IN VITRO Volume 13, No. 7, 1977 All rights reserved 9

ORGAN CULTURE OF HUMAN ARTICULAR CARTILAGE: S T U D I E S ON S U L P H A T E D G L Y C O S A M I N O G L Y C A N S Y N T H E S I S L. S. McKENZIE, B. A. HORSBURGH, P. GHOSH, ANDT. K. F. TAYLOR

Raymond Purves R esearch Laboratories (The Universityof Sydney), The Royal North Shore Hospital of Sydney, St. Leonards, N.S. IV., 2065Australia

SUMMARY An organ culture system is described for adult human articular cartilage obtained from joints after femoral head replacement operations. Cartilage slices maintain maximal viability for 2 days in culture as assessed by uptake of [3H]uridine and [3H]leucine into whole tissue, and 3sS04 into sulphated glycosaminoglycans (GAGs). Since GAGs are the components of cartilage matrix, the depletion of which is associated with osteoarthrosis, a method for measuring sulphated GAG synthesis in culture has been investigated.

Key words: organ culture; articular cartilage; glycosaminoglycan synthesis. INTRODUCTION

GAGs in adult human cartilage. This technique Articular cartilage is characterized by its low has been utilized successfully for comparative cellularity and low metabolic activity compared to studies of GAG synthesis in normal and osteomost other tissues. In mature cartilage, the arthrosic human articular cartilage (11), and for chondrocytes produce collagen and proteogly- investigating the effect of anti-inflammatory cans, which constitute the intercellular matrix, at drugs on GAG synthesis in these tissues (12). a rate sufficient to balance the normal rate of Similar culture methods have been reported returnover. The proteoglycan aggregates of the ma- cently for studies of calf articular cartilage (13) trix consist of glycosaminoglycan (GAG) chains and human-knee articular cartilage from patients (mainly chondroitin and keratin sulphates) at- with rheumatoid arthritis (14). tached to a protein core. Maroudas (1) has reMATERIAI~ AND METHODS ported a half-life of about 800 days for sulfated GAGs of adult human articular cartilage; and Sampling of cartilage. Human articular cartiturnover rates for collagen in adult cartilage are lage was obtained from femoral heads removed also slow (2). surgically following subcapital fract~e of the A survey of the literature shows that metabolic femur, or for severe osteoarthrosis. As far as could studies on articular cartilage have used, in gen- be ascertained, patients admitted for fracture of eral, short-term cultures in which pieces of freshly the femur had not taken anti-inflammatory drugs dissected cartilage are immediately placed into sa- 1 week prior to surgery. Ostcoarthrosics, howline or medium containing the radioactive precur- ever, were known to use anti-inflammatory drugs sor for periods of hours. Such methods have been on a regular basis. used to follow, autoradiographically, the uptake For "healthy" femoral heads, cartilage was of 35SO4by chondrocytes (3, 4). Biochemical stud- sampled from the superior surface, which is consiies on 3sS04 uptake into sulphated GAGs have dered to be weight-bearing (15, 16). However, the been carried out by several workers (5-8). Pro- experiments of Maroudas, Evans and Almeida tein, DNA and RNA synthesis in cartilage in (16} showed no significant differences in GAG vitro also has been studied by following the up- content over the area of the femoral head, suggesttake of [3H]glycine, [3H]thymidine and [3H]ttriing that the area of cartilage sampled is not critidine, respectively (9, 10). cal. Osteoarthrosic femoral heads could not alThe purpose of the present paper is to report a ways be sampled from the superior surface, beculture technique which has been developed to en- cause cartilage here often was eroded or severely able studies to be conducted on the synthesis of damaged. 423

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McKENZIE ET AL.

As the GAG content and metabolism of articular cartilage vary with depth from the articular surface (1), it was considered important to sample cartilage to its full depth, so that each slice encompassed the full range of cellular activities. Cartilage slices of uniform thickness were obtained with a specially devised tool which maintained, by metal washers, four parallel razor blades 1 mm apart. New razor blades were used for each experiment to minimize tissue and cell damage (1, 8). Slices were cut perpendicularly down to the subchondral bone and then carefully dissected free at the cartilage-bone interface. Each slice was divided into smaller lengths of I to 2 mm for culture. Trowell (17) demonstrated that the maximum thickness of tissue slices with low oxygen consumption for organ culture should be 2 mm; otherwise inadequate exchange of dissolved gases and nutrients could lead to necrosis at the interior. Maroudas and Evans (8) reported that for a cartilage slice of 1-mm thickness, 11 minutes were necessary to enable full equilibration of 3ss04 across the slice. This equilibrium time was found to increase markedly for thicker slices. A choice of 1 mm for tissue thickness was made, taking both these factors into account. Culture method. Explants were set up in culture within 2 hr of surgical removal of the femoral head; considerable care was taken to keep the cartilage moist with Hank's balanced salt solution at all stages. The culture technique used is a modification of that of Trowell (17) and Jensen and Castellano (18}. Stainless-steel grids, 20- by 20- by 2-ram, were placed into Falcon plastic Petri dishes of 35mm diameter. Exactly 2 ml of culture medium in the dish was just sufficient to bathe the cartilage slices placed on the grid. Petri dishes were incubated at 37~ in a humidified atmosphere of 5% CO2 in air. Dulbecco's modified Eagle's Minimum Essential Medium (Commonwealth Serum Laboratories) supplemented with 10% fetal bovine serum (Commonwealth Serum Laboratories} and antibiotics (sodium penicillin G, 40 units per ml, and streptomycin sulphate, 50 ~g per ml) was used as the culture medium and was changed daily. Preliminary experiments showed that the presence of no less than 10% serum was necessary for maximal viability of cultures. The viability of the cartilage slices over a number of days in culture was followed by measuring the uptake of [3H]uridine and 3sS04 by cartilage slices over a 3day period (Fig. 1}. Autoradiographs were prepared using the conventional dipping techniques

with Ilford K5 nuclear track emulsion diluted with distilled water (1:1.7 v/v) to establish that uptake of radioactivity was into the chondrocytes rather than nonspecific binding to the matrix as a whole. Sections were exposed for 3 weeks at 5~ developed in Kodak D19B, fixed in Hypam rapid fixer (Ilford and Co.), washed for 30 min, dried and stained with hematoxylin and eosin.

Incubation of cartilage with radioactively labeled precursors. Cartilage slices were incubated with 3sSO4 (sodium [3sS]sulphate up to I00 mCi per mmol; The Radiochemical Centre, Amersham, England) at 40/~Ci per ml in culture medium for 6-hr periods, removing triplicate slices at hourly intervals. Controls were incubated at 0~ a temperature at which negligible biosynthesis of GAGs was observed. The control slices were prechilled for 30 min in ice-cold culture medium before exposure to 3sSO4. Studies utilizing [3H]uridine ([5-3H]uridine, 5 Ci per mmol) and [3H]leucine (L-[4,5-3H]leucine, 38 Ci per mmol) (both from The Radiochemieal Centre, Amersham, England) employed concentrations of 20 pCi per ml of culture medium. Removal of unbound radioactivity. At completion of the incubation periods, thorough washing of cartilage slices was performed to remove free isotope. Each slice was soaked for 10 min in 5 ml of ice-cold saline containing nonradioactive precursor (I mg per ml) as a chaser, and the process repeated in fresh solutions every 10 min until unbound radioactivity was removed. Solubilization of the tissue. After washing, cartilage slices were dehydrated in acetone and alcohol and then dried under vacuum at 60~ and dry weights were determined. Solubilization of dried tissue for scintillation counting was achieved by two methods: Acid hydrolysis--heating with 300/~I 6 N HCI per cartilage slice (approximately 1 mg dry weight) for 16 hr at 60~ followed by neutralization with 6 N NaOH. This method allowed estimation of uptake of radioactivity into the tissue as a whole and was used routinely for studies using [3Hlleucine and [3H]uridine. Enzymic digestion--with 1.5 mg crystalline papain (Merck) per ml of acetate buffer (0.1 M), pH 5.6, containing E D T A (0.05 M) and cysteine (0.005 M) (total 1 ml per slice) at 60~ for 16 hr. After papain digestion, direct counts of radioactivity in whole tissue were made. A portion of the digest was dialyzed overnight against water to remove any 3sSO4 not incorporated into the macromolecular GAG fraction, and the dialysate re-

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CULTURE OF HUMAN CARTILAGE counted after making up to a standard volume. This method was used only for studies on 3sSO4 uptake, Radioactivity of samples was determined by standard liquid-scintillation counting techniques, using a Triton X-100/toluene scintillation mixture, and expressed as cpm per mg dry weight of cartilage. An estimate of chondroitin sulphate concentration in dialyzed papain digests of cartilage was made from the uronic acid levels as determined by the method of Bitter and Muir (19).

4oooo

cm,.o oar wt~.T 20000

10

20 WASHING

RESULTS

Viability of adult human articular cartilage in culture. The viability of cartilage in culture declined after 1 to 2 days. Fig. 2 shows results of experiments in which uptake of 3sso4, [3H]leucine and [3H]uridine was measured daily. On the initial day of culture (day 0), [3H]uridine, [3H]leucine and 3sso, uptake all appeared lower than on day 1, corresponding with the initial "shock phase" described by Biggers (20). However, ttests showed a significant difference for [3H]uridine (P

Organ culture of human articular cartilage: studies on sulphated glycosaminoglycan synthesis.

IN VITRO Volume 13, No. 7, 1977 All rights reserved 9 ORGAN CULTURE OF HUMAN ARTICULAR CARTILAGE: S T U D I E S ON S U L P H A T E D G L Y C O S A M...
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