Calcif. Tissue Int. 29, 89-94 (1979)

Calcified Tissue International c 1979 by Springer-Verlag

Change in Response With Age of Human Articular Cartilage to Plasma Somatomedin Activity I . K . A s h t o n a n d J.A. M a t h e s o n Nuffield Department of Orthopaedic Surgery, Nutfield Orthopaedic Centre, Headington, Oxford. OX3 7LD, England

Summary. N o r m a l male a r t i c u l a r cartilage (34 speci m e n s , age range 1-30 years) has b e e n e x a m i n e d in vitro for r e s p o n s e to s o m a t o m e d i n (SM) a c t i v i t y . Basal a H - t h y m i d i n e a n d 35S-sulfate i n c o r p o r a t i o n b o t h d e c r e a s e d with i n c r e a s i n g age o f the cartilage d o n o r . H o w e v e r , e n h a n c e m e n t of isotope i n c o r p o r a t i o n w h i c h was a t t a i n e d o n a d d i t i o n of 10% normal p l a s m a ( c o n t a i n i n g I U SM/ml) was greatest in cartilage from a d o l e s c e n t s in the age range 12-17 years. T h e m e a n e n h a n c e m e n t o f 3 H - t h y m i d i n e inc o r p o r a t i o n ( e x p r e s s e d as % basal) was as follows: age 1-10 y e a r s = 184 _+ 28 (SE), N = 9; 12-17 y e a r s = 436 _+ 101 (11); 18-30 years = 231 -+ 49 (8); a n d for :~sS-sulfate i n c o r p o r a t i o n was 1-10 years = 389 _+ 100 (8); 12-17 years = 824 _+ 273 (11); a n d 18-30 years = 572 _+ 56 (8). The i n c r e a s e d r e s p o n s e o f cartilage in the 12-17 y e a r g r o u p suggests that a g r e a t e r s e n s i t i v i t y to the s o m a t o m e d i n s m a y c o n t r i b u t e to the i n c r e a s e d skeletal g r o w t h d u r i n g adolescence. Key words: A r t i c u l a r cartilage - - S o m a t o m e d i n - Skeletal g r o w t h .

Introduction Clinical e v i d e n c e suggests that the c i r c u l a t i n g g r o w t h - p r o m o t i n g peptides c o l l e c t i v e l y k n o w n as the s o m a t o m e d i n s are i m p o r t a n t for n o r m a l g r o w t h a n d skeletal d e v e l o p m e n t in c h i l d r e n [1-4]. A l t h o u g h m a n y in vitro studies have s h o w n that the s o m a t o m e d i n s stimulate cell p r o l i f e r a t i o n a n d c o l l a g e n a n d p r o t e o g l y c a n s y n t h e s i s in cartilage from m a n y a n i m a l species [5, 6], t h e i r effects o n hum a n cartilage have r e c e i v e d little a t t e n t i o n . In two studies w h i c h have b e e n r e p o r t e d , h u m a n n e o n a t a l

Send offprint requests to Dr. I.K. Ashton at the above address.

a n d fetal cartilage o n l y has b e e n used [6, 7]. It is clear from p r e v i o u s reports that the r e s p o n s e o f rat a n d rabbit a n d p o r c i n e cartilage to the s o m a t o m e dins d e p e n d s o n the age o f the a n i m a l [8-10]. T h e p r e s e n t w o r k e x a m i n e s the effects of p l a s m a s o m a t o m e d i n a c t i v i t y o n p o s t n a t a l h u m a n cartilage a n d c o m p a r e s the r e s p o n s e o f h u m a n cartilage o f different ages.

Materials and Methods Materials The Waymouth's medium (Flow Laboratories) was supplemented with 20 mM HEPES (N-2-hydroxyethyl piperazine-Nethane sulfonic acid) buffer, 10 mM sodium bicarbonate, 0.5% BSA, 1.8 mM serine, and 1.1 mM glutamine and adjusted to pH 7.4 with NaOH, and contained 0.4 mM penicillin and I).2 mM streptomycin. It was sterilized before use by passage through millipore filters (0.45 gm).

Preparation o f Cartilage Articular cartilage was obtained from 34 male patients either as a result of elective orthopedic surgery 128 patients) or as a consequence of accidental injury (6 patients). Patients with endocrine disorders, degenerative and inflammatory joint disease, or growth disturbances, e.g., scoliosis, were excluded. A total of six different joints were used as shown in Table 1. Cartilage was placed in sterile 0.9% saline containing 0.8 mM penicillin, 0.4 mM streptomycin, and 8 x 10.~units gentamicin/liter immediately it was removed and stored at 4~ until used. The mean time interval from surgery to the start of the incubation was 3 h, and the maximum was 10 h as shown in Table 1. All subsequent procedures were conducted aseptically. The specimens of cartilage were cleaned of adherent connective tissue and the remainder sliced into pieces approximately 1 mm2 and 0.5 mm thick [11] and placed in fresh 0.9% saline containing 0.4 mM penicillin and 0.2 mM streptomycin. Cartilage was retained from some specimens for routine histological examination. The number of cells in an area of 0.025 mm~ was determined in 7 ~m sections by use of a calibrated eyepiece.

0171-967X/79/0029-0089 $01.20

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Table

I.K Ashton and J.A. Matheson: Human Articular Cartilage and Somatomedin

1. Source of articular cartilage

Age (years)

1-10 12-17 18-30

Source"

A E A E A E

No. of patients

0 11 1 12 5 5

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S ite

Patella

MTP joints ~

--1 2 4 2

. 2 . 7

Tarsal joints .

.

Radial head

IP joints

3

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--

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--

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4.0

--

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2

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(1-7)

8 .

.

1~

~ A, accidental; E, elective t, Metatarsophalangeal ': Interphalangeal joint Itoe) '~ Interphalangeal joint tfinger) ~' Time interval between removal and incubation (mean and range) f No significant difference by Student's t test

Incubation Procedure The cartilage pieces w'ere incubated in 2 ml aliquots of supplemented Waymouth's medium containing either 3H-thymidine ~1 ~Ci/ml) or asS-sulfate (1 p.Ci/ml). The human plasma standard was added to half of the tubes to a final concentration of 10%. Three or four pieces of cartilage were added to each tube so that there were four replicate tubes with or without plasma for each isotope, i.e., eight tubes for each isotope. The tubes were stoppered, mixed, and incubated with shaking at 37~ for 48 h. Sufficient material was usually obtained from each patient to determine the incorporation of both isotopes in the same experiment. In the presentation of data, N is the number of patients from whom cartilage was used in the experiment described. After incubation, the tubes were boiled for 3 rain. the medium was decanted, and the cartilage pieces washed overnight with three changes of distilled water. The cartilage from each tube was then transferred to a glass coverslip in a Petri dish, dried at room temperature overnight, and weighed ~_+ 1 ~g). Dry weight range was 1-4 rag. The cartilage was then transferred to a glass scintillation vial and hydrolyzed at 85~ overnight in 0.5 ml 23N formic acid. Next 15 ml of scintillation fluid was added (l 1 toluene: 500 ml ethoxyethanol: 7.5 g PPO) and the incorporated isotope was measured in a scintillation counter.

Assessment of Skeletal and Sexual Maturity Pubertal development was assessed by physical examination of the patient, the rating being an average for genital and pubic hair development as described by Tanner [12]. For ethical reasons, only existing radiographs could be used in this study. When hand X-rays were available, skeletal age was determined by comparison with the standards of Greulich and Pyle [13]: otherwise, an estimate of skeletal age was made according to Sutton and Grainger [14]. Chronological age was the age attained at the nearest birthday.

Standard Plasma The standard plasma was prepared from a pool of plasma from

six adult male volunteers. It was necessary to prepare two subsequent standards; each time the same donors were used and the potency of the new standard checked against the previous one with a rabbit chondrocyte assay for somatomedin activity [15]. No significant difference in activity of the standards was found.

Results

Basal Isotope Incorporation T h e i n c o r p o r a t i o n o f b o t h a H - t h y m i d i n e a n d 3'~S-sulfate per dry weight of cartilage was significantly g r e a t e r in t h e s p e c i m e n s in t h e 1 - 1 0 y e a r a g e g r o u p ( F i g . 1). C e l l d e n s i t y w a s a l s o g r e a t e r in t h e s p e c i m e n s f r o m y o u n g e r i n d i v i d u a l s ( F i g . 2). D i f f e r e n c e s in p r o t e o g l y c a n s y n t h e s i s as w e l l a s in c e l l u l a r i t y h a v e b e e n r e p o r t e d in a r t i c u l a r c a r t i lage f r o m d i f f e r e n t j o i n t s [16, 17]. I n o r d e r t o d e t e r m i n e w h e t h e r t h e h i g h e r b a s a l i n c o r p o r a t i o n in t h e younger specimens was due to the predominance of articular cartilage from the tarsal joints, mean basal i n c o r p o r a t i o n o f b o t h 3 H - t h y m i d i n e a n d 3'~S-sulfate in c a r t i l a g e f r o m d i f f e r e n t s i t e s in t h e 1 - 1 0 y e a r a n d 1 2 - 1 7 y e a r a g e g r o u p s w e r e c o m p a r e d ( T a b l e 2), b u t no significant difference between these sites was observed. All s p e c i m e n s in t h e 1 - 1 0 y e a r g r o u p w e r e o b tained as a result of elective surgery: however, the mean time interval between removal of the tissue a n d i n c u b a t i o n w a s n o t s i g n i f i c a n t l y s h o r t e r t h a n in t h e 1 1 - 1 7 y e a r a g e g r o u p , a l t h o u g h it w a s l e s s ( P < 0 . 0 5 ) t h a n in t h e 1 8 - 3 0 y e a r g r o u p w h i c h i n c l u d e d some specimens received after accidental injury ( T a b l e 1). N o c o r r e l a t i o n c o u l d b e e s t a b l i s h e d b e t w e e n t i m e i n t e r v a l a n d b a s a l i n c o r p o r a t i o n o f eit h e r i s o t o p e w i t h i n a n y a g e g r o u p ( F i g . 3). T h e r e w a s a l s o n o a p p a r e n t d i f f e r e n c e in a c t i v i t y in t h e 1 8 -

I.K A s h t o n and J.A. Matheson: H u m a n Articular Cartilage and S o m a t o m e d i n

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Change in response with age of human articular cartilage to plasma somatomedin activity.

Calcif. Tissue Int. 29, 89-94 (1979) Calcified Tissue International c 1979 by Springer-Verlag Change in Response With Age of Human Articular Cartila...
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