The effects of matrix compression on proteoglycan metabolism in articular cartilage explants

Osteoarthritis and Cartilage (1994) 2, 91-101 © 1994 Osteoarthritis Research Society

1063-4584/94/020091 + 11 $08.00/0

OSTEOARTHRITIS and CARTILAGE The effects o f m a t r i x c o m p r e s s i o n o n p r o t e o g l y c a n m e t a b o l i s m in articular cartilage explants BY FARSHID GUILAK, B. CHRISTOPH MEYER, ANTHONY RATCLIFFE AND VAN C. MOW

Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Columbia University, New York 10032, U.S.A.

Summary The effects of compressive stress on the rate of proteoglycan synthesis and release were determined in bovine articular cartilage from 4-5-month-old animals. Full depth cartilage explants were compressed in an unconfined configuration at various stresses ranging up to 1.0 MPa. At mechanical equilibrium (after 24 h), no significant changes were detected in the rate of [asS]-sulfate (35SO4) incorporation at the low level of compressive stresses used (less than 0.057 MPa). At an intermediate level of compressive stress (0.057, 0.1, 0.5 MPa), 35SO4 incorporation rates were reduced to ~ 60~/o of control values. At the highest level compressive stress (1.0 MPa) studied, 35SO4 incorporation rates were further reduced to ~ 20~/o that of controls. Recovery experiments at intermediate stress levels showed increased rates of 35SO4 incorporation at 24 h after compression. In explants loaded for 24 h at stresses of 0.1 MPa or higher, there was a stressdose dependent inhibition of proteoglycan release into the media (up to 61% at 1.0 MPa), and proteoglycan release rates did not return to control values following a 24 h recovery period. While cartilage composition and biosynthetic activity were found to vary significantly with depth in control cartilage, the observed suppression (~/o change) in biosynthetic activity was relatively uniform with depth in both loading and recovery experiments. The study indicates that compression of the tissue to physiological strain magnitudes serves as a signal to modulate chondrocyte biosynthetic and catabolic responses through the depth of cartilage, while prolonged compression at higher strains may be responsible for tissue and cell damage. Key words: Chondrocyte, Biosynthesis, Biomechanics, Mechanical signal transduction.

general, static compression can suppress proteog l y c a n s y n t h e s i s r a t e s in a r t i c u l a r c a r t i l a g e e x p l a n t s [9, 12] a n d e p i p h y s e a l c a r t i l a g e e x p l a n t s [6, 7], w h i l e c y c l i c c o m p r e s s i o n a t specific f r e q u e n cies c a n s t i m u l a t e p r o t e o g l y c a n m e t a b o l i s m [10, 11, 14]. I n t h e p r e s e n t s t u d y , we w i s h to f u r t h e r o u r u n d e r s t a n d i n g o f t h e effects of m e c h a n i c a l l o a d i n g o n c a r t i l a g e m e t a b o l i s m in a n in vitro explant experiment. N o r m a l c h o n d r o c y t e m e t a b o l i c a c t i v i t y is r e g u lated by both genetic and environmental factors which include soluble mediators (cytokines, g r o w t h f a c t o r s a n d p h a r m a c e u t i c a l a g e n t s ) [15-17] a n d m a t r i x c o m p o s i t i o n [18], as well as m e c h a n i c a l f a c t o r s [1-14] w h i c h a r e a s s o c i a t e d w i t h f u n c tional joint loading. Under physiologic loading c o n d i t i o n s , p e a k s t r e s s e s as h i g h as 18 M P a h a v e been reported during dynamic loading of the joint [19]. H o w e v e r , t h e in situ e q u i l i b r i u m c o m p r e s s i v e s t r a i n in c a r t i l a g e h a s b e e n o b s e r v e d to be n o m o r e t h a n 20% [20]. F u r t h e r m o r e , it is l i k e l y t h a t u n d e r in vivo c o n d i t i o n s , i n t e r s t i t i a l fluid p r e s s u r i z a t i o n in c a r t i l a g e p l a y s a m a j o r r o l e in l o a d s u p p o r t in t h e j o i n t [21, 22]. T h u s , in vivo t h e m e c h a n i c a l e n v i r o n m e n t a r o u n d a c h o n d r o c y t e is l i k e l y t o b e v e r y d i f f e r e n t f r o m t h a t o b t a i n e d in e x p l a n t

Introduction IT is well a c c e p t e d t h a t m e c h a n i c a l f o r c e s a n d deformations can alter the metabolic activity of c h o n d r o c ~ t e s in a r t i c u l a r c a r t i l a g e . F o r e x a m p l e , in vivo s t u d i e s h a v e f o u n d s i g n i f i c a n t c h a n g e s in the composition, structure and mechanical properties o f a r t i c u l a r c a r t i l a g e e x p o s e d to disuse, o v e r u s e , or a l t e r e d j o i n t l o a d i n g c o n d i t i o n s [1-5]. To b e t t e r u n d e r s t a n d the.- m e c h a n i s m s t h r o u g h which mechanical loading regulates the catabolic a n d a n a b o l i c e v e n t s i n v o l v e d in t h e m a i n t e n a n c e of t h e e x t r a c e l l u l a r m a t r i x , a n u m b e r of in vitro s t u d i e s h a v e a t t e m p t e d to i s o l a t e specific f a c t o r s i n v o l v e d in t h i s t r a n s d u c t i o n p r o c e s s [6-14]. As a n a d v a n t a g e o v e r t h e in vivo c o n d i t i o n s , t h e s e in culture experiments offer a vitro e x p l a n t controlled biomechanical and biochemical environment for determining the response of a r t i c u l a r c a r t i l a g e to m e c h a n i c a l f o r c e s o r d e f o r m a t i o n s . T h e s e s t u d i e s i n d i c a t e t h a t in Submitted 12 November 1993; accepted 21 January 1994. Address correspondence and reprint requests to: Dr Farshid Guilak, Musculo-Skeletal Research Laboratory, Health Sciences Center T18-030, State University of New York at Stony Brook, Stony Brook, New York 11794-8181, U.S.A. 91

92

Guilak e t a l . : Matrix c o m p r e s s i o n a n d p r o t e o g l y c a n m e t a b o l i s m

studies, except perhaps for the compressive strain. In addition, other mechanical, physicochemical and electrical effects m a y result from loading of cartilage, including interstitial fluid flow, osmotic pressure, static and dynamic m a t r i x deformation, streaming potentials, changes in the tissue fixed charge density or pH, and deformation of the chondrocytes [8, 9, 12, 21-25]. Currently, there is evidence t h a t any of these phenomena could be involved in the signal t r a n s d u c t i o n process of the chondrocyte. Isolation of the specific regulatory signals derived from the mechanical and~-physicochemical environment of the t i s s u e is f u r t h e r complicated by the inhomogeneity of the chondrocytes and extracellular matrix in the different zones of the tissue [26-29]. To better understand the mechanism(s) t h r o u g h which mechanical, physicochemical and electrical signals are translated into biochemical events by the chondrocyte, it is a d v a n t a g e o u s to examine the response of chondrocytes to each of these events separately, whenever possible. At compressive equilibrium, no complicating effects will appear from interstitial fluid flow, fluid pressurization, deformation rates, streaming potentials or other electrokinetic phenomena [24]. The objective of this study was to q u a n t i f y zonal variations in the metabolic response of cartilage explants to equilibrium compressive stresses. Rates of proteoglycan synthesis and release were determined for the explants as a function of the equilibrium stress. The recovery response of the biosynthetic activity was also examined upon unloading of the explants. Finally, considering t h a t both tissue composition [18] and mechanical load [1-12, 14] are known regulators of cartilage metabolic activity, the biochemical composition of the tissue and the compression-induced changes in biosynthesis rates were examined in the different zones of the tissue.

Materials

and methods

I N S T R U M E N T A T I O N FOR E X P L A N T C O M P R E S S I O N A N D MECHANICAL TESTING

To prescribe the load or displacement on a single specimen during an explant culture study and for mechanical testing of the specimens, a load and displacement controlled device (LODEC) was designed and constructed in our laboratory [30]. This closed-loop device uses feedback control on a force signal from an in-line load cell (Model #31, Sensotec Inc., Columbus, OH) m o u n t e d on the loading shaft (Fig. 1). M o t i o n of the loading shaft is achieved by a computer-controlled motor-

Stepp, Motori

5 L

mmputer :ontrol

Speci~ Cultur Chaml ~nslation

FIG. 1. Diagram of the computer-controlled loading apparatus (LODEC). This instrument was used for the control of displacement and load on cartilage explants for the mechanical tests and tissue culture experiments. Displacements were applied through a sintered stainless steel porous filter using a motorized micrometer. The force on the sample was monitored using an in-line load cell. A feedback loop on the force signal was used to maintain a constant stress on the explant for the entire duration of the experiment. ized micrometer (StepperMike, Oriel Corp., Stratford, CT) mounted rigidly on a t r a n s l a t i o n stage above the explant culture dish. This system, which has a force resolution of 0.01 N and a displacement resolution of 1 pm, was used for both mechanical testing of the explants and for the tissue culture experiments. Once the initial protocol for the explant compression studies was developed, a specially designed rack was built to allow testing of multiple samples simultaneously. With this instrument, six samples were tested simultaneously using dead weights. Compressive loading was applied to samples in individual chambers t h r o u g h steel shafts aligned by linear bearings. To facilitate diffusion of n u t r i e n t s to the chondrocytes in the tissue during compression, the sample was loaded in a state of unconfined compression using porous-permeable, sintered stainless steel platens. The platens have an average pore size of 50 #m and a porosity (ratio of void volume to total volume) of 0.4-0.6. Due to the high permeability of these platens ( ~ 10 -11 m4/Ns, approximately 1000 x the tissue permeability), the

O s t e o a r t h r i t i s a n d Cartilage Vol. 2 No. 2

unconfined configuration permitted all surfaces of the specimen free access to the culture medium. From previous studies on the diffusion rates of solutes, low molecular weight solutes diffuse rapidly into the tissue [7, 12, 25, 31]. Assuming t h a t the porous platens are freely permeable, a sample thickness of 1 mm has a governing distance of 0.5 mm, corresponding to a diffusion time of less t h a n a few minutes [7, 25, 31].

EXPLANT

HARVESTING

AND

CULTURE

Full-thickness discs of articular cartilage ( 1-1.5 mm thick) were harvested from the central region of carpometacarpal joints of 4-5-month-old calves within 3-4 h post mortem. In a sterile environment, samples were punched out using a 5 mm-diameter stainless steel trephine and the cartilage was removed from the underlying bone using a scalpel. Explants were m a i n t a i n e d in culture for up to 6 days in Dulbecco's Modified Eagle Medium supplemented with 0.1% bovine serum albumin, 50 U/ml penicillin/streptomycin, and 2 mM nonessential amino acids and glutamine, at 37°C, 5% CO 2. The media were changed daily and stored for analysis.

MECHANICAL

TESTING

PROTOCOL

The equilibrium stress-strain behavior of the calf articular cartilage was determined on a set of 12 independent samples using the unconfined loading configuration. Full-thickness cartilage discs were compressed between the permeable loading platens in the LODEC under unconfined compression. Seven different loads were applied sequentially to each specimen, yielding 0.001, 0.01, 0.028, 0.057, 0.1, 0.5 or 1.0 MPa. Strains were recorded once the tissue had reached equilibrium at each level of stress, which occurred rapidly at the lower levels of applied stress. At the higher levels of applied stress, equilibrium was reached within 3600 s. Because large deformations were produced at the 0.5 and 1.0 M P a levels, the equilibrium strain was also calculated using the standard finite deformation formula given by e = (,~2_1)/2 [32], where the stretch ,~ = h / h o, h is the equilibrium compressed thickness and h o is the initial thickness of the specimen. Thickness measurements were made using an apparatus which utilizes the conductivity of the tissue to indicate contact between the tissue and a measuring probe [27].

EFFECT

93

OF STATIC

LOAD ON PROTEOGLYCAN

SYNTHESIS

RATES

In preliminary studies, [35S]-sulfate (35SO4) incorporation and sulfated glycosaminoglycan (S-GAG) release of the expiants were measured for 7 days after harvesting to assess the stability of the culture system. Compression experiments were performed on days 2-6, at which time, the rates of 35SO4 incorporation and S-GAG release in control cultures did not vary significantly (see Results). Prior to each experiment, the loading platens were ultrasonically cleaned and flame-sterilized. Explants were compressed in individual petridishes in 4 ml of media, and control samples, t a k e n from the same joint surface, were cultured unloaded in an otherwise identical experiment. Step loads were applied to the explants to yield stresses of 0.001, 0.01, 0.028, 0.057, 0.1, 0.5 or 1.0 MPa. To determine changes in the rate of proteoglycan synthesis, the tissue was compressed for 24 h and incubated in media containing 20 pCi/ml of 35SO4 during the last 4 h of each compression. To determine the rate of proteoglycan synthesis, specimens were digested with papain overnight at 60°C, and the amount of 35SO4 t h a t eluted in the Vo of a PD-10 column (Pharmacia) was determined. 35SO4 incorporation was normalized to the total S-GAG content of the tissue and expressed as a percentage of the rate of 35SO4 incorporation in the control explants. In separate tests, to determine the ability of the tissue to recover from the compression effects, the samples were compressed for 24 h and then removed from the loading apparatus and cultured unloaded for 20 h before radiolabeling for 4 h in the presence of 20 ttCi/ml of a5SO4. At compressive stresses which resulted in significant changes in the biosynthetic activity of the whole explant, the zonal variation in the response to compression was determined. Following compression, radiolabeled explants were divided into three layers using a specially designed cutting jig. The top 100-150 pm (surface zone) was first removed, and the remaining piece was then divided into two equal sections (middle and deep zones) with thickness ranging from 400-800 #m. Each zone was analyzed separately and compared to the same zone from control tissue.

EFFECT

OF STATIC

LOAD

RELEASE

ON PROTEOGLYCAN RATES

To determine the effect of static load on the rate of release of proteoglycan from articular cartilage,

94

Guilak e t a l . : Matrix c o m p r e s s i o n a n d p r o t e o g l y c a n m e t a b o l i s m

media were collected for t he day before compression (day 1), after t he 24 h period of compression (day 2), and after the 24 h following compression (day 3). The porous filters were stirred for 2 min in the medium before collection to release any glycosaminoglycan still r e m a i n i n g in t he filter pores. To assess p r o t e o g l y c a n release, the aliquots of the collected media were analyzed for S-GAG c o n t e n t and normalized to t he t ot a l S-GAG c o n t e n t of the specimen. Similar results were obtained by expressing synthesis and release rates normalized to the h y d r o x y p r o l i n e c o n t e n t of t he cartilage. The results for S-GAG release from compressed samples on day x(x = 1,2,3) (denoted by R(test)dayx) w e r e normalized to the m e a n release of control samples on the same day (denoted by R((control)dayx). In order to a c c o u n t for nor m a l variation between samples, these values for each day are expressed as p e r c e n t a g e of t he release on day 1 of the same sample, using the following equation: Normalized release

=

(R(test)dayx/R(c°ntr°l)dayx))<100

0

1.2

Engineering strain e = (h-ho)/ho 0.1 0.2 0.3 0.4 0.5 0.6 I

I

I

0.7

I

I

I

I

i

i

I

I

0.8

1.0 f

[

d9 0.6

0.4 0.2 •

~

~--~---i]-~--~

0 I

i

I

I

0 0.10 0.18 0.26 0.32 0.37 0.42 0.46 0.48 Finite deformation strain e = ()~L1)/2, A = h/ho

FIG. 2. The equilibrium stress-strain curve of calf metacarpal cartilage measured in unconfined compression. This stress-strain curve was determined on a separate set of 12 explants. Step loads corresponding to stresses of 0.001-1.0 MPa were sequentially applied to each explant, permitting the sample to creep to equilibrium at each stress. The applied stress was calculated from the applied load and the initial cross-sectional area. The tissue exhibited a linear stress-strain behavior at lower strains (e < 0.25) with a characteristic nonlinear stiffening effect at higher strains (e = 0.25-0.45). Values shown are m e a n + S . D . , N = 12.

DETERMINATION OF TISSUE COMPOSITION

STA'I~ISTICAL ANALYSIS

Samples were weighed before and after lyophilization at - 5 0 ° C for 48 h to det er m i ne t he w a t e r c o n t e n t (water weight/wet weight of tissue). The tissue was th en digested with papai n (1.25 mg/100 mg tissue) for 16 h at 60°C, and aliquots of this digest were t a k e n for s epar a t e analyses to determine h y d r o x y p r o l i n e and S-GAG contents. To determine the h y d r o x y p r o l i n e c o n t e n t of this tissue, as a m e a s ur e of collagen content, the papain digest was hydrolyzed with HC1 at 107°C overnight, dried, and t h e n analyzed by a colorimetric p r o c e d u r e [33]. The S-GAG content, a m e a s u r e of t he proteoglycan content, was det er m i ned using t he dye 1,9d i m e t h y l m e t h y l e n e blue [34]. T he dye was used at a c o n c e n t r a t i o n of 16 mg/1 in f o r m a t e buffer, pH 3.5. F o r t y microliters of sample or s t a n d a r d (diluted in phosphate buffered saline, pH 7.2) was added to wells of a m i c r o t i t e r plate, 250/~l of dye r e a g e n t was added, and the a b s o r b a n c e at 530 nm and 600 nm was m eas ur ed rapidly (within 5 min) using a Bio-Tek EL309 m i c r o p l a t e a u t o r e a d e r . A n eg ativ e ab s o r b a nc e c h a n g e is o b t a i n e d at 600 nm and a positive a b s o r b a n c e c ha nge is given at 530 nm. Th e total change in a b s o r b a n c e was determined as the sum of the c h a n g e in a b s o r b a n c e at 530 nm and the change in a b s o r b a n c e at 600 nm [35]. C h o n d r o i t i n sulfate from s h a r k c a r t i l a g e was used as a s t a n d a r d b e t w e e n 5 and 50 pg/ml.

I n c o r p o r a t i o n of 3 5 8 0 4 and release of S-GAG of each explant was normalized to t he total S-GAG c o n t e n t of t he sample. Data are r e p o r t e d as th e rat i o of the m e a n of the experi m ent al to the m e a n of the cont rol results. Statistical comparisons bet w een samples compressed at different stresses were made using one-way analysis of v a r i a n c e (ANOVA). Two-way ANOVA was used to assess differences b e t w e e n release rat es on different days vs different stresses. Post hoc comparisons were made using t he T u k e y Studentized Range M e t h o d and the S t u d e n t - N e w m a n - K e u l s Multiple R a n g e test. St at i st i cal analysis was carri ed out using the B M D P statistical p a c k a g e (BMDP Statistical Software Inc., Los Angeles, CA), w hi ch re p o rts statistical significance at the 90%, 95% and 99% confidence levels.

Results

MECHANICAL TESTING Samples w hi ch were excised from the j o i n t swelled in c u l t u r e with a mean i n c r e a s e in thickness of 9.4+2.6%. T he equilibrium s t r e s s - s t r a i n dat a of the cal f c a r p o m e t a c a r p a l a r t i c u l a r cartilage obt ai ned on the LODEC for t he applied compressive stresses (0.001-1.0 MPa) in unconfined compression are shown in Fig. 2. F o r strains

Osteoarthritis

and Cartilage

95

l i n e a r s t i f f e n i n g effect, w h i c h is c o n s i s t e n t w i t h p r i o r o b s e r v a t i o n s [6, 10, 21, 28]. T h e m e a n equilib r i u m s t r a i n , e= ( s u r f a c e - t o - s u r f a c e s t r a i n ) , of t h i s t i s s u e r a n g e d f r o m less t h a n 0.01 a t 0.001 M P a to 0.45 a t 1.0 M P a . A t l o w e r s t r e s s e s , m e c h a n i c a l e q u i l i b r i u m w a s r e a c h e d w i t h i n 1200 s w h i l e a t h i g h e r s t r e s s e s (0.5-1.0 M P a ) a n d w i t h t h i c k e r s a m p l e s , e q u i l i b r i u m w a s r e a c h e d w i t h i n 3600 s. R e m o v a l o f c o m p r e s s i v e s t r e s s r e s u l t e d in n e a r l y c o m p l e t e r e s w e l l i n g of t h e t i s s u e to its o r i g i n a l t h i c k n e s s (95% r e c o v e r y w i t h i n 3600 s), e x c e p t a t 1.0 M P a , w h e r e t h e t i s s u e r e s w e l l e d t o a p p r o x i m a t e l y 75~/o of its o r i g i n a l t h i c k n e s s a t 24 h.

Surface Zone

Middle Zone

Deep Zone

'~"

Vol. 2 No. 2

100 pm

FIG. 3. Histologic mierograph of bovine articular cartilage. Cylindrical samples of articular cartilage from the carpometacarpal joints of 4-5-month-old calves were fixed in glutaraldehyde in the presence of ruthenium hexammine trichloride as described previously [49]. This procedure maintains cell shape and prevents separation of the cell membrane from the extracellular matrix. Analysis showed that cell density and structure of the tissue were fairly constant with depth (scale bar 100 pm).

TISSUE COMPOSITION AND BIOSYNTHETIC RATES

H i s t o l o g i c a l a n a l y s i s of c o n t r o l ( u n l o a d e d ) s a m p l e s i n d i c a t e d few a p p a r e n t d i f f e r e n c e s i n t h e u l t r a s t r u c t u r e of t h e d i f f e r e n t z o n e s o f t h e tissue. W h i l e cells a t or n e a r t h e s u r f a c e of t h e t i s s u e a p p e a r e d m o r e f l a t t e n e d as h a s b e e n d e s c r i b e d in a d u l t c a r t i l a g e [29], cell d e n s i t y a p p e a r e d to be f a i r l y u n i f o r m w i t h d e p t h (Fig. 3). H o w e v e r , s i g n i f i c a n t d i f f e r e n c e s w e r e n o t e d in t h e bioc h e m i c a l c o m p o s i t i o n a n d b i o s y n t h e t i c r a t e s of t h e s u r f a c e z o n e t i s s u e as c o m p a r e d to t h e m i d d l e a n d d e e p z o n e s (Fig. 4(a)-(d)). S - G A G c o n t e n t o n a

less t h a n 0.25, t h e s t r e s s - s t r a i n b e h a v i o u r o f cartil a g e w a s f o u n d to be n e a r l y linear. A t h i g h e r loads, t h e t i s s u e e x h i b i t e d t h e c h a r a c t e r i s t i c n o n -

0.25

012[ (a)

b),

0.2 0.15 006

O

R ~0.03

0.05

T

0

0

Surface 100 80

8

40

(c)

Middle

Deep 200

, __

f f / 7 7 J J f J /

150 O <

/ / f / i JJ~JJ I I I J i

O3

// // // // / / / / / / / / f f / J

~

N 20 0

0.1

l/z// Middle

Middle

Deep

(d)

T

100 50

/] /] /~/ [/ [ #//// Sur~ce

iiiiiiiiii'~i!iii Surface

0 Deep

Surface

Middle

Deep

Fro. 4. Zonal variations in tissue composition. Biochemical analyses were performed to examine zonal variations in tissue composition, including hydroxyproline, S-GAG and water contents. While no zonal differences were noted in hydroxyproline content (a), a significant difference was observed in the S-GAG content (b), water content (c), and sulfate incorporation rates (d) of the surface zone as compared to the middle and deep zones. Values shown are mean±s,D., N--- 10, *P < 0.01. El: dry weight basis; m: wet weight basis.

96

Guilak e t a l . : Matrix c o m p r e s s i o n and p r o t e o g l y c a n m e t a b o l i s m 120 .~

100

(a) 300 t

o~, 8 0 8~

60

d~

40

~

T

209

20 0 7 6

2

3

4

5

6 m 100

(b)

0

1 0

2

3

4 5 Days in culture

6

FIG. 5. a5SO4 incorporation and S-GAG release rates in control samples. Preliminary tests were performed for 7 days following harvesting of the explants to assess the stability of the culture system. All compression experiments were performed between days 2-6 following explant harvesting, during which proteoglycan synthesis rates (a) and release rates (b) did not vary significantly.

w e t w e i g h t b a s i s w a s s i g n i f i c a n t l y l o w e r ( P < 0.01) and water content was significantly higher ( P < 0.01) in t h e s u r f a c e zone. N o z o n a l d i f f e r e n c e s w e r e d e t e c t e d for t h e h y d r o x y p r o l i n e c o n t e n t o n a w e t or d r y w e i g h t basis, o r for S - G A G c o n t e n t o n a d r y w e i g h t basis. P r o t e o g l y c a n b i o s y n t h e t i c r a t e s ( d p m / p g S-GAG) w e r e s i g n i f i c a n t l y h i g h e r in t h e s u r f a c e z o n e as c o m p a r e d to t h e m i d d l e a n d d e e p z o n e s ( P < 0.01). EFFECT OF STATIC COMPRESSION ON PROTEOGLYCAN SYNTHESIS RATES

All e x p e r i m e n t s w e r e p e r f o r m e d o n d a y s 2 - 6 f o l l o w i n g h a r v e s t i n g , w h e r e it w a s s h o w n t h a t proteoglycan synthesis and release rates of control s a m p l e s w e r e c o n s t a n t (Fig. 5(a)). I n p r e l i m i n a r y studies, t h e effects o f p r e s e n c e o f t h e s t a i n l e s s s t e e l filter for 24 h ( w i t h a 4 h r a d i o l a b e l i n g period) were examined, and no differences were f o u n d b e t w e e n s y n t h e s i s r a t e s in f r e e - s w e l l i n g c o n t r o l s a m p l e s a n d t h o s e m a i n t a i n e d in c o n t a c t w i t h t h e filter o n b o t h sides ( e x p e r i m e n t a l - 92 ± 37% of f r e e - s w e l l i n g c o n t r o l s , P = 0.57, t-test). T h u s all e x p e r i m e n t a l r e s u l t s a r e s h o w n n o r m a l ized to t h e m e a n s y n t h e s i s r a t e s o f f r e e - s w e l l i n g control samples. In samples compressed at stresses less t h a n 0.057 M P a , t h e r a t e o f ~5SO4 i n c o r p o r a -

Control

0.057

0.100 0.500 Stress (MPa)

1.000

FIG. 6. 35SO4 incorporation rates with compression and recovery. ~5SO4 incorporation rates, normalized to tissue S-GAG content, are presented for cartilage explants compressed at different stresses after 24 h of load and with recovery at 24 h following removal of the load. Compressive stress did not affect 35SO4 incorporation rates at stresses of 0.001, 0.01 or 0.028 MPa. A significant decrease in s~SO4 incorporation rates was observed in samples which were compressed at 0.057 MPa and higher. Recovery experiments suggested a 'rebound' phenomenon following loading at stresses of 0.1 and 0.5 MPa, where 3~SO4 incorporation rates were significantly elevated in the 24 h following compression. At 1.0 MPa, incomplete recovery was observed, suggesting tissue damage or cell death. []: compression; []: recovery. Values shown are mean_+s.D., N = 10-12, *P < 0.05 vs control, **P < 0.01 vs control. t i o n w a s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e r a t e in u n l o a d e d c o n t r o l s a n d t h u s s t r e s s levels l o w e r t h a n 0.057 M P a a r e n o t s h o w n . A t 0.057, 0.1 a n d 0.5 M P a , i n c o r p o r a t i o n r a t e s w e r e r e d u c e d to a p p r o x i m a t e l y 60% t h a t o f c o n t r o l v a l u e s (Fig. 6). T h i s i n h i b i t i o n did n o t v a r y s i g n i f i c a n t l y a m o n g t h e s e t h r e e s t r e s s e s . A t 1.0 M P a , 35SO4 i n c o r p o r a t i o n w a s f u r t h e r r e d u c e d to o n l y ~ 20% t h a t o f control samples. EFFECT OF STATIC COMPRESSION ON PROTEOGLYCAN RELEASE RATES

I n c o n t r o l c u l t u r e s , t h e r e w a s a slow b u t s t e a d y release of S-GAG into the media at a rate of 3-5~/o/day o f t h e t o t a l S - G A G in t h e c a r t i l a g e e x p l a n t s (Fig. 5(b)). As w i t h t h e s y n t h e s i s s t u d i e s , p r e l i m i n a r y s t u d i e s w e r e p e r f o r m e d to e n s u r e t h a t t h e filters u s e d to c o m p r e s s t h e t i s s u e did n o t a l t e r the pattern of S-GAG exchange with surrounding m e d i a . T h e s e s t u d i e s s h o w e d n o s i g n i f i c a n t differe n c e s in S - G A G r e l e a s e b e t w e e n f r e e - s w e l l i n g c o n t r o l s a n d s a m p l e s in c o n t a c t w i t h t h e p o r o u s filter on b o t h sides ( e x p e r i m e n t a l = 1 1 4 ± 4 3 % of f r e e - s w e l l i n g c o n t r o l s , P = 0.79, t-test). E x p l a n t s

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2o0t 150 ~

T;

T

c~

8 100

O

**

o O9

50

0

0.001

0.01

0.028

0.057 S t r e s s (MPa)

0.1

0.5

1.0

FIG. 7. S-GAG release rates with compression and recovery. S-GAG release rates from cartilage explants are presented for different compressive stresses under free-swelling (control) conditions, during 24 h of compression, and for 24 h after removal of the load. Values are normalized to the release of S-GAG of each sample on Day 1 as described in the text. At stresses of 0.1 MPa and higher, a significant suppression of S-GAG release rates was observed. In recovery experiments at 0.5 and 1.0 MPa, an increase in S-GAG release rates relative to compressed values was observed, although these values were still significantly lower than release rates observed in free-swelling samples. Values shown are mean+s.D., N = 10-12, *P < 0.05 vs Day 1, e P < 0.01 vs Day 1, **P < 0.05 vs Day 1, Day 2. f~: Day 1 (free swelling); m: Day 2 (compressed); m: Day 3 (unloaded). w h i c h w e r e c o m p r e s s e d a t 0.057 M P a a n d l o w e r d e m o n s t r a t e d n o c h a n g e s in S - G A G r e l e a s e f o l l o w i n g c o m p r e s s i o n or r e c o v e r y . I n s a m p l e s w h i c h w e r e c o m p r e s s e d a t 0.1 M P a a n d h i g h e r , a d o s e - d e p e n d e n t i n h i b i t i o n of S - G A G r e l e a s e w a s o b s e r v e d d u r i n g t h e 24 h l o a d i n g p e r i o d (Fig. 7). A t 0.1 M P a , S - G A G r e l e a s e i n t o t h e m e d i a w a s s i g n i f i c a n t l y i n h i b i t e d b y 41~/o, a t 0.5 M P a b y 54% a n d a t 1.0 M P a , S - G A G r e l e a s e w a s i n h i b i t e d by 61~/o. E F F E C T OF S T A T I C C O M P R E S S I O N ON R E C O V E R Y O n PROTEOGLYCAN SYNTHESIS RATES

In cartilage that was compressed and then u n l o a d e d a n d a l l o w e d to r e c o v e r , n o d i f f e r e n c e s w e r e n o t e d in t h e r a t e o f 3~SO4 i n c o r p o r a t i o n c o m p a r e d to c o n t r o l s a t s t r e s s e s less t h a n 0.057 M P a (Fig. 6). M e c h a n i c a l r e c o v e r y o f t h e t i s s u e o c c u r r e d in less t h a n 3600 s, a l t h o u g h a t 1.0 M P a , recovery was not complete. At two intermediate s t r e s s e s (0.1 a n d 0.5 M P a ) , r e c o v e r y e x p e r i m e n t s s h o w e d a s i g n i f i c a n t m a r k e d i n c r e a s e in t h e r a t e of 35SO4 i n c o r p o r a t i o n 24 h f o l l o w i n g r e m o v a l o f t h e compression. At the highest stress level of the test (1.0 M P a ) , i n c o r p o r a t i o n a f t e r 24 h o f r e c o v e r y r e m a i n e d i n h i b i t e d to ~ 40% t h a t o f c o n t r o l s .

EFFECT OF STATIC COMPRESSION ON RECOVERY OF PROTEOGLYCAN RELEASE RATES I n s a m p l e s c o m p r e s s e d a t 0.057 M P a a n d l o w e r , t h e r a t e of r e l e a s e of S - G A G i n t o t h e m e d i a w a s unaffected by the compression, during and after t h e c o m p r e s s i o n (Fig. 7). I n s a m p l e s c o m p r e s s e d a t 0.1 M P a a n d a b o v e , a n d t h e n a l l o w e d to r e c o v e r , t h e r a t e of S - G A G r e l e a s e did n o t r e t u r n to c o n t r o l v a l u e s d u r i n g t h e 24 h of r e c o v e r y f o l l o w i n g c o m p r e s s i o n . A t 0.1 M P a , t h e r a t e o f S - G A G release remained at ~ 60% c o n t r o l v a l u e s . H o w e v e r , a t 0.5 a n d 1.0 M P a t h e r e w a s a signific a n t r e c o v e r y of S - G A G r e l e a s e r a t e s , a l b e i t incomplete.

EFFECT OF COMPRESSION ON PROTEOGLYCAN SYNTHESIS RATES WITH DEPTH I n c o n t r o l s a m p l e s , 35SO4 i n c o r p o r a t i o n r a t e s ( n o r m a l i z e d to t i s s u e S - G A G c o n t e n t ) w e r e h i g h e s t in t h e s u r f a c e l a y e r o f t h e t i s s u e as c o m p a r e d t o t h e m i d d l e a n d d e e p l a y e r s (Fig. 4(d)). I n t h e c o m p r e s s i o n e x p e r i m e n t s , t h e d e c r e a s e d 35SO4 incorporation rates were observed throughout the d e p t h of t h e t i s s u e (Fig. 8(a)). T h e suppression in s y n t h e s i s r a t e s s e e m e d to be g e n e r a l l y g r e a t e r in

98

Guilak e t a l . : Matrix c o m p r e s s i o n and p r o t e o g l y c a n m e t a b o l i s m

150 ](a)

/00 r 0

0.1 MPa

0.5 MPa

1.0 MPa

3OO (b

~

250 200

~

150 100 50

0

0.1 MPa (Recovery)

1.0 MPa 0.5 MPa (Recovery) (Recovery)

FIG. 8. Zonal variations in the biosynthetic response to compression (a) and recovery following compression (b). Values represent 35SO4 incorporation rates in compressed tissue normalized to ~5SO4 incorporation rates in the same zone of control tissue. While increased suppression was consistently observed in the surface and deep zones, this trend was not statistically significant (P > 0.05). With recovery, no zonal patterns were apparent in the alterations of proteoglycan synthesis rates. Values show mean_+s.D., N = 10-12, *P < 0.05 vs other zones. [2]: surface zone; m: middle zone; ~]: deep zone; []: whole tissue.

the surface and deeper layers as com pa r ed to the middle layer; however, this t r e n d was n o t statistically significant. T h e one except i on to this finding was at 1.0 MPa, w he r e t h e synthesis r a t e in t he surface zone was significantly lower t h a n t h a t of the middle or deep zones ( P < 0.05). T he changes in 35SO4 i n c o r p o r a t i o n r at es observed following r e c o v e r y from compression did not v a r y significantly t h r o u g h the dept h of t he tissue (Fig. 8(b)). Discussion

Def o r matio n of t he e x t r a c e l l u l a r matrix, and t h e associated changes in tissue h y d r a t i o n , macrom olecu lar c o n c e n t r a t i o n , i n t e r s t i t i a l pH and cell shape h a v e been suggested as m e c ha ni sm s by which th e c h o n d r o c y t e s m a y per c e i ve changes in their mech an ic al e n v i r o n m e n t u n d e r static compression [6, 9,12, 13,23]. In t he unconfined

compression configuration, the d e f o r m a t i o n field within t he expl ant is highly n o n u n i f o r m at the onset of loading, but as creep progresses with time, an equilibrium state is r e a c h e d as i n t e r s t i t i a l fluid flow ceases r~m ~ ~" " , ~qu,,,,Hurn, __. :,_.L_:_.. the [u,j. At m~clmnica~ effects of m at ri x d e f o r m a t i o n can be studied w i t h o u t t he c o n f o u n d i n g f a c t o r s caused by fluid flow, h y d r a u l i c fluid pressure, s t r e a m i n g potentials or o t h e r k i n e t i c phenomena. At lower stresses, w here assumptions of l i n e a r elasticity and infinitesimal s t r a i n are still valid ( < 20% strain), t he equilibrium s t r e s s - s t r a i n distrib u tio n is n e a r l y uniform t h r o u g h o u t t he radius up to a t hi n b o u n d a r y l ayer n e a r t he p e r i p h e r y of the specimen and n e a r t he tissue-filter i n t e r f a c e [36]. At h i g h e r stresses w hi ch resul t in finite deformation of t he m a t r i x , t he st rai n d i s t r i b u t i o n within the tissue is more inh0mogeneous, c h a r a c t e r i z e d by h i g h e r d i l a t a t i o n and s h e a r strains at the t i s s u e - f i l t e r i nt erface due to radial expansion and frictional effects from t he porous filters (F Guilak and J K Suh, unpublished results). Because complex stress and s t r a i n fields are developed within t he explants, i n t e r p r e t a t i o n of changes in metabolic act i vi t y due to compressive loading is difficult since it is n o t k n o w n which of the m echani cal l y-i nduced signals the c h o n d r o c y t e s are a c t u a l l y responding to, even in the equilibrium condition. Our results showed a decrease in p r o t e o g l y c a n synthesis and p r o t e o g l y c a n release ra te s with increasing compressive stress, similar to previ ousl y r e p o r t e d results [6, 9, 11, 12, 14]. These findings are consi st ent with several possible m e c h a n i s m s of cel l ul ar t r a n s d u c t i o n , including decreased i nt erst i t i al w a t e r content, in c re a se d c o n c e n t r a t i o n s of tissue m a c r o m o l e c u l e s and a l t e r a t i o n s in the p e r i c e l l u l a r pH [6, 9, 12, 13]. This hypot hesi s is consistent with t he o b s e r v a t i o n t h a t a decrease in p r o t e o g l y c a n c o n t e n t of th e tissue results in increased p r o t e o g l y c a n synthesis rates [37]. T hese findings suggest t h a t c h o n d r o c y t e s m a y have t he ability to utilize load-induced changes in t he p h y s i c o c h e m i c a l e n v i r o n m e n t of t he tissue as a r e g u l a t o r y signal for c o n t r o l of t h e i r metabolic activity, and ultimately, t he composition of t h e i r s u r r o u n d i n g e x t r a c e l l u l a r m a t r i x [18]. M e c h a n i c a l l y - i n d u c e d a l t e r a t i o n s in chondrocyte shape h a v e also been proposed as a possible r e g u l a t o r of cartilage a c t i v i t y [23, 38-40]. In cell c u l t u r e studies, c h o n d r o c y t e shape has been show n t o play a role in gene expression, and cell size as well as spheri ci t y h a v e been associated with chondrogenesi s [41]. Cellular d e f o r m a t i o n has been hypothesized to r e g u l a t e metabolic a c t i v i t y t h r o u g h several possible mechanisms

Osteoarthritis and Cartilage Vol. 2 No. 2

[42, 43], such as stretch-activated ion channels [40, 44] and cytoskeletally-mediated deformation of intracellular organelles [45]. While previous studies have shown that chondrocytes undergo both shape and volume changes during compression of the extracellular matrix [39], the p a t h w a y through which these phenomena are transduced to a biochemical signal which regulates specific gene transcription is yet to be determined. Cartilage explant recovery following removal of load after a period of compression also showed a dose dependency effect for both synthesis and proteoglycan release. In previous studies, suppression of proteoglycan synthesis rates was noted in the first 4-5 h following removal of compression [7, 10], followed by increased synthesis rates from 8-48 h following removal of compression [9, 10]. The findings of the present study are consistent with this 'rebound' phenomenon, which was observed 20-24 h following removal of compression at stresses of 0.1-0.5 MPa. Although previous studies have noted an increase in proteoglycan release rates during recovery from static compression [9, 11], in this study continued suppression of proteoglycan release was shown. One potentially important difference is that previous investigations have studied the release of newly synthesized proteoglycan, whereas our study examined the effect on the total proteoglycan population At 1.0 M P a compression, proteoglycan synthesis rates were greatly inhibited, and removal of this stress for 24 h did not result in complete recovery. Incomple6e reswelling of the tissue following this level of stress implies that u l t r a s t r u c t u r a l damage may have occurred to the extracellular matrix and/or to t h e chondrocytes, and may indicate a sustained decrease in tissue pore size after removal of compression. While chondrocytes have been shown to recover their morphology following 20-30°//o compressive strain [23, 38, 39], preliminary~ studies in: our l a b o r a t o r y suggest that at this higher level of strain, a decrease in cell viability may have been responsible for the continued suppression of proteoglycan synthesis rates. One suggested mechanism for these observed effects is that compression of cartilage will impede solute and proteoglycan diffusion in cartilage, as the diffusion coefficient of small solutes in the cartilage matrix has been shown to depend on both interstitial ion concentration and matrix porosity [24, 25]. For uncompressed explants, the diffusion time constant for small solutes is related to the square of the characteristic distance (tissue thickness) [25, 31]. For explants which are ~ 1 mm thick, this time constant will be of the order of

99

several minutes, which will have little effect on the labeling procedure [7, 25], even in compressed tissue [12]. As regards proteoglycan degradation, the continued suppression of S-GAG release on removal uL ~he compressive load at ~ high end ne our loading range suggests that altered diffusion rates were not responsible for the observed effects. Rather, this finding suggests that an alternative, chondrocyte-mediated mechanism may have been involved at 0.1-0.5 MPa, where nearly complete reswelling of the tissue occurred. The mechanism used for the b r e a k d o w n :of aggregating proteoglycans in cartilage is likely to be, at least in part, caused by the activities of matrix metalloproteinases that are secreted by the cells [46]. A molecular mechanism that results in proteoglycan loss from cartilage is a cleavage of the protein core of aggregating proteoglycan between the G1 domain (involved in aggregation) and the glycosaminoglycan containing region [16, 17, 47, 48]. Thus it seems likely that compression of the tissue at intermediate stress levels (0.1-0.5 MPa) results in a cellular response which reduces the rate of this mechanism, possibly by reducing the synthesis or activation of the metalloproteinases. At higher stresses (1.0 MPa), there is potential for physical damage of the cells, which would result in the release of proteases from the cells into the extracellular matrix and thus lead to accelerated proteoglycan release. Although a suppression of proteoglycan release was still detected at 1.0 MPa, the true effect of reduced specific proteoglycan b r e a k d o w n may be somewhat masked by the potentially increased release of proteases from lysed cells. Interestingly, we found that the changes in the rate of proteoglycan synthesis as caused by equilibrium compression did not vary significantly with depth through the tissue. Yet the structure and composition of the extracellular matrix, as well as the biosynthetic rates, morphology and density of the chondrocytes, do vary at different depths in the cartilage, especially in adult tissue [25, 26, 29]. Cartilage permeability [25] and tensile properties [27] also vary with depth, and there is evidence that, in adult tissue, the compressive modulus is lower in the surface and deep zones as compared to the middle zone [28]. Further, at higher applied stresses, frictional effects will result in larger strains at the interface b e t w e e n the tissue and the loading platen. In nearly all samples compressed at 0.1 and 0.5 MPa, a w e a k b u t consistent trend of higher suppression in the surface and deep zones was observed. These findings together support the notion that increased strain or dilatation in the surface and deep zones m a y be responsible for the

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Guilak et al.: Matrix compression and proteoglycan metabolism

observed effects. At 1.0 MPa, the significant suppression of 35S04 i n c o r p o r a t i o n rates of the s u r f a c e zone seems to be due to cell necrosis and tissue damage at the surfaces in c o n t a c t with the porous loading platen. While specific zonal v a r i a t i o n s in t he composition and biosynthetic r at es of t h e c o n t r o l tissue were observed with depth, these p a t t e r n s did not c o r r e l a t e with t he observed p a t t e r n s of suppression of p r o t e o g l y c a n synthesis rates in the compressed tissue. These findings suggest that, in y o u n g cartilage, tissue composition is not a strong d e t e r m i n a n t of c h o n d r o c y t e response to load. Alternatively, the zonal v a r i a t i o n s in t he properties of the e x t r a c e l l u l a r m a t r i x and the chondrocytes of y o u n g car t i l a ge m a y be such t ha t , u n d e r static compression, the m e c h a n i c a l e n v i r o n m e n t of the c h o n d r o c y t e remains r e l a t i v e l y uniform in the different zones. In previous studies, it has been shown t h a t c h o n d r o c y t e response to compression is significantly g r e a t e r in t he middle-zone of adult a r t i c u l a r cartilage, w he r e tissue fixed charge density is th e highest [12]. These differences may be due to the m or e p r o n o u n c e d h e t e r o g e n e i t y of adult cartilage [29] or c h o n d r o c y t e s [26], and thus an i m p o r t a n t c o n s i d e r a t i o n is t h a t t he present study was performed on a r t i c u l a r car t i l a ge from r elativ ely y o u n g animals (4-5 m o n t h s old). Extrapolation of the d a t a to m a t u r e a r t i c u l a r c art i l age should be done with caution, since the activities of c h o n d r o c y t e s from car t i l a ge of different ages can vary. At present, it is u n c l e a r how physiological loading of a r t i c u l a r car t i l a ge will affect t he stress, strain, pressure (hydraulic and osmotic), fluid flow and e l e c t r o k i n e t i c e n v i r o n m e n t a r o u n d the individual chondrocyte, or how individual c h o n d r o c y t e s metabolically respond to c ha nge s of these physical quantities. One possibility is to exam i ne changes in the c h o n d r o c y t e m e c h a n i c a l e n v i r o n m e n t and response to m e c h a n i c a l s t i m u l a t i o n on a single cell basis [23, 40]. With this know l edge one m ay hypothesize t h a t physiological changes in quantities such as i n t r a c e l l u l a r strain, m e m b r a n e stretch, fluid s h e a r stresses or changes in t h e pericellular fixed charge density m a y serve as signals to m o d u l a t e c h o n d r o c y t e m et abol i c response. These precisely defined physical q u a n t i t i e s (and others) offer new a ve nue s in s e a r c h of the m e c h a n i c a l and p h y s i c o c h e m i c a l t r a n s d u c t i o n signals responsible for m o d u l a t i n g c h o n d r o c y t e activity. Acknowledgments We wish to t h a n k Ms F a t e m e h Saed-Nejad and Ms Wenda S h u r e t y for t h e i r e x c e l l e n t t e c h n i c a l

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