of matrix molecules and superficial zone protein (SZP). Results: The cartilaginous constructs formed from superficial zone chondrocytes exhibited less matrix growth and lower compressive properties than constructs from middle zone chondrocytes, with the stratified superficial-middle constructs exhibiting intermediate properties. Expression of SZP was highest at the construct surfaces, with the localization of SZP in superficial-middle constructs being concentrated at the superficial surface. Conclusions: Manipulation of subpopulations of chondrocytes can be useful in engineering cartilage tissue with a biomimetic approach, and in fabricating constructs that exhibit stratified features of normal articular cartilage. © 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Cartilage, Tissue engineering, Superficial zone protein.
and allow more rapid patient remobilization4,5. A common design goal for such constructs is to form tissue with properties similar to those of immature articular cartilage, which has a capacity to repair6. The alginate-recoveredchondrocyte method recapitulates certain features of immature cartilage, including a high cell density in a tissue free of exogenous scaffolds, by first culturing cells in a three-dimensional gel to generate a pericellular matrix, then releasing these cells and their newly formed matrix from the gel, and seeding them on cell culture inserts at a high density7. The cell phenotype and matrix properties of articular cartilage normally vary with depth from the articular surface. This stratification confers specialized functional properties to the superficial, middle, and deep zones of cartilage8. The superficial zone has relatively low sulfatedglycosaminoglycan (GAG) and aggrecan content8 with a correspondingly low compressive modulus9, allowing the superficial zone to conform to the opposing tissue surface and thereby distribute stress under compressive load. In contrast, collagen content is similar among the zones8. Differences in the phenotype of chondrocytes, the cells within articular cartilage, appear to be responsible primarily for these variations. The chondrocytes of the superficial
Introduction Articular cartilage normally functions as the low-friction, wear-resistant, load-bearing tissue at the ends of bones in synovial joints. Unfortunately, articular cartilage becomes degenerate frequently and increasingly with age, and has minimal capacity for self-repair1. To address this problem, tissue engineering approaches to repair cartilage defects are under active investigation. Clinical therapies currently range from transplanting osteochondral allografts with mature articular cartilage2 to isolating autogeneic chondrocytes from a biopsy of cartilage, proliferating these cells in culture, and then injecting them into the defect site under a periosteal patch3. Recent efforts have focused on fabricating cartilaginous tissue in vitro with the view that such constructs could circumvent the lack of donor supply for allografts, be implanted with a simpler surgical procedure, This work was supported by grants from NASA, NIH, and NSF. *Address correspondence and reprint requests to: Robert L. Sah, Department of Bioengineering, University of California-San Diego, 9500 Gilman Dr., Mail Code 0412, La Jolla, CA, USA. Tel: 858-534-0821; Fax: 858-534-6896; E-mail: [email protected] Received 5 November 2002; revision accepted 15 April 2003.
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Fig. 1. Schematic of methods used to engineer cartilage tissue using cells isolated separately from superficial and middle zones of articular cartilage. Homogeneous constructs were generated from chondrocytes of the superficial (S) or middle (M) zones. Stratified constructs (S/M) were formed by sequentially seeding different chondrocyte subpopulations, with time allowed between seedings for the initial layer to coalesce. Superficial (white) and middle (gray) zone chondrocytes and the tissues derived from them are shaded differently to emphasize their location in culture and in the engineered tissue.
zone secrete GAG at a relatively low rate10–12. Conversely, chondrocytes of the superficial zone secrete superficial zone protein (SZP) at a high rate, and biosynthesis of SZP by articular chondrocytes has been used to define this population of cells and also demarcate the superficial zone of cartilage13–15. SZP appears to have important mechanical properties, being identical or closely related to a molecule termed lubricin16 and imparting lubrication properties to the articular surface17–19. The superficial zone of cartilage also appears to be typically the region damaged first, during age-associated weakening and the early stages of osteoarthritis6. Despite the marked zonal variations in cartilage biology and function, and the importance of the superficial zone, few attempts have been made previously to fabricate articular cartilage tissue with stratification. Previous studies have focused on the differences between chondrocytes in the deeper regions of cartilage20. The objectives of this study were to determine if subpopulations of chondrocytes from the superficial or middle zones (1) would differentially express SZP, collagen, GAG, and compressive properties in engineered cartilage tissue, and (2) could be used to fabricate cartilage constructs with normal physiological stratification, including differential expression of SZP.
Materials and methods CARTILAGE HARVEST, CHONDROCYTE ISOLATION AND CULTURE, AND CONSTRUCT FORMATION
Osteochondral blocks were harvested from the patellofemoral groove of bovine calf knees using a scalpel. The blocks were from six animals, 1–3 weeks old, with the blocks from each animal treated separately. Blocks were then mounted on a sledge microtome and sectioned in 0.2 mm slices from the articular surface. Slices were pooled from the superficial (0–0.2 mm) and middle (0.4–1.6 mm) zones. Superficial and middle zone chondrocyte subpopulations were isolated by sequential digestion for 1 h with 0.2% pronase (Sigma–Aldrich, St. Louis, MO) and 12 h with 0.025% collagenase P (Roche Diagnostics, Indianapolis, IN)21. Chondrocytes were washed and resus-
pended in 1.2% alginate (Keltone LV, Kelco, Chicago, IL) in 150 mM NaCl at 4 million cells/ml, and then expressed from a 22-gage needle into a 102 mM CaCl2 solution to polymerize the alginate as small beads (∼2.7 mm in diameter) thus entrapping the chondrocytes22. The alginate beads were then transferred to T-175 flasks containing DMEM/ F12 with additives (10% fetal bovine serum, 25 µg/ml ascorbic acid, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml Fungizone, 0.1 mM MEM non-essential amino acids, 0.4 mM L-proline, 2 mM L-glutamine) and cultured for 7 days with daily medium changes (1 ml per million cells). Cells and their associated matrix (CM) were released from alginate using 55 mM sodium citrate in 150 mM NaCl. Selected cells were labeled by incubating for 2 h in medium containing 10 µM CellTracker™ Orange CMTMR (middle) or Green CMFDA (superficial) (Molecular Probes, Eugene, OR). CM were then seeded onto 6.5 or 12 mm diameter cell inserts with a 0.4 µm pore size polyester membrane (Corning Inc., Corning, NY) to achieve a final density of 5 million cells per cm2 membrane area. Three types of cartilage constructs were formed: homogeneous superficial (S), homogeneous middle (M), and stratified with superficial layered atop middle (S/M) (Fig. 1). S and M constructs were formed by seeding CM at one time, while S/M constructs were formed by seeding middle CM (2.5 million/cm2) followed 18 h later by superficial CM. All groups were incubated overnight, to allow the CM to coalesce, prior to flooding the tissue culture well with medium. Constructs were cultured for 1 or 2 weeks with daily changes of medium (DMEM with additives). At the end of culture, constructs were removed from the membrane, measured for thickness with a current sensing micrometer, and weighed wet. To assess the stability of cell stratification, some samples were sectioned after 1 week, and fluorescence and phase contrast photomicrographs of sections were overlayed using Adobe Photoshop (Adobe Systems, San Jose, CA).
BIOCHEMICAL ANALYSIS
Certain constructs (N⫽6–19 constructs per type per time point) were used for biochemical analyses. A single 3 mm
Osteoarthritis and Cartilage Vol. 11, No. 8 diameter disk, centered 1.5 mm from the center of the construct, was punched from each construct. The disks were then solubilized with proteinase K (Roche Diagnostics), and analyzed for DNA23, for collagen as hydroxyproline24 assuming 7.25 g collagen/g hydroxyproline25, and for GAG26. MECHANICAL TESTING
Other constructs (N⫽6–10 constructs per type per time point) were punched to a diameter of 4.8 mm, then transferred to a radially confining chamber filled with phosphate buffered saline (PBS) and subjected to compression tests essentially as described previously27. To increase the sensitivity of the system, a load cell with 1 mN resolution (Model 31/1435-03, Sensotec, Columbus, OH) was used. This allowed determination of the equilibrium confined compressive modulus, HA0, at a resolution of 0.1 kPa for the sample geometry and test protocol used. IMMUNOHISTOCHEMISTRY
A third set of constructs were treated for 16 h with 0.1 µM monensin (Sigma–Aldrich) in medium to prevent secretion of SZP from the Golgi apparatus14, frozen in O.C.T. compound (Fisher Scientific Co., Tustin, CA), and cut normal to the construct surface into 40 µm thick sections. Sections were probed for SZP and Col II using the Vectastain ABC Elite Kit (Vector Laboratories, Burlingame, CA) as per manufacturer’s instructions, employing an anti-bovine SZP monoclonal antibody (mAb), 3-A-4 (a generous gift from Dr Bruce Caterson, Wales, UK)14, a mouse mAb anti-Col II antibody cocktail (Chondrex, Redmond, WA), or mouse IgG (Vector Labs) as control. Full-thickness bovine articular cartilage treated with monensin served as a positive (in the superficial zone) and negative control (in the deeper zones) for SZP. Bovine cartilage and tendon served as positive and negative controls for Col II, respectively. Sections were counterstained with Methyl Green (Vector Labs) for 2–5 min at room temperature and mounted in Crystal Mount (Biomeda, Foster City, CA). Serial sections were stained with 0.1% Alcian Blue in 0.4 M MgCl2, 0.025 M sodium acetate, pH 5.6 to stain GAG11,28. Positive reactivity with SZP and Col II, and GAG staining were documented by photomicroscopy using brightfield illumination. SZP positive and negative cells were marked using Adobe Photoshop and analyzed to determine the percentage of positive cells as a function of depth from the top and bottom surfaces, using NIH Image 1.62. ELISA FOR SZP
Portions of medium from days 12 to 14 of culture (9–13 constructs per type) were analyzed for SZP by ELISA. Medium was allowed to bind to the wells of 96-well plates overnight at 4°C in PBS containing 0.1 M guanidine-HCl, pH 7.4. Conditioned medium from cartilage explants, which was standardized against purified SZP13,14, was also assayed in twofold dilutions to generate a sigmoidal standard curve. Plates were washed extensively with PBS containing 0.1% Tween-20 (PBS-T) between all steps. Non-specific binding was blocked with 5% non-fat milk in PBS for 1 h. Plates were then incubated for 1 h with a 1:1000 dilution of primary mAb 3-A-4 in PBS with 0.1% bovine serum albumin (Sigma–Aldrich), followed by an additional 1 h incubation with a 1:1000 dilution of a horseradish peroxidase conjugated goat-anti-mouse IgG (Pierce,
597 Rockford, IL) secondary antibody. ABTS substrate (Roche Diagnostics, Indianapolis, IN) was applied for 30 min and the reaction was stopped by the addition of 1% SDS in water. Optical density measurements were made at 405 nm on an EMAX microplate reader (Molecular Devices, Menlo Park, CA). Optical densities at three dilutions were used to calculate sample SZP concentration using the standard curve. WESTERN BLOT FOR SZP
Portions of medium from days 12 to 14 of culture were pooled from five constructs per experimental condition and analyzed for SZP by Western Blot. Pooled medium and purified SZP13,14 were digested with chondroitinase ABC (Seikagaku America, Rockville, MD) in sample buffer (0.1 M Tris and 0.05 M sodium acetate, pH 8.0) for 2 h at 37°C. Samples were mixed with an equal volume of 2× Laemmli sample buffer and boiled for 3 min immediately prior to electrophoresis on a 5% SDS-polyacrylamide gel. Separated proteins were transferred to an Immobilon-P membrane (Millipore, Burlington, MA) overnight at 4°C with 1.4 mA/cm2 in a transfer buffer containing 12 mM Tris, 0.3 mM EDTA, and 6 mM sodium acetate, pH 7.429. The membrane was then removed and blocked for 30 min with 5% non-fat dry milk in PBS-T. After rinsing, the membrane was incubated for 1 h with mAb 3-A-4 diluted 1:200 in PBS-T, followed by incubation with a secondary antibody, as above, for 2 h. The proteins were visualized with hyperfilm—ECL (Amersham Pharmacia, Piscataway, NJ). The approximate molecular weight of the SZP bands was determined using rainbow molecular weight standards (Amersham Pharmacia). STATISTICAL ANALYSIS
Data are expressed as mean±S.E.M. The effects of construct type and culture duration were assessed by two-way ANOVA (α⫽0.05) and appropriate Tukey post-hoc tests, with animal treated as a random variable, using Systat 5.2.1 (Systat Software, Inc., Richmond, CA).
Results CONSTRUCT PHYSICAL PROPERTIES
Cohesive cartilage constructs that could withstand manual manipulation after removal from the membrane were fabricated from chondrocytes, isolated separately from superficial and middle zones of bovine calf cartilage, using the alginate-recovered-chondrocyte method7 (Fig. 1). The cells isolated from the superficial and middle zones were relatively enriched and devoid, respectively, in the proportion of cells expressing SZP (data not shown). In S/M constructs, the tissue layers with cells of distinct origin were created and maintained, as demonstrated by localization of fluorescent-labeled superficial and middle zone cells (data not shown). Construct thickness varied with construct type (P<0.001) and increased with culture duration (P<0.001). After 1 and 2 weeks of culture, M constructs were significantly thicker than S/M constructs, which were in turn thicker than S constructs [Fig. 2(A)]. The wet weight of constructs varied in a manner consistent with thickness data [Fig. 2(B)]. Cell proliferation was also evident, as DNA content increased from 1 to 2 weeks (P<0.01), but
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Fig. 2. Structural, compositional, and functional properties of three types of cartilage constructs formed from superficial and/or middle zone chondrocytes after 1 and 2 weeks in culture. Wet weight (WW), DNA, GAG, and collagen (Col) were all normalized to the cross-sectional area of the construct. (F) The equilibrium modulus, HA0, was measured in confined compression. Data are expressed as mean±S.E.M. (P<0.001 (↔), P<0.01 (+), P<0.05 (♦)).
was similar for the three types of constructs (P⫽0.12) [Fig. 2(C)]. CARTILAGE MATRIX DEPOSITION AND LOCALIZATION, AND COMPRESSIVE PROPERTIES
Cartilage matrix constituents, GAG and collagen, were deposited at different rates by the various types of constructs. GAG content increased 51% from 1 to 2 weeks (P<0.001) and was significantly greater in M than in S and S/M constructs [each, P<0.001, Fig. 2(D)]. Collagen content tripled from 1 to 2 weeks (P<0.001) and was significantly greater in M than S constructs (P<0.001), and at an intermediate level in S/M constructs [P<0.001 vs S, P<0.05 vs M, Fig. 2(E)]. The equilibrium confined compressive modulus tended to increase with time (P⫽0.06), and was greater in M constructs than in S and in S/M constructs [P<0.01 and P<0.05, respectively, Fig. 2(F)]. Matrix constituents typical of normal articular cartilage were detected throughout the engineered constructs. Sulfated GAG was visualized throughout the thickness of all constructs with Alcian Blue dye, using conditions specific for chondroitin sulfate and keratan sulfate [Fig. 3(A, E, H)]. While GAG was present throughout the thickness of all sections, Alcian Blue staining of the S/M constructs was heavier in the bottom portion [Fig. 3(E)]. Collagen type II (Col II), a marker of hyaline cartilage, was also localized throughout the thickness of all types of constructs by immunohistochemistry [Fig. 3(B, F, I)]. SZP IMMUNOLOCALIZATION AND SECRETION
SZP, a molecule that is normally a product of chondrocytes in the superficial region of cartilage, was synthesized variably by S, S/M, and M constructs. In S and S/M constructs, SZP was immunolocalized within a high pro-
portion of the chondrocytes [Fig. 3(D, G)]. However, in M constructs SZP was localized within a low proportion of chondrocytes [Fig. 3(J)]. Cells near the top (free) and bottom (on the membrane) surfaces of S constructs stained more frequently than cells of the interior, while cells at the free surface stained more frequently than other cells in S/M constructs. After 2 weeks of culture, the percentage of cells positive for SZP averaged 52% in the top 0.2 mm, and then dropped to 36% in the next 0.2 mm, and only 13% in the remainder of the tissue [Fig. 4(A)]. In contrast, S constructs showed similarly high percentages of SZP-positive cells at both surfaces (48% in both the top 0.2 mm and the bottom 0.2 mm) and lower percentages in the interior (20% at 0.2–0.4 mm, 29% in the remainder of tissue). M constructs had a low proportion of SZP-positive cells throughout, ranging from 2% in the top 0.2 mm to 15% in the bottom 0.2 mm [Fig. 4(A)]. The extent of SZP secretion into the medium varied between the different constructs. S constructs secreted 21.5±4.0 µg/[cm2 day], a rate approximately 3.4-fold higher than both S/M and M constructs [P<0.001, Fig. 4(B)]. On Western blot, the SZP in media pooled from days 12 to 14 of culture had the same electrophoretic mobility as SZP secreted by chondrocytes in cartilage explants, with an approximate molecular weight of 345 kD (data not shown).
Discussion The above results indicate that chondrocyte subpopulations can play an important role in determining the structure, composition, metabolism, and mechanical function of tissue-engineered cartilage constructs, and that multiple chondrocyte populations may be used to form cartilage with stratification features that are typical of normal articular cartilage. A variety of cell sources could be used to impart
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Fig. 3. Localization of matrix and secreted molecules in S, S/M, and M cartilage constructs cultured for 2 weeks. Some tissue sections (A, E, H) were stained with Alcian Blue to detect glycosaminoglycan. Other sections were immunostained for (B, F, I) type II collagen or (D, G, J) SZP, and counterstained with Methyl Green. Positive immunoreactivity is indicated by a purple color. Counterstaining results in a blue–green color. For clarity, higher power images (D1, G1, J1) of the top and (D2, G2, J2) of the middle of each construct are shown. To show specificity of immunostaining, high power images (C1, C2) of a section treated with non-specific control IgG are shown. Bar, 100 µm.
Fig. 4. Depth-dependent SZP expression in each construct type after 2 weeks of culture. (A) Cells were counted in 0.2 mm bins, with cells between 0.4 mm from the top surface and 0.2 mm from the bottom surface combined ({) to allow for variations in construct thickness. (B) SZP measured in spent medium from days 12 to 14 of construct culture by indirect ELISA, normalized to construct area and culture duration (P<0.001 (↔)).
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on the engineered tissue the phenotypic zonal characteristics of articular cartilage, such as lubrication at the surface19, and higher resistance to compression in the deeper layers9. Like the distinctive superficial and middle zones of normal articular cartilage30, S constructs, formed from superficial zone chondrocytes, were different from M constructs, formed from middle zone chondrocytes, with the former having relatively low GAG content and compressive modulus (Fig. 2). Additionally, like articular cartilage with normal stratification14, S/M constructs, formed by layering chondrocytes from the superficial zone atop those from the middle zone, exhibited expression of SZP by cells within a few cell layers of the free surface of the tissue, whereas S and M constructs did not exhibit normal stratification (Figs. 3 and 4). The biomimetic approach of engineering cartilage tissue to have functional stratification may be useful with a variety of methods of forming cartilaginous tissue in vitro. The tissue engineering method used here, involving preincubation of chondrocytes within a three-dimensional gel, facilitates formation of cartilaginous matrix even of adult chondrocytes31. The method of seeding cells in biodegradable scaffolds32 could also be modified to form cartilage layers sequentially and with distinct cell populations. Important criteria for the cells of each layer of the construct may include SZP and collagen production in the superficial layer, and collagen expression in addition to a greater amount (relative to the superficial layer) of aggrecan production with little SZP expression in the deeper layers. Since there is a limited supply of primary chondrocytes from different zones, additional expansion of the few cells or an alternative cell source may be required for clinical application. Chondrocytes from the superficial zone can be expanded and maintain expression of SZP in monolayer culture33. Also, chondrocytes can be expanded and then induced to redifferentiate by three-dimensional culture34. Certain synovial cells may be useful as an alternative source of cells for the superficial layer of cartilage constructs, as they also express SZP14. Further, the individual layers may be composed of heterogeneous cell populations, rather than a single population. The degree of in vitro maturation of engineered cartilage typically varies with the duration of culture. The short-term in vitro studies, described here, have resulted in stratified tissue constructs that recapitulate certain features of immature, fetal-like cartilage tissue, including high cellularity and a low compressive modulus, while exhibiting SZP stratification akin to both immature and mature normal articular cartilage. While the compressive modulus of the constructs [4 kPa, Fig. 2(F)] fabricated in this study was ∼20 times lower than that of the top 1 mm of third trimester fetal bovine cartilage (80 kPa)35, the compressive modulus of other constructs become similar to that of even adult articular cartilage with extended culture duration, and stimulation with growth factors and bioreactor culture36–38. Both immature and mature cartilaginous tissue may be useful for repair of cartilage defects. The degree of construct maturation may affect the therapeutic efficacy of the engineered cartilage following transplantation. Immature cartilage, when injured, apparently has the intrinsic capacity to heal39, whereas adult cartilage does not40 and integrates poorly with biological grafts41. Engineered cartilage with immature mechanical properties also has a greater integration potential than engineered cartilage with more fully developed matrix properties42. In addition, immature cartilage constructs are likely to have a high growth potential and allow for enhanced filling of a defect
with irregularities. On the other hand, implants with fully functional articular cartilage, such as osteochondral allografts, may also be advantageous, and more effectively and quickly contribute to mechanical load bearing and a relatively short duration of post-operative rehabilitation. While formation of stratified cartilaginous tissue was achieved, refinement of the engineering methodology and biological understanding is likely to be useful for scientific and possible clinical applications. More complete characterization of the enriched subpopulations used in this study and mixed populations used in others could be achieved using zonal molecular markers such as SZP14 and clusterin43. Purification of cell subpopulations may allow generation of cartilage constructs with a greater degree of stratification and enable more detailed experimental studies. The mechanisms of regulation of chondrocyte phenotype and tissue growth during extended in vitro culture remain to be fully elucidated. Here, the number and proportion of cells in S constructs expressing SZP, as judged by immunohistochemistry of the parent population and the grown construct, declined with culture duration. This could be due to a relatively rapid proliferation of an initially small population of middle zone cells, which would tend to decrease the apparent population of SZP-positive cells in long term cultures. Alternately, the pattern of maintenance of SZP expression by cells near the surfaces of S constructs could suggest a phenotypic dependence on nutrient levels or chemical gradients in the tissue, possibly like those established in vivo due to the avascular nature of cartilage. Additionally, SZP expression could be regulated by cell–cell or cell–matrix interactions.
Acknowledgements This work was supported by grants from NASA, NIH, and NSF.
References 1. Mankin HJ. The response of articular cartilage to mechanical injury. J Bone Joint Surg Am 1982; 64:460–6. 2. Bugbee WD, Convery FR. Osteochondral allograft transplantation. Clin Sports Med 1999;18:67–75. 3. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994;331:889–95. 4. Sah RL, Chen AC, Chen SS, Li KW, DiMicco MA, Kurtis MS, et al. Articular cartilage repair. In: Koopman WJ, Ed. Arthritis and Allied Conditions. A Textbook of Rheumatology. Philadelphia: Lippincott, Williams and Wilkins 2001;2264–78. 5. Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage 2002; 10:432–63. 6. Sah RL. The biomechanical faces of articular cartilage. In: Kuettner KE, Hascall VC, Eds. The Many Faces of Osteoarthritis. New York: Raven Press 2002;409–22. 7. Masuda K, Sah RL, Hejna MJ, Thonar EJ. A novel two-step method for the formation of tissueengineered cartilage by mature bovine chondrocytes:
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8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
the alginate-recovered-chondrocyte (ARC) method. J Orthop Res 2003;21:139–48. Maroudas A. Physico-chemical properties of articular cartilage. In: Freeman MAR, Ed. Adult Articular Cartilage. Tunbridge Wells: Pitman Medical 1979;215–90. Schinagl RM, Gurskis D, Chen AC, Sah RL. Depthdependent confined compression modulus of fullthickness bovine articular cartilage. J Orthop Res 1997;15:499–506. Wong M, Wuethrich P, Eggli P, Hunziker E. Zonespecific cell biosynthetic activity in mature bovine articular cartilage: a new method using confocal microscopic stereology and quantitative autoradiography. J Orthop Res 1996;14:424–32. Aydelotte MB, Greenhill RR, Kuettner KE. Differences between sub-populations of cultured bovine articular chondrocytes. II. Proteoglycan metabolism. Connect Tissue Res 1988;18:223–34. Zanetti M, Ratcliffe A, Watt FM. Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate. J Cell Biol 1985; 101:53–9. Schumacher BL, Block JA, Schmid TM, Aydelotte MB, Kuettner KE. A novel proteoglycan synthesized and secreted by chondrocytes of the superficial zone of articular cartilage. Arch Biochem Biophys 1994; 311:144–52. Schumacher BL, Hughes CE, Kuettner KE, Caterson B, Aydelotte MB. Immunodetection and partial cDNA sequence of the proteoglycan, superficial zone protein, synthesized by cells lining synovial joints. J Orthop Res 1999;17:110–20. Flannery CR, Hughes CE, Schumacher BL, Tudor D, Aydelotte MB, Kuettner KE, et al. Articular cartilage superficial zone protein (SZP) is homologous to megakaryocyte stimulating factor precursor and Is a multifunctional proteoglycan with potential growthpromoting, cytoprotective, and lubricating properties in cartilage metabolism. Biochem Biophys Res Commun 1999;254:535–41. Swann DA, Silver FH, Slayter HS, Stafford W, Shore E. The molecular structure and lubricating activity of lubricin isolated from bovine and human synovial fluids. Biochem J 1985;225:195–201. Jay GD, Tantravahi U, Britt DE, Barrach HJ, Cha CJ. Homology of lubricin and superficial zone protein (SZP): products of megakaryocyte stimulating factor (MSF) gene expression by human synovial fibroblasts and articular chondrocytes localized to chromosome 1q25. J Orthop Res 2001;19:677–87. Jay GD, Haberstroh K, Cha CJ. Comparison of the boundary-lubricating ability of bovine synovial fluid, lubricin, and Healon. J Biomed Mater Res 1998; 40:414–8. Schmid TM, Su JL, Lindley KM, Soloveychik V, Madsen L, Block JA, et al. Superficial zone protein (SZP) is an abundant glycoprotein in human synovial fluid with lubricating properties. In: Kuettner KE, Hascall VC, Eds. The Many Faces of Osteoarthritis. New York: Raven Press 2002;159–61. Yu H, Grynpas M, Kandel RA. Composition of cartilagenous tissue with mineralized and non-mineralized zones formed in vitro. Biomaterials 1997; 18:1425–31. Mok SS, Masuda K, Ha¨uselmann HJ, Aydelotte MB, Thonar EJ. Aggrecan synthesized by mature bovine
601
22.
23. 24.
25.
26.
27.
28. 29. 30.
31.
32.
33.
34. 35.
36.
37.
chondrocytes suspended in alginate. Identification of two distinct metabolic matrix pools. J Biol Chem 1994;269:33021–7. Ha¨uselmann HJ, Aydelotte MB, Schumacher BL, Kuettner KE, Gitelis SH, Thonar EJ-MA. Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads. Matrix 1992;12:116–29. Kim YJ, Sah RLY, Doong JYH, Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 1988;174:168–76. Woessner JF. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys 1961; 93:440–7. Pal S, Tang L-H, Choi H, Habermann E, Rosenberg L, Roughley P, et al. Structural changes during development in bovine fetal epiphyseal cartilage. Collagen Rel Res 1981;1:151–76. Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 1986;883:173–7. Chen AC, Bae WC, Schinagl RM, Sah RL. Depth- and strain-dependent mechanical and electromechanical properties of full-thickness bovine articular cartilage in confined compression. J Biomech 2001;34:1–12. Scott JE, Dorling J. Differential staining of acid glycosaminoglycans (mucopolysaccharides) by alcian blue in salt solutions. Histochemie 1965;5:221–33. Schwartz NB. Synthesis and secretion of an altered chondroitin sulfate proteoglycan. J Biol Chem 1990; 254:2271–7. Hunziker EB, Quinn TM, Hauselmann H. Quantitative structural organization of normal adult human articular cartilage. Osteoarthritis Cartilage 2002; 10:564–72. Hauselmann HJ, Masuda K, Hunziker EB, Neidhart M, Mok SS, Michel BA, et al. Adult human chondrocytes cultured in alginate form a matrix similar to native human articular cartilage. Am J Physiol 1996; 271:C742–52. Freed LE, Marquis JC, Nohria A, Emmanual J, Mikos AG, Langer R. Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers. J Biomed Mater Res 1993;27:11–23. Schmidt TA, Schumacher BL, Klein TJ, Voegtline MS, Sah RL. Resurfacing articular cartilage with chondrocytes expressing superficial zone protein. Trans Orthop Res Soc 2003;28:137. Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 1982;30:215–24. Williamson AK, Chen AC, Sah RL. Compressive properties and function-composition relationships of developing bovine articular cartilage. J Orthop Res 2001;19:1113–21. Mauck RL, Soltz MA, Wang CC, Wong DD, Chao PH, Valhmu WB, et al. Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J Biomech Eng 2000;122:252–60. Martin I, Obradovic B, Treppo S, Grodzinsky AJ, Langer R, Freed LE, et al. Modulation of the mechanical properties of tissue engineered cartilage. Biorheology 2000;37:141–7.
602
T. J. Klein: Tissue engineering of stratified articular cartilage from chondrocyte subpopulations
38. Vunjak-Novakovic G, Martin I, Obradovic B, Treppo S, Grodzinsky AJ, Langer R, et al. Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J Orthop Res 1999;17:130–9. 39. Namba RS, Meuli M, Sullivan KM, Le A, Adzick NS. Spontaneous repair of superficial defects in articular cartilage in a fetal lamb model. J Bone Joint Surg Am 1998;80:4–10. 40. Hunter W. On the structure and diseases of articulating cartilage. Philos Trans R Soc Lond 1743;42:514–21.
41. DiMicco MA, Waters SN, Akeson WH, Sah RL. Integrative articular cartilage repair: dependence on developmental stage and collagen metabolism. Osteoarthritis Cartilage 2002;10:218–25. 42. Obradovic B, Martin I, Padera RF, Treppo S, Freed LE, Vunjak-Novakovic G. Integration of engineered cartilage. J Orthop Res 2001;19:1089–97. 43. Khan IM, Salter DM, Bayliss MT, Thomson BM, Archer CW. Expression of clusterin in the superficial zone of bovine articular cartilage. Arthritis Rheum 2001; 44:1795–9.