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The connective tissue response to immobility: An accelerated ageing response?
Exp. Geront. Vol. 3. pp. 289-301. Pergamon Press 1968. Printed in Great Britain
t22
T H E CONNECTIVE T I S S U E RESPONSE TO I M M O B I L I T Y : AN ACCELERATED A G E I N G RESPONSE? W. H. AKESON,*D. AMIEL,D. LAVIOLETTEand D. SECRIST Department of Orthopedics, University of Washington, School of Medicine, Seattle, Washington 98105, U.S.A. (Received 26 February 1968) INTRODUCTION THERE IS a strong human intuition that exercise is good for us. Beneficial effects of such physical activity as competitive sports, hiking, running and climbing have been accepted since the beginning of history. Similarly, restricted activity has been thought to have detrimental effects on musculoskeletal physiology. Details of such effects are not known except that severely reduced activity produces muscle wasting and acute osteoporosis. Ageing is associated with some superficially similar changes in the appendicular skeleton. In particular, muscle wasting and osteoporosis are commonly observed in older individuals. The acute response of fibrous connective tissue to immobility has been described in a model system consisting of the immobilized canine knee (Akeson, Amiel and LaViolette, 1967) (Akeson, 1961). Collagen turnover was not accelerated in this system and total collagen and saline soluble collagen were not increased on the immobilized side (Brooke and Slack, 1959) (Slack, 1954). The single exception to this was described by Peacock who stated that immobilization of the dog by internal fixation in the manner described by the present author resulted in increased collagen concentration in the popliteal connective tissue (Peacock, 1963). Peacock stated that contractures were the result of increased collagen synthesis occurring at strategic sites. Work by the present authors demonstrated that water concentration, hyaluronic acid and chondroitin-4 and 6-sulfate concentration were appreciably reduced in immobilized fibrous connective tissue (Akeson, Amiel and LaViolette, 1967; Akeson, Amiel and LaViolette, 1966). The relationship between these changes in water and acid mucopolysaccharide concentration and the resultant contracture has raised interesting questions about the functional role of water and acid mucopolysaccharides as plasticizers of collagen matrices, but a conclusive definition of the functional interaction between collagen, water and acid mucopolysaccharide changes in the system is not possible at the present time. The relationship of the acute immobility to ageing is raised on two principle points. In certain tissues a change in the "gel-fibre" ratio occurs with ageing (Sobel and Marmorston, 1956; Boas and Foley, 1954; Hallen, 1958; Davison and Small, 1963). It is also generally accepted in clinical orthopedics that immobilization of extremities in the aged is apt to result in contractures more frequently, after less trauma, and after shorter periods of time than in younger individuals. Supported by: National Institutes of Health Grant No. AM-06363. * Associate Professor, Department of Orthopedics. 289
290
W. H. AKESON, D. AMIEL, D. LAVIOLETTEAND D. SECRIST
T h e p r e s e n t work was u n d e r t a k e n to s t u d y the q u e s t i o n of the c o n n e c t i v e tissue r e s p o n s e to i m m o b i l i t y i n greater detail, a n d i n p a r t i c u l a r to e x a m i n e w h e t h e r the resp o n s e is a generalized p h e n o m e n o n c o m m o n to all fibrous c o n n e c t i v e tissue structures, e.g. t e n d o n s , l i g a m e n t s , capsule, or w h e t h e r it is restricted to specific fibrous c o n n e c t i v e tissue s t r u c t u r e s , as Peacock postulated.
METHODS
AND
PROCEDURES
A n i m a l s used i n this s t u d y were m o n g r e l dogs of a d u l t size b u t of u n k n o w n age. Stainless steel t h r e a d e d wires u s e d for i n t e r n a l fixation were ~ in. i n diameter. T h e y were of a s t a n d a r d t y p e u s e d i n o r t h o p e d i c s u r g e r y a n d were o b t a i n e d f r o m the Z i m m e r M a n u f a c t u r i n g Co. C h e m i c a l s for analytical use were as described in a r e c e n t p a p e r (Akeson, A m i e l a n d LaViolette, 1967).
Animal preparations T h e experimental model used for the present study is one that has been developed by us. It provides immobilization by internal fixation of the knee by two threaded wires. The wires are inserted through the proximal tibia into the femur. The course of the wires is inside the skin fold, behind the knee. They are placed as far posterior to the posterior capsule as possible and still remain within the skin fold (Akeson, Amiel and LaViolette, 1967). After a 10- to 12-week period of immobility, the animals were killed by an overdose of intravenous nembutal. Animals which did not maintain their preoperative weight, animals in whom internal fixation failed, or animals in whom infection occurred were not used in this study. Twelve animals were operated for the first portion of this study. Of this group, eight animals were successfully followed for the immobilization period. After the animals were killed, control and immobilized knees were dissected in a standard way. The periarticular connective tissue was separated anatomically as follows. T e n d o n structures included the quadriceps and patellar tendons. The sample labeled anterior capsule and synovium included the fibrous reinacula extending on either side of the quadriceps and patellar tendons as well as the capsule itself and the underlying synovial membrane. This sample was triangular and extended about two-thirds of the way toward the posterior aspect of the knee joint on either side. The medial and lateral collateral ligaments are discrete structures which were dissected cleanly from their proximal and distal attachments to bone. Cruciate ligaments are also very discrete structures which were easily detached from their origin and insertion. The sample labeled posterior capsule was prepared as follows. The medial and lateral margins of this sample were defined as the portion of the capsule between the medial and lateral sesamoid bone on the posterior aspect of the knee. The proximal attachment was to the femur and the distal attachment to the tibia. This specimen was roughly rectangular. Care was taken with this specimen not to include any structures in the popliteal fossa. The region of the popliteal fossa contains the threaded wires and a certain amount of scar tissue which inevitably develops secondary to trauma resulting from insertion of the threaded wire. Skin, bone, articular cartilage, fibrocartilage and muscle were discarded. The final samples from the control and immobilized knees included (1) quadriceps and patellar tendon, (2) anterior capsule and synovial membrane, (3) posterior capsule, (4) collateral ligaments, and (5) cruciate ligaments. These structures were placed in individual vials of acetone for 4 days at 4°C with several changes of acetone. The tissue was air dried and ground in a Wiley Mill to pass through a 60 mesh screen. T h e fat was extracted with a 50/50 mixture of acetone-ether for 24 hr. The tissue was again air dried and then dried to constant weight at 80°C and processed in a manner detailed below. T e n animals were operated in an identical manner for the purpose of determining limiting viscosity numbers of the hyaluronic acid fraction. Seven of these animals were followed successfully for 10-12 weeks. The animals were killed as described above. Periarticular connective tissue of the knee joints of control and immobilized knees was dissected as described in a previous paper (Akeson, Amiel and LaViolette, 1967). The sample included collateral ligaments, capsule, synovial membrane, cruciate ligaments, quadriceps tendon, patellar tendon and tendons of origin
CONNECTIVE TISSUE RESPONSE TO IMMOBILITY:
AN ACCELERATEDAGEING RESPONSE?
291
of the gastrocnemius muscle, all in one vial. This type of dissection and pooling was necessary in order to obtain enough tissue to provide a hyaluronic acid fraction sufficiently large for viscosity studies. Subsequent treatment of the tissue was identical to that described above.
Crude acid mucopolysaccharide extraction After an aliquot of the dry fat-free material was taken for total hexosamine and total collagen determination, the rest of each dried sample was weighed and suspended in 20 ml acetate buffer and 2 mg papain per gram of sample as previously detailed. Digestion was accomplished in 30 hr. Subsequently, the sample was cooled to 4°C and cold trichloroacetic acid was added to 10 per cent final concentration. Celite was added and the sample was filtered through a M fritted disc. T h e precipitate was washed with cold 10 per cent v/v trichloroacetic acid. T h e clear filtrate was dialyzed, concentrated in a rotary evaporating flask and passed over Dowex-50 8X 200-400 mesh (H ÷ ) as previously described (Akeson, Amiel and LaViolette, 1967). T h e crude A M P * collected in the water effluent from the Dowex columns was neutralized with 1 N N aO H , concentrated in a rotary evaporating flask, dialyzed, concentrated again to a final concentration of 1 mg AMP/m1 and an aliquot of the final crude A M P extract was taken for the purposes of uronic acid and hexosamine analysis before column separation on cellulose-CPC.~
Cellulose-CPC microcolumn procedure T h e crude A M P was fractionated on cellulose-CPC microcolumns (Antonopoulos, Gardell, Szirmai and DeTyssonsk, 1964). Minor modifications of the technique have been recently described (Akeson, Amiel and LaViolette, 1967). A 40/zl extract of crude acid mucopolysaccharide containing 3 mg of A M P / m l was applied to the top of each cellulose column. T h e A M P was solubilized from the column using stepwise increments of salt concentration starting with 1 per cent CPC and following with 0" 3 N NaC1, 0" 9 M MgC12 in 0" 1 N acetic acid, 0" 75 M MgCI2 in water. All of the salt solutions were made up in 0"05 per cent CPC. 6 - N HC1 was used to elute any remaining A M P at the conclusion of the run. Uronic acid was determined by the borate procedure (Bitter and Muir, 1962). T h e 6 N HC1 fraction was first dried in a vacuum desiccator over soda lime. When dry, 1 ml of water was added and the uronic acid was determined as above. Standard glucuronolactone was made up in each of the salt solutions for comparison with the unknown in the respective salt fractions. Similarly, blanks were made using 1 ml of each of the salt solutions for comparison with the standard and unknowns in the respective salt solutions. T h e microcolumn fractionation procedure was run in triplicate on each crude A M P extract. Agreement within 5 per cent was usually obtained. If not, the procedure was repeated.
CeUulose-CPC macrocolumn fractionation T h e validity of the microcolumn separation depends upon characterization of each fraction on a macro scale as a preliminary step. This characterization has previously been performed for the tissue under discussion (Akeson, Amiel and LaViolette, 1967) and was not repeated at this time. Previous characterization included uronic acid by the borate and Dische methods expressed as molar ratios to hexosamine, sulfate expressed as a molar ratio to hexosamine, electrophoresis on Sepraphore III, paper chromatography and Gardell column chromatography of hexosamine, turbidity reduction and infra-red spectra. On the basis of these procedures, it was concluded that the 0.3 M NaC1 fraction contained chiefly hyaluronic acid, the 0.9 M MgC12 in 0.1 M acetic acid contained chiefly chondroitin-4 and 6-sulfate and the 0.75 M MgC12 fraction contained chiefly dermatan sulfate.
Analytical procedures Details have been given in recent reports (Akeson, Amiel and LaViolette, 1967; Akeson, Amiel and LaViolette, 1966). Uronic acid was estimated according to the Dische procedure (Dische, 1947) and also by the borate procedure (Bitter and Muir, 1962). Tw o aliquots of the dried, ground material from each sample were weighed and hydrolyzed in a sealed tube. Hydrolysis for hexosamine was accomplished in 6-N HC1 in a 100°C oven for 5 hr. Subsequently, the acid * Acid mucopolysaccharide. t Cetyl pyridinium chloride.
292
W. H. AKESON, D. AMIEL, D. LAVIOLETTE AND D. SECRIST
was quickly evaporated over N a O H and calcium chloride in vacuo. T h e dried hydrolysate was dissolved in water and hexosamine was estimated by the procedures of Boas (Boas, 1953). Hydroxyproline was determined by the method of Woessner (Woessner, 1961). 5 mg dry material was weighed and hydrolyzed in 6 N HCI for 3 hr at 130°C.
Limiting viscosity numbers T h e limiting viscosity numbers were determined in a Cannon-Ubbelohde semi-micro dilution viscometer at 20°C using a 0.01 M phosphate buffer in 0.38 M NaCI at p H 7.0.
RESULTS
Total amount of tissue T h e total f a t - f r e e d r y w e i g h t of t h e d i s s e c t e d s t r u c t u r e s f r o m c o n t r o l a n d i m m o b i l i z e d l i m b s is g i v e n in T a b l e 1. M e a n v a l u e s a n d s t a n d a r d d e v i a t i o n s a r e g i v e n f o r the posterior capsule, collateral ligaments, cruciate ligament, anterior capsule and p a t e l l a r a n d q u a d r i c e p s t e n d o n . T h e r e was n o significant d i f f e r e n c e b e t w e e n t h e c o n t r o l a n d i m m o b i l i z e d side in a n y of t h e s e g r o u p s . T h i s s u g g e s t s t h a t significant a t r o p h y o f t h e f i b r o u s c o n n e c t i v e t i s s u e s t r u c t u r e s a b o u t t h e i m m o b i l i z e d k n e e d i d n o t occur.
Total collagen and total hexosamine T h e v a l u e s for t o t a l c o l l a g e n in t h e v a r i o u s s t r u c t u r e s f r o m c o n t r o l a n d i m m o b i l i z e d k n e e s are g i v e n in T a b l e 2. N o significant d i f f e r e n c e was o b s e r v e d b e t w e e n c o n t r o l a n d TABLE 1. TOTAL FAT-FREE DRY WEIGHT (g) OF DISSECTED STRUCTURES
Dog J1 Jz J4 J~ K7 Ks K9 K10
Mean
Posterior Capsule
Collateral Ligament
Cruciate Ligament
Anterior Capsule
Patellar Quadriceps Tendon
Control Immobilized Control Immobilized Control Immobilized Control Immobilized Control Immobilized Control Immobilized Control Immobilized Control Immobilized
0" 870 0- 500 0" 840 1" 180 0" 900 0" 580 0"660 0- 600 0- 405 0- 308 0"535 0" 328 0"461 0" 445 0" 544 0" 643
0" 173 0' 203 0" 234 0" 184 0' 134 0' 137 0"144 0" 131 0" 094 0" 105 0"092 0" 074 0' 078 0" 064 0" 106 0" 099
• 123 0" 109 0" 132 0" 127 0' 056 0" 063 0"077 0" 075 0" 057 0" 057 0"059 0" 057 0" 045 0" 055 0"096 0- 076
1" 430 0" 770 1 •480 1" 780 1-480 1" 930 2'190 1' 480 0" 931 1 •472 0-880 1" 380 1" 014 1" 380 1" 122 1 •234
1' 500 1" 700 1"450 1" 610 1" 520 1" 460 1-430 1" 460 0- 990 1" 361 1-011 1" 115 0" 993 0" 977 1" 133 1- 610
Control SD
0'652 0"195
0"132 0'052
0.081 0"033
1"316 0-431
1-253 0.242
Immobilized SD
0"573 0"274
0.125 0"050
0"077 0'027
1"428 0"350
1"412 0"252
Knee
Values
T h e r e was no significant difference in the amount of fat-free tissue on the two sides in any of the tissues studied.
293
CONNECTIVE TISSUE RESPONSE TO IMMOBILITY: AN ACCELERATED AGEING RESPONSE~ TABLE 2. TOTAL COLLAGEN IN STRUCTURES FROM CONTROL AND IMMOBILIZED TISSUE
Dog
Knee
Posterior Capsule
Collateral Ligament
% mg/g~
Control
Changet
533
J1
%
553 544
Immobilized Control
545 383
Immobilized Control
497 612
Immobilized Control
572 595
Immobilized Control
637 530
Immobilized Control
613 601
Immobilized Control
493 670
Immobilized
637
J2
K~
893
855
612
813
+ 1.6
-
6.5
-
-
-
+ 7.0
--12.2 678 776
+16.8
1-6
-
-- 9.1
-- 2.3
3.2
751 673
529 738
862
+12.0 786 772
670 582
824 882
873
6.4
+21.5
-1-9
-- 2.0 871 816
-
738 582
826 837
+29.0 837 702
554 579 + 3.2
+ 6.4 887 889
-15.5
4.8
865 842
+ 9-6 728 649
500 592 -
7-4
-11.1
7.7
824 838
-15.7 685 664
570 582 -
3"1
834 836
-- 4.9
Control SD Mean Values Immobilized SD
+ 2.1
741 866
925 901
+ 3.4 633 641
887 803 + 0.3
-18.0
K10
2.7
916 955
+15-7
K9
+ 6.2 908 869
843 913
+ 7.1
Ks
% mg/g* Changer
907 866
-
% mg/g* Changer
+29.8
J6
%
Patellar & Quadriceps Tendon
mg/g* Changer
+ 0.2
J4
Anterior Capsule & Fascia
mg/ge Changer
+ 3.8 Immobilized Control
Cruciate Ligament
+10.8 746 794
-
3.3
691
-- 6-0 746
558.5 85.28
883.6 43.96
849.0 24-62
613.5 54.63
730.4 65.26
568.4 57.30
882.0 33.13
842.1 51.29
610.6 84.91
744.6 51-49
* Results expressed as mg/g fat-free dry weight. t Immobilized-Control × 100. Control No significant difference was observed between control and immobilized sides in any of the groups. i m m o b i l i z e d sides i n a n y of the s t r u c t u r e s studied. T o t a l h e x o s a m i n e for the c o n t r o l a n d i m m o b i l i z e d s t r u c t u r e s is g i v e n i n T a b l e 3. T h i s v a l u e was o b t a i n e d o n dry, fat-free material p r i o r to A M P extraction. T h e i m m o b i l i z e d tissue h a d a significantly lower c o n c e n t r a t i o n of h e x o s a m i n e i n all cases. T h i s c o n f i r m s p r e v i o u s results o n pooled tissue f r o m c o n t r o l a n d i m m o b i l i z e d tissue (Akeson, A m i e l a n d LaViolette, 1967) a n d suggests that the r e s p o n s e seen i n r e d u c t i o n of h e x o s a m i n e o n t h e i m m o b i l i z e d side is a u n i f o r m r e s p o n s e t h r o u g h o u t the tissues s t u d i e d .
294
w.H.
AKESON, D. AMIEL, D. LAVIOLETTE AND D. SECRIST
TABLE 3. TOTAL HEXOSAMINE IN STRUCTURES FROM CONTROL AND IMMOBILIZED KNEES
Dog
Knee
Posterior Capsule~
Collateral Ligament~
% nag/g* Change?
Control
4"8
J,
%
4"1 4"4
Immobilized Control
2"8 2"7
Immobilized Control
2.7 3"1
Immobilized Control
2"6 3"2
Immobilized Control
2"8 2.8
Immobilized Control
2"2 3,2
Immobilized Control
3"1 4"3
Irnmobilized
3 "1
J2
5.0
7" 3
3 "7
4.1
~ 2"0 5"7 5'7 -
2"0
-31"6
-37"1
-12"5
-21"4
-15.4 2'2 7'6
K9
-
3'1
43"4
K10
- 18 '6
5"9
-18"6
-20"6
- 4 3 -5
17"1
3.5 4"9
2.7 3-6
3 "5
-
3"4 4"3 -
-22"7
- 36"8 2'4
31 "0
3"2 3'4
3"4 6.2
-41"8 3'2 4"1
-
-35"3
4"3 3-8
3'4
2-9 3'4
3-3 4"4
-19.1 3"8 5"5
-
-40"9
-25'0
3.6
2"8 4"2
3"9 5'1
9"8
3 "9 4"7 -
-32'7
-44'2
5.9
2'7 2"9
3"5 6'6
2"9 2"6
Ks
-
-48'6
3"7 5"2
3.2 2'8
3"6 5'2
3'5 5"2 12"5
-18'9 3"0 3"4
3 '9 7"0
2"2 4'0 -16"1
Control SD Mean Values Immobilized SD
-21"9
5 -0 3"5
-
% rag/g* Change?
0
K7
% mg/g* Change?
-36"4
J6
%
Patellar & Quadriceps Tendon §
mg/g* Change?
5"1 5'1
J4
Anterior Capsule & Fascia~
rag/g* Change?
--14"6 Immobilized Control
Cruciate Ligament §
--18"4 4'0 4"1
- 16"7 3 '0
- 17" 1 3 '4
3"563 0.809
4'60 1.511
5"938 1.011
3"425 0"443
4"613 0'549
2"925 0"555
3"45 1-218
3"850 0'778
2'938 0"200
3"613 0.280
* Results expressed as mg/g fat-free dry weights. t Immobilized-Control ×
100
Control
P < 0.05. § P < 0.01. The immobilized knee was significantly different from the control knee in all cases.
Acid mucopolysaccharide content T h e acid m u c o p o l y s a c c h a r i d e c o n t e n t of c o n t r o l a n d i m m o b i l i z e d s t r u c t u r e s is g i v e n i n T a b l e s 4 - 8 . I n T a b l e 4, the A M P c o n t e n t of the patellar a n d q u a d r i c e p s t e n d o n is detailed. Values are p r e s e n t e d for the c r u d e A M P c o n t e n t a n d the A M P c o n t e n t i n 0.3 M NaC1, 0.9 M MgCI2 i n acetic acid a n d 0.75 M MgCI2 a q u e o u s solution. T h e o t h e r fractions did n o t c o n t a i n significant a m o u n t s of A M P and, therefore, are n o t recorded. M e a n values of the A M P c o n t e n t of the c r u d e material a n d of the various fractions are g i v e n at the foot of the table. All values are significantly r e d u c e d o n the
CONNECTIVE TISSUERESPONSE TO IMMOBILITY:AN ACCELERATEDAGEINGRESPONSE.~
295
TABLE 4. ACID MUCOPOLYSACCHARIDECONTENT OF CONTROL AND IMMOBILIZED PATELLAR AND QUADRICEPSTENDONS Dog
Knee Control
Crude AMP % 0.3 M before Changer NaCI* Fractionation* 2"66
J1
% Changer
0"93 -33.8
0"9 M % 0"75 M % MgCI~ Changer MgCI2* Changer in HAc* 1 "05
-46"2
1 "76 2.45
Immobilized Control
2-10 2"79
Immobilized Control
2.09 2.41
Immobilized Control
1"38 2.55
Immobilized Control
1 "61 2"11
Immobilized Control
1.69 2.40
Immobilized Control
2.12 3"42
Immobilized
2.34
0"76
0.93
0'48
2"60 0"388
0.867 0.155
1-050 0.166
0.528 0.117
-14"3
J4
-20"0 0"52 0"90
-25.1
J6
Ks
K10
-
-- 9-7
-33"9
-14"6 0"41 0"73
-30"1
--35"8
-34"2
--24"9 0.788 0.170
3"0
0"34 0"48
0.84 1 "33
0"555 0"129
- 34"6 0"34 0"33
-23.0
-23"7
--27.0 1 "89 0"324
- 35.9
0,67 0,93
0"71 1"15
-38.0 0"31 0.52
0.66 0,87 -28.4
-31"6
Control SD Mean Values Immobilized SD
-37"8
- 45 "3
0'58 0'93
- 13.5 0'45 0'50
0,56 1 '03
0"47 0"81
-37"5 0"40 0'52
-21.4
-48"6
-11"7
3-9
0"99 0,90
0"36 0.86
-19"9
K9
-
-40.0
- 36"9
0"34 0"64
0"99 1 '26
0.54 0-70 -42"7
K7
0"66 1 "03
-32.0
Immobilized Control J~
0"50 0"65
0"50 -37"1
--25.2 0"384 0-061
* Results expressed in micrograms of uronic acid per gram of fat-free connective tissue. t Immobilized-Control x 100 Control All values were significantly different P < 0"01. e x p e r i m e n t a l side. T h i s f i n d i n g is i n c o n t r a d i c t i o n w i t h p r e v i o u s results o n pooled m a t e r i a l w h e r e a l t h o u g h r e d u c t i o n s were f o u n d i n the 0.75 M MgCI2 f r a c t i o n the c h a n g e s were n o t statistically significant. I n T a b l e 5, t h e A M P c o n t e n t of c o n t r o l a n d i m m o b i l i z e d a n t e r i o r capsule a n d synovial m e m b r a n e is given. Significant r e d u c t i o n i n the A M P c o n t e n t was f o u n d i n the c r u d e m a t e r i a l before f r a c t i o n a t i o n a n d i n the 0"3 M NaC1 a n d the 0.9 M MgC12 i n acetic acid fractions. T h e f r a c t i o n c o n t a i n e d i n the 0.75 M MgC12 i n a q u e o u s s o l u t i o n was n o t significantly different f r o m the c o n t r o l values. T h i s result c o r r e s p o n d s to p r e v i o u s f i n d i n g s w i t h pooled s t r u c t u r e s . T a b l e s 6, 7 a n d 8 c a n n o t b e assessed statistically b e c a u s e of p o o l i n g of tissue f r o m several animals. T h e values for
296
W. H. AKESON, D. AMIEL, D. LAVIOLETTE AND D. SECRIST
T ~ L E 5.
A C I D MUCOPOLYSACCHARIDE CONTENT OF CONTROL AND IMMOBILIZED ANTERIOR CAPSULE AND SYNOVIAL MEMBRANE
Dog
Knee
Crude AMP before Fraetionatione,a~
% Changer
J1 Control
% Changer
SAMPLE LOST 0"40
1.95
J2
0.3 M NaCI *(~
-
32"8
-45
0.9 M MgCI~ in HAc*cbj
0"89
"0
0.75 M % MgCI~*lc~ Changer
0"54 -
37"1
1.31 1 "30
Immobilized Control
0.95 1.33
Immobilized Control
1.12 1.51
Immobilized Control
1.16 1-45
Immobilized Control
1.43 1.72
Immobilized Control
1.24 1.87
Immobilized
1.55
0"40
0"59
0"34
1.50 0.259
0.444 0.139
0.671 0.120
0"373 0.100
-26.9
J6
-32.1 0"19 0.34
-15"8
K7
K9
-
-11"7 -27"9
-10"3 0"26 0"39
-18"1
--31"4 0.299 0"081
+ 6"5 0"49 0"29
0-49 0"72 -32"2
--20"7 1.251 0.200
-- 17"9 0"23 0'46
0-53 0"68 -40-3
--17"1
Control SD Mean Values Immobilized SD
-- 20"3
9"1
0"37 0"59
+10"7 0"31 0.28
0'47 0"60
0-30 0"62 -27"9
K10
+ 7"1
-- 34"5
1"4
+ 2.7 0"38 0"28
0"75 0.59
0.36 0"33 -
-13.5
-26"5
--12.8
--17"4 0"549 0"102
2 5 "9
0"40 0"37
0"45 0"70
0"25 0-55 -- 23 "2
Ks
0"56 0"52
-
Immobilized Control J4
0"22 0.28
% Changer
-- 6"7 0.344 0"088
* Results expressed in micrograms of uronic acid per gram of fat-free connective tissue. t Immobilized-Control × 100. Control (a) All values were significantly different P < 0"01. (b) 0"9 M M g C h values were significantly different P < 0. (c) T h e control and immobilized values were not significantly different.
A M P c o n t e n t o f p o s t e r i o r capsule, c r u c i a t e l i g a m e n t a n d collateral l i g a m e n t s t r u c t u r e s are g i v e n in t h e s e t a b l e s in t h e m a n n e r s i m i l a r to t h e tables p r e v i o u s l y d e s c r i b e d . P o o l i n g was n e c e s s a r y b e c a u s e o f t h e s m a l l a m o u n t s o f tissue available for analysis. T h e results shown suggest that the changes were consistent with those previously noted. The d i r e c t i o n o f c h a n g e s e e m e d t h e s a m e in all cases. T h e n o t a b l e q u e s t i o n arises w i t h r e s p e c t to t h e f r a c t i o n in 0"75 M M g C I 2 in a q u e o u s s o l u t i o n w h e r e in s o m e cases t h e c h a n g e s w e r e g r e a t e r t h a n in others. T h e significance o f this f i n d i n g is u n c e r t a i n at t h e present time.
CONNECTIVE TISSUE RESPONSE TO I M M O B I L I T Y : AN ACCELERATED AGEING RESPONSE~
297
T A B L E 6. A C I D MUCOPOLYSACCHARIDE CONTENT OF CONTROL AND IMMOBILIZED POSTERIOR CAPSULE Dog
Knee
Control
Crude AMP before Fractionation*
% Changet
1.84
J~
0.3 M NaCI #
0"36 - 21 "7
Immobilized Control KT,Ks (pooled) Immobilized Control K%K10 (pooled) Immobilized
% Changet
1.44 1-82 -34.6
--39"0
--36.6
- 28-0 0-36 0-33
-- 9"2
-31"6
% Changer
0"50
0"89 1.21
0-39
0-75 M MgCh ~
- 16"2 0"83 0"98
0"25 0"57
1 "54
% Changet
0"99 - 30" 5
0"25 0"41
1.19 2"43
0.9 M MgCh in HAe#
-- 6-1 0.31 0"55
--31"4 0"83
-- 7"0 0.51
* Results e x p r e s s e d in m i c r o g r a m s of u r o n i c acid p e r g r a m of fat-free c o n n e c t i v e tissue. t Immobilized-Control x 100. Control
T A B L E 7. A C I D MUCOPOLYSACCHARIDE CONTENT OF CONTROL AND IMMOBILIZED CRUCIATE LIGAMENTS Dog
Knee
Control All Dogs Pooled Immobilized
Crude AMP before Fractionation °
% Changet
4"0
0.3 M NaCI #
% Changer
0"89 - 45 "8
2'17
0.9 M MgCh in HAe#
% Changet
2"66 -- 53 "9
0"41
0-75 M MgCh #
% Changet
0"54 - 48" 1
1 "38
- 37 "0 0"34
* Results expressed in m i c r o g r a m s of u r o n i c acid p e r g r a m of fat-free c o n n e c t i v e tissue. t Immobilized-Control x 100. Control
T A B L E 8.
ACID
MUCOPOLYSACCHARIDE CONTENT OF CONTROL AND IMMOBILIZED COLLATERAL LIGAMENTS
Dog
Knee
Control All Dogs Pooled Immobilized
Crude AMP before Fractionationo
% Changer
2"09
0-3 M NaC1#
% Changer
0.57 -13.4
1 "81
0"9 M MgCll in HAc*
1 "8 -33"3
0"38
% Changet
0.75 M MgCla °
0"58 - 1 1 "1
0"96
% Changet
0"0 0-58
* Results e x p r e s s e d in m i c r o g r a m s of u r o n i c acid p e r g r a m of fat-free c o n n e c t i v e tissue. t" I m m o b i l i z e d - C o n t r o l × 100. Control
298
W. H. AKESON, D. AMIEL, D. LAVIOLETTE AND D. SECRIST
TABLE 9.
LIMITING VISCOSITY NUMBERS I/z] OF HYALURONICACID OBTAINED FROM PERIARTICULAR CONNECTIVE TISSUE OF CONTROL AND IMMOBILIZEDKNEES OF DOGS
Animal # 23 24 27 28 29 30 Mean Values
Control 5'39 4.15 2' 90 4" 44 3" 02 2.62 3' 75
Immobilized 2-44 3' 62 2' 70 1 •88 3- 57 3' 21 2' 90
There is no significant difference in the control and immobilized values when paired observations are compared by the T test.
Limiting viscosity numbers The results of viscosity determination are given in Table 9 where they are presented as limiting viscosity number (LVN). The mean value on the experimental side was 23 per cent less than on the control side. However, a significant difference was not observed. A typical plot of the limiting viscosity number for pooled material is given in Fig. 1. ~.©
3'00-
2"50 n-n s nsc 2'00
j/
1.50-
o.65 o.io 0% o.~o o.~5 Concentration ( G m / l O O m I )
FIG. 1.
LVN of hyaluronic acid from control and immobilized canine periarticular connective tissue.
DISCUSSION A superficial similarity between immobility and ageing includes prominent atrophy of bone and muscle in the extremities of older individuals. In addition, certain chemical changes in the gel-fibre ratio have been described in such tissue as skin (Sobel and Marmorston, 1956) (Boas and Foley, 1954) and nucleus pulposus (Hallen, 1958) (Davidson and Small, 1963). However, the nature of the gel-fibre ratio changes is not fully established in human material, particularly in fibrous connective tissue structures and the full
CONNECTIVE TISSUE RESPONSE TO IMMOBILITY: AN ACCELERATED AGEINGRESPONSE?
299
implications of the changes described in the paper as they relate to ageing cannot be appreciated until such studies have been completed. The present study was undertaken to determine whether the connective tissue response to immobility is a uniform response involving all the fibrous connective tissue structures or whether the changes are restricted to strategic sites as Peacock has suggested (Peacock, 1963). The results indicate that the response is indeed a nearly uniform one. There was a uniformly negative finding in so far as total collagen concentration was concerned. No statistically significant changes in collagen concentration were found in any of the structures studied, including the posterior capsule. The results of Peacock which are at variance with these in so far as the structures of the posterior aspect of the knee are concerned are probably best explained as simply an artefact resulting from scar formed secondary to the insertion of the threaded wire in the popliteal region. The tissue sampling in this study was a different one, and attention was paid to including none of the tissue in the immediate vicinity of the threaded wire used for internal fixation. The posterior capsule structures themselves which would be the structures ordinarily involved in a flexion contracture of the knee showed no increase in collagen concentration. All of the structures studied, namely tendon, capsule, collateral ligaments and cruciate ligaments showed changes in the same direction and of the same order of magnitude in so far as total hexosamine and total acid mucopolysaccharide concentration is concerned. The hyaluronic acid and chondroitin-4 and 6-sulfate changes confirm the previous findings on pooled periarticular connective tissue of the canine knee. Dermatan sulfate changes were less uniform. Dermatan sulfate was significantly reduced in patellar and quadriceps tendon but not in the anterior capsule. Statistical analysis was not possible in the case of the ligamentous and posterior capsule structures because pooling was necessary to provide sufficient material for AMP separation. A previous study on pooled periarticular connective tissue showed that dermatan sulfate concentration was reduced, but the change was not statistically significant (Akeson, Amiel and LaViolette, 1967). Presumably, the change in quadriceps and patellar tendon was masked by the pooling procedure. The changes were sufficiently large in the cruciate ligament and posterior capsular material to suggest that they could be statistically significant. The determination of limiting viscosity numbers on a hyaluronic acid fraction showed small changes between the control and experimental sides but these were not statistically significant. These findings suggest that depolymerization of ground substance is probably not an important mechanism in the decrease in AMP concentration observed. Previous studies in which half-time of turnover of hyaluronic acid was determined in this experimental model suggested that accelerated breakdown of hyaluronic acid did not occur. The present findings on limiting viscosity number are consistent with that conclusion. From the available information, the reduced concentration of hyaluronic acid is probably the result of reduced synthesis rather than accelerated breakdown. These findings point to the importance of activity in the maintenance of normal connective tissue homeostasis. They raise questions as to whether certain changes associated with ageing might be secondary to reduced physical stress. The final interpretation of the question of acid mucopolysaccharide and water influence on connective tissue pliability awaits further elucidation of the organization of fibrous connective tissue on a molecular level. In the meantime, considerable work of a descriptive type remains to be done in anticipation of the time when molecular organization is sufficiently understood to relate changes such as those just described to the ageing process as a whole.
300
W. H. AKESON, D. AMIEL, D. LAVIOLETTE AND D. SECRIST REFERENCES
AKESON, W. H. (1961)07" Bone ~ 07oint Surg. 43-A, 1022. AKESON, W. H., AMIEL, D. and LAVIOLETTE, D. (1966) I n Biochimie et Physiologie du Tissue Conjonctlf (Edited by COMTE, P.), pp. 679-689, Societe Ormeco et Imprimerie, Lyon, France. AKESON, W. H., AMmL, D. and LAVIOLETTE, D. (1967) Clin. Orthop. 51, 183. ANTONOPOULOS, C. A., GARDELL, S., SZlRMAI, J. A. and DE TYSSONSK, E. R. (1964) Biochim. biophys. Acta 83, 1. BITTER, T. and MUIR, H. M. (1962) Anal. Biochem. 4, 330. BOAS, N. F. (1953)07. biol. Chem. 204, 553. BoAs, N. F. and FOLEY, J. B. (1954) Proc. Soc. exp. Biol. ivied. 86, 690. BROOKE, J. W. and SLACK, H. G. B. (1959) Ann. rheumat. Dis. 18, 129. DAVlDSON,E. A. and SMALL, W. (1963) Biochim. biophys. Acta 69, 445. DlSCHE, Z. (1947)07. biol. Chem. 167, 189. HALLEN, A. (1958) Acta chem. Scand. 12, 1869. PEACOCK, E. E. (1963) Surg. Forum 14, 440. SLACK,n . G. B. (1954)Biochem. 07. 60, 112. SOBEL, H. and MARMORSTON,J. (1956)07- Gerontol. U , 2. WOESSNER, J. F. (1961) Arch. Biochem. 93, 440. S u m m a r y - - A certain similarity between changes secondary to lack of extremity function and those of ageing has stimulated the present inquiry. The chemical changes occurring in immobility are described and compared with available information on such changes in the ageing process. A change in the gel-fibre ratio was noted to be a uniform process which occurred in all fibrous connective tissue structures of the immobilized canine knee. There was no change in total collagen to support the hypothesis that contractures occur as a result of stimulated collagen synthesis in strategic sites. Acid mucopolysaccharides on the immobilized side were uniformly reduced in terms of concentration of hyaluronic acid and chondroitin-4 and 6-sulfate. Changes in the dermatan sulfate fraction were less consistent. R 6 s u m 6 - - U n e certaine similitude entre les alt6rations consfcutives g u n manque de fonctionnement des extr6mit6s et celles provoqu6es par le vieillissement a conduit la pr6sente recherche. Les modifications chimiques se manifestant en cas d'immobilisation sont d6crites et comp~r6es avec l'information disponible concernant de teUes modifications au cours du processus de vieillissement. On a constat6 qu'une modification de la proportion gel/fibres est un processus g6n6ralis6 se d6roulant dans routes les structures de tissu conjonctif fibreux du genou immobilis6 chez le chien. I1 n'y avait pas de variation du collagbne total pouvant 6tayer l'hypothfise que des contractures seraient la cons6quence d'une stimulation de la synthbse du collagbne g des points strat6giques. Au c6t6 immobilis6, les mucopolysaccharides 6taient g6n6ralement r6duits en ce qui concerne la concentration de l'acide hyaluronique et le sulfate -4 et -6 de chondro/tine. Les variations de la fraction sulfate de dermatan 6taient moins constantes. Z u s a m m e n f a s s u n g - - D i e vorliegende Untersuchung wurde wegen einer gewissen •~hnlichkeit zwischen den Ver~inderungen nach dem Verlust einer Extremit~itenfunktion und den Altersver~inderungen unternommen. Die chemischen ~ n d e r u n g e n bei der Immobilit~t werden beschrieben und mit den vorhandenen Ergebnissen tiber derartige ~ n d e r u n g e n im AltersprozeB verglichen. Die Ver~inderung in dem GelFaser-Verh~iltnis zeigte uniformes Verhalten bei allen Faserstrukturen des Bindegewebes eines stillgelegten Hundeknies. Eine ~ n d e r u n g im Gesamtkollagen war nicht zu sehen, welche die Hypothese gestiJtzt h~itte, dab als Ergebnis angeregter Kollagensynthese an besonders wichtigen Stellen Kontrakturen entstehen. Saure Mukopolysaccharide wie Hyalurons~iure une Chondroitin 4- und 6-sulfat waren auf der stillgelegten Seite in ihrer Konzentration vermindert. Die ,~nderungen in der Dermatansulfatfraktion waren weniger eindeutig.
CONNECTIVE TISSUE RESPONSE TO I M M O B I L I T Y : AN ACCELERATED AGEING RESPONSE? Pe310Me---HacToflLuee HCCJIe~OBaHHC 6a3HpoBa.~OCb Ha TOM, ~ITO cyuleCTByeT HeKOTODOe CXO~CTBO M e ) I ~ y H3MeHeHHflMH~ KOTOpbIe I'IpOHCXO~flT rIocJle HOTepH ~)yHKIII~ B KOHe~IHOo CTflX, H TaKOBbIMH, KOTOpbIe BBI3bIBaIOTC~I ¢TapeHHeM. OIIHCbIBaIOTCR XHMII~IeGKHe H3MeHeHHfl~ CBff3aHI-Ible C HerIO~BH>KHOCTbtO~ H OHH cpaBHHBaIOTCff C I42vfetOHMHCfl ~aHHbIMH OTHOCHTeYlbHO TaKHx H3MeHeHHI~ B n p o t ~ e c c e CTapeHILq. O 6 H a p y ~ o c b , ~ITO OTHOmeHHe reIlbBOYlOKHO gBngeTCH paBHOMepHblM HpoIIeCCOM, KOTOpbll~ n p o H c x O ~ H T BO Bcex BOHOKHHCTblX coe~I4HHTeYlbHblX TKaH~X B HMMO6HJIH3HpOBaHHOM r o n e H e y c 0 6 a r m H e ~bI~lO HnKaKHx H3MeHeHI~ B O611IeM r o Y m a r e H c , KOTOp/~Ie MOFJ/H ~bI IIO~TBep~HTb rHIIOTe3y, corYlacHo KOTOp o ~ KOHTpaKTypbI BbI3bmalOTCg c T r r M y a g t m e ~ CICHTe3a r o y t n a r e n a B cTpaTerHtlecKHx nyHKTaX. H a HMMO6HYlH314pOBaHHOI~ CTOpOHe M y K O n O Y m c a x a p I ~ b I 6bIY[H paBHOMepHo CHH~eHbI B CMbICne romaeHTpamm r H a ~ y p o H o B O f i XHCnOTbI H XOH~pOHTHHa-4 ~ 6 - c y n b q b a T a . I/I3MeHeHI4~I B ~epMaTaH-cyYlbtbaTHOl~ q b p a ~ 6b~nH MeHee 3aKOHOMepHblMtt.
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T H E CONNECTIVE T I S S U E RESPONSE TO I M M O B I L I T Y : AN ACCELERATED A G E I N G RESPONSE? W. H. AKESON,*D. AMIEL,D. LAVIOLETTEand D. SECRIST Department of Orthopedics, University of Washington, School of Medicine, Seattle, Washington 98105, U.S.A. (Received 26 February 1968) INTRODUCTION THERE IS a strong human intuition that exercise is good for us. Beneficial effects of such physical activity as competitive sports, hiking, running and climbing have been accepted since the beginning of history. Similarly, restricted activity has been thought to have detrimental effects on musculoskeletal physiology. Details of such effects are not known except that severely reduced activity produces muscle wasting and acute osteoporosis. Ageing is associated with some superficially similar changes in the appendicular skeleton. In particular, muscle wasting and osteoporosis are commonly observed in older individuals. The acute response of fibrous connective tissue to immobility has been described in a model system consisting of the immobilized canine knee (Akeson, Amiel and LaViolette, 1967) (Akeson, 1961). Collagen turnover was not accelerated in this system and total collagen and saline soluble collagen were not increased on the immobilized side (Brooke and Slack, 1959) (Slack, 1954). The single exception to this was described by Peacock who stated that immobilization of the dog by internal fixation in the manner described by the present author resulted in increased collagen concentration in the popliteal connective tissue (Peacock, 1963). Peacock stated that contractures were the result of increased collagen synthesis occurring at strategic sites. Work by the present authors demonstrated that water concentration, hyaluronic acid and chondroitin-4 and 6-sulfate concentration were appreciably reduced in immobilized fibrous connective tissue (Akeson, Amiel and LaViolette, 1967; Akeson, Amiel and LaViolette, 1966). The relationship between these changes in water and acid mucopolysaccharide concentration and the resultant contracture has raised interesting questions about the functional role of water and acid mucopolysaccharides as plasticizers of collagen matrices, but a conclusive definition of the functional interaction between collagen, water and acid mucopolysaccharide changes in the system is not possible at the present time. The relationship of the acute immobility to ageing is raised on two principle points. In certain tissues a change in the "gel-fibre" ratio occurs with ageing (Sobel and Marmorston, 1956; Boas and Foley, 1954; Hallen, 1958; Davison and Small, 1963). It is also generally accepted in clinical orthopedics that immobilization of extremities in the aged is apt to result in contractures more frequently, after less trauma, and after shorter periods of time than in younger individuals. Supported by: National Institutes of Health Grant No. AM-06363. * Associate Professor, Department of Orthopedics. 289
290
W. H. AKESON, D. AMIEL, D. LAVIOLETTEAND D. SECRIST
T h e p r e s e n t work was u n d e r t a k e n to s t u d y the q u e s t i o n of the c o n n e c t i v e tissue r e s p o n s e to i m m o b i l i t y i n greater detail, a n d i n p a r t i c u l a r to e x a m i n e w h e t h e r the resp o n s e is a generalized p h e n o m e n o n c o m m o n to all fibrous c o n n e c t i v e tissue structures, e.g. t e n d o n s , l i g a m e n t s , capsule, or w h e t h e r it is restricted to specific fibrous c o n n e c t i v e tissue s t r u c t u r e s , as Peacock postulated.
METHODS
AND
PROCEDURES
A n i m a l s used i n this s t u d y were m o n g r e l dogs of a d u l t size b u t of u n k n o w n age. Stainless steel t h r e a d e d wires u s e d for i n t e r n a l fixation were ~ in. i n diameter. T h e y were of a s t a n d a r d t y p e u s e d i n o r t h o p e d i c s u r g e r y a n d were o b t a i n e d f r o m the Z i m m e r M a n u f a c t u r i n g Co. C h e m i c a l s for analytical use were as described in a r e c e n t p a p e r (Akeson, A m i e l a n d LaViolette, 1967).
Animal preparations T h e experimental model used for the present study is one that has been developed by us. It provides immobilization by internal fixation of the knee by two threaded wires. The wires are inserted through the proximal tibia into the femur. The course of the wires is inside the skin fold, behind the knee. They are placed as far posterior to the posterior capsule as possible and still remain within the skin fold (Akeson, Amiel and LaViolette, 1967). After a 10- to 12-week period of immobility, the animals were killed by an overdose of intravenous nembutal. Animals which did not maintain their preoperative weight, animals in whom internal fixation failed, or animals in whom infection occurred were not used in this study. Twelve animals were operated for the first portion of this study. Of this group, eight animals were successfully followed for the immobilization period. After the animals were killed, control and immobilized knees were dissected in a standard way. The periarticular connective tissue was separated anatomically as follows. T e n d o n structures included the quadriceps and patellar tendons. The sample labeled anterior capsule and synovium included the fibrous reinacula extending on either side of the quadriceps and patellar tendons as well as the capsule itself and the underlying synovial membrane. This sample was triangular and extended about two-thirds of the way toward the posterior aspect of the knee joint on either side. The medial and lateral collateral ligaments are discrete structures which were dissected cleanly from their proximal and distal attachments to bone. Cruciate ligaments are also very discrete structures which were easily detached from their origin and insertion. The sample labeled posterior capsule was prepared as follows. The medial and lateral margins of this sample were defined as the portion of the capsule between the medial and lateral sesamoid bone on the posterior aspect of the knee. The proximal attachment was to the femur and the distal attachment to the tibia. This specimen was roughly rectangular. Care was taken with this specimen not to include any structures in the popliteal fossa. The region of the popliteal fossa contains the threaded wires and a certain amount of scar tissue which inevitably develops secondary to trauma resulting from insertion of the threaded wire. Skin, bone, articular cartilage, fibrocartilage and muscle were discarded. The final samples from the control and immobilized knees included (1) quadriceps and patellar tendon, (2) anterior capsule and synovial membrane, (3) posterior capsule, (4) collateral ligaments, and (5) cruciate ligaments. These structures were placed in individual vials of acetone for 4 days at 4°C with several changes of acetone. The tissue was air dried and ground in a Wiley Mill to pass through a 60 mesh screen. T h e fat was extracted with a 50/50 mixture of acetone-ether for 24 hr. The tissue was again air dried and then dried to constant weight at 80°C and processed in a manner detailed below. T e n animals were operated in an identical manner for the purpose of determining limiting viscosity numbers of the hyaluronic acid fraction. Seven of these animals were followed successfully for 10-12 weeks. The animals were killed as described above. Periarticular connective tissue of the knee joints of control and immobilized knees was dissected as described in a previous paper (Akeson, Amiel and LaViolette, 1967). The sample included collateral ligaments, capsule, synovial membrane, cruciate ligaments, quadriceps tendon, patellar tendon and tendons of origin
CONNECTIVE TISSUE RESPONSE TO IMMOBILITY:
AN ACCELERATEDAGEING RESPONSE?
291
of the gastrocnemius muscle, all in one vial. This type of dissection and pooling was necessary in order to obtain enough tissue to provide a hyaluronic acid fraction sufficiently large for viscosity studies. Subsequent treatment of the tissue was identical to that described above.
Crude acid mucopolysaccharide extraction After an aliquot of the dry fat-free material was taken for total hexosamine and total collagen determination, the rest of each dried sample was weighed and suspended in 20 ml acetate buffer and 2 mg papain per gram of sample as previously detailed. Digestion was accomplished in 30 hr. Subsequently, the sample was cooled to 4°C and cold trichloroacetic acid was added to 10 per cent final concentration. Celite was added and the sample was filtered through a M fritted disc. T h e precipitate was washed with cold 10 per cent v/v trichloroacetic acid. T h e clear filtrate was dialyzed, concentrated in a rotary evaporating flask and passed over Dowex-50 8X 200-400 mesh (H ÷ ) as previously described (Akeson, Amiel and LaViolette, 1967). T h e crude A M P * collected in the water effluent from the Dowex columns was neutralized with 1 N N aO H , concentrated in a rotary evaporating flask, dialyzed, concentrated again to a final concentration of 1 mg AMP/m1 and an aliquot of the final crude A M P extract was taken for the purposes of uronic acid and hexosamine analysis before column separation on cellulose-CPC.~
Cellulose-CPC microcolumn procedure T h e crude A M P was fractionated on cellulose-CPC microcolumns (Antonopoulos, Gardell, Szirmai and DeTyssonsk, 1964). Minor modifications of the technique have been recently described (Akeson, Amiel and LaViolette, 1967). A 40/zl extract of crude acid mucopolysaccharide containing 3 mg of A M P / m l was applied to the top of each cellulose column. T h e A M P was solubilized from the column using stepwise increments of salt concentration starting with 1 per cent CPC and following with 0" 3 N NaC1, 0" 9 M MgC12 in 0" 1 N acetic acid, 0" 75 M MgCI2 in water. All of the salt solutions were made up in 0"05 per cent CPC. 6 - N HC1 was used to elute any remaining A M P at the conclusion of the run. Uronic acid was determined by the borate procedure (Bitter and Muir, 1962). T h e 6 N HC1 fraction was first dried in a vacuum desiccator over soda lime. When dry, 1 ml of water was added and the uronic acid was determined as above. Standard glucuronolactone was made up in each of the salt solutions for comparison with the unknown in the respective salt fractions. Similarly, blanks were made using 1 ml of each of the salt solutions for comparison with the standard and unknowns in the respective salt solutions. T h e microcolumn fractionation procedure was run in triplicate on each crude A M P extract. Agreement within 5 per cent was usually obtained. If not, the procedure was repeated.
CeUulose-CPC macrocolumn fractionation T h e validity of the microcolumn separation depends upon characterization of each fraction on a macro scale as a preliminary step. This characterization has previously been performed for the tissue under discussion (Akeson, Amiel and LaViolette, 1967) and was not repeated at this time. Previous characterization included uronic acid by the borate and Dische methods expressed as molar ratios to hexosamine, sulfate expressed as a molar ratio to hexosamine, electrophoresis on Sepraphore III, paper chromatography and Gardell column chromatography of hexosamine, turbidity reduction and infra-red spectra. On the basis of these procedures, it was concluded that the 0.3 M NaC1 fraction contained chiefly hyaluronic acid, the 0.9 M MgC12 in 0.1 M acetic acid contained chiefly chondroitin-4 and 6-sulfate and the 0.75 M MgC12 fraction contained chiefly dermatan sulfate.
Analytical procedures Details have been given in recent reports (Akeson, Amiel and LaViolette, 1967; Akeson, Amiel and LaViolette, 1966). Uronic acid was estimated according to the Dische procedure (Dische, 1947) and also by the borate procedure (Bitter and Muir, 1962). Tw o aliquots of the dried, ground material from each sample were weighed and hydrolyzed in a sealed tube. Hydrolysis for hexosamine was accomplished in 6-N HC1 in a 100°C oven for 5 hr. Subsequently, the acid * Acid mucopolysaccharide. t Cetyl pyridinium chloride.
292
W. H. AKESON, D. AMIEL, D. LAVIOLETTE AND D. SECRIST
was quickly evaporated over N a O H and calcium chloride in vacuo. T h e dried hydrolysate was dissolved in water and hexosamine was estimated by the procedures of Boas (Boas, 1953). Hydroxyproline was determined by the method of Woessner (Woessner, 1961). 5 mg dry material was weighed and hydrolyzed in 6 N HCI for 3 hr at 130°C.
Limiting viscosity numbers T h e limiting viscosity numbers were determined in a Cannon-Ubbelohde semi-micro dilution viscometer at 20°C using a 0.01 M phosphate buffer in 0.38 M NaCI at p H 7.0.
RESULTS
Total amount of tissue T h e total f a t - f r e e d r y w e i g h t of t h e d i s s e c t e d s t r u c t u r e s f r o m c o n t r o l a n d i m m o b i l i z e d l i m b s is g i v e n in T a b l e 1. M e a n v a l u e s a n d s t a n d a r d d e v i a t i o n s a r e g i v e n f o r the posterior capsule, collateral ligaments, cruciate ligament, anterior capsule and p a t e l l a r a n d q u a d r i c e p s t e n d o n . T h e r e was n o significant d i f f e r e n c e b e t w e e n t h e c o n t r o l a n d i m m o b i l i z e d side in a n y of t h e s e g r o u p s . T h i s s u g g e s t s t h a t significant a t r o p h y o f t h e f i b r o u s c o n n e c t i v e t i s s u e s t r u c t u r e s a b o u t t h e i m m o b i l i z e d k n e e d i d n o t occur.
Total collagen and total hexosamine T h e v a l u e s for t o t a l c o l l a g e n in t h e v a r i o u s s t r u c t u r e s f r o m c o n t r o l a n d i m m o b i l i z e d k n e e s are g i v e n in T a b l e 2. N o significant d i f f e r e n c e was o b s e r v e d b e t w e e n c o n t r o l a n d TABLE 1. TOTAL FAT-FREE DRY WEIGHT (g) OF DISSECTED STRUCTURES
Dog J1 Jz J4 J~ K7 Ks K9 K10
Mean
Posterior Capsule
Collateral Ligament
Cruciate Ligament
Anterior Capsule
Patellar Quadriceps Tendon
Control Immobilized Control Immobilized Control Immobilized Control Immobilized Control Immobilized Control Immobilized Control Immobilized Control Immobilized
0" 870 0- 500 0" 840 1" 180 0" 900 0" 580 0"660 0- 600 0- 405 0- 308 0"535 0" 328 0"461 0" 445 0" 544 0" 643
0" 173 0' 203 0" 234 0" 184 0' 134 0' 137 0"144 0" 131 0" 094 0" 105 0"092 0" 074 0' 078 0" 064 0" 106 0" 099
• 123 0" 109 0" 132 0" 127 0' 056 0" 063 0"077 0" 075 0" 057 0" 057 0"059 0" 057 0" 045 0" 055 0"096 0- 076
1" 430 0" 770 1 •480 1" 780 1-480 1" 930 2'190 1' 480 0" 931 1 •472 0-880 1" 380 1" 014 1" 380 1" 122 1 •234
1' 500 1" 700 1"450 1" 610 1" 520 1" 460 1-430 1" 460 0- 990 1" 361 1-011 1" 115 0" 993 0" 977 1" 133 1- 610
Control SD
0'652 0"195
0"132 0'052
0.081 0"033
1"316 0-431
1-253 0.242
Immobilized SD
0"573 0"274
0.125 0"050
0"077 0'027
1"428 0"350
1"412 0"252
Knee
Values
T h e r e was no significant difference in the amount of fat-free tissue on the two sides in any of the tissues studied.
293
CONNECTIVE TISSUE RESPONSE TO IMMOBILITY: AN ACCELERATED AGEING RESPONSE~ TABLE 2. TOTAL COLLAGEN IN STRUCTURES FROM CONTROL AND IMMOBILIZED TISSUE
Dog
Knee
Posterior Capsule
Collateral Ligament
% mg/g~
Control
Changet
533
J1
%
553 544
Immobilized Control
545 383
Immobilized Control
497 612
Immobilized Control
572 595
Immobilized Control
637 530
Immobilized Control
613 601
Immobilized Control
493 670
Immobilized
637
J2
K~
893
855
612
813
+ 1.6
-
6.5
-
-
-
+ 7.0
--12.2 678 776
+16.8
1-6
-
-- 9.1
-- 2.3
3.2
751 673
529 738
862
+12.0 786 772
670 582
824 882
873
6.4
+21.5
-1-9
-- 2.0 871 816
-
738 582
826 837
+29.0 837 702
554 579 + 3.2
+ 6.4 887 889
-15.5
4.8
865 842
+ 9-6 728 649
500 592 -
7-4
-11.1
7.7
824 838
-15.7 685 664
570 582 -
3"1
834 836
-- 4.9
Control SD Mean Values Immobilized SD
+ 2.1
741 866
925 901
+ 3.4 633 641
887 803 + 0.3
-18.0
K10
2.7
916 955
+15-7
K9
+ 6.2 908 869
843 913
+ 7.1
Ks
% mg/g* Changer
907 866
-
% mg/g* Changer
+29.8
J6
%
Patellar & Quadriceps Tendon
mg/g* Changer
+ 0.2
J4
Anterior Capsule & Fascia
mg/ge Changer
+ 3.8 Immobilized Control
Cruciate Ligament
+10.8 746 794
-
3.3
691
-- 6-0 746
558.5 85.28
883.6 43.96
849.0 24-62
613.5 54.63
730.4 65.26
568.4 57.30
882.0 33.13
842.1 51.29
610.6 84.91
744.6 51-49
* Results expressed as mg/g fat-free dry weight. t Immobilized-Control × 100. Control No significant difference was observed between control and immobilized sides in any of the groups. i m m o b i l i z e d sides i n a n y of the s t r u c t u r e s studied. T o t a l h e x o s a m i n e for the c o n t r o l a n d i m m o b i l i z e d s t r u c t u r e s is g i v e n i n T a b l e 3. T h i s v a l u e was o b t a i n e d o n dry, fat-free material p r i o r to A M P extraction. T h e i m m o b i l i z e d tissue h a d a significantly lower c o n c e n t r a t i o n of h e x o s a m i n e i n all cases. T h i s c o n f i r m s p r e v i o u s results o n pooled tissue f r o m c o n t r o l a n d i m m o b i l i z e d tissue (Akeson, A m i e l a n d LaViolette, 1967) a n d suggests that the r e s p o n s e seen i n r e d u c t i o n of h e x o s a m i n e o n t h e i m m o b i l i z e d side is a u n i f o r m r e s p o n s e t h r o u g h o u t the tissues s t u d i e d .
294
w.H.
AKESON, D. AMIEL, D. LAVIOLETTE AND D. SECRIST
TABLE 3. TOTAL HEXOSAMINE IN STRUCTURES FROM CONTROL AND IMMOBILIZED KNEES
Dog
Knee
Posterior Capsule~
Collateral Ligament~
% nag/g* Change?
Control
4"8
J,
%
4"1 4"4
Immobilized Control
2"8 2"7
Immobilized Control
2.7 3"1
Immobilized Control
2"6 3"2
Immobilized Control
2"8 2.8
Immobilized Control
2"2 3,2
Immobilized Control
3"1 4"3
Irnmobilized
3 "1
J2
5.0
7" 3
3 "7
4.1
~ 2"0 5"7 5'7 -
2"0
-31"6
-37"1
-12"5
-21"4
-15.4 2'2 7'6
K9
-
3'1
43"4
K10
- 18 '6
5"9
-18"6
-20"6
- 4 3 -5
17"1
3.5 4"9
2.7 3-6
3 "5
-
3"4 4"3 -
-22"7
- 36"8 2'4
31 "0
3"2 3'4
3"4 6.2
-41"8 3'2 4"1
-
-35"3
4"3 3-8
3'4
2-9 3'4
3-3 4"4
-19.1 3"8 5"5
-
-40"9
-25'0
3.6
2"8 4"2
3"9 5'1
9"8
3 "9 4"7 -
-32'7
-44'2
5.9
2'7 2"9
3"5 6'6
2"9 2"6
Ks
-
-48'6
3"7 5"2
3.2 2'8
3"6 5'2
3'5 5"2 12"5
-18'9 3"0 3"4
3 '9 7"0
2"2 4'0 -16"1
Control SD Mean Values Immobilized SD
-21"9
5 -0 3"5
-
% rag/g* Change?
0
K7
% mg/g* Change?
-36"4
J6
%
Patellar & Quadriceps Tendon §
mg/g* Change?
5"1 5'1
J4
Anterior Capsule & Fascia~
rag/g* Change?
--14"6 Immobilized Control
Cruciate Ligament §
--18"4 4'0 4"1
- 16"7 3 '0
- 17" 1 3 '4
3"563 0.809
4'60 1.511
5"938 1.011
3"425 0"443
4"613 0'549
2"925 0"555
3"45 1-218
3"850 0'778
2'938 0"200
3"613 0.280
* Results expressed as mg/g fat-free dry weights. t Immobilized-Control ×
100
Control
P < 0.05. § P < 0.01. The immobilized knee was significantly different from the control knee in all cases.
Acid mucopolysaccharide content T h e acid m u c o p o l y s a c c h a r i d e c o n t e n t of c o n t r o l a n d i m m o b i l i z e d s t r u c t u r e s is g i v e n i n T a b l e s 4 - 8 . I n T a b l e 4, the A M P c o n t e n t of the patellar a n d q u a d r i c e p s t e n d o n is detailed. Values are p r e s e n t e d for the c r u d e A M P c o n t e n t a n d the A M P c o n t e n t i n 0.3 M NaC1, 0.9 M MgCI2 i n acetic acid a n d 0.75 M MgCI2 a q u e o u s solution. T h e o t h e r fractions did n o t c o n t a i n significant a m o u n t s of A M P and, therefore, are n o t recorded. M e a n values of the A M P c o n t e n t of the c r u d e material a n d of the various fractions are g i v e n at the foot of the table. All values are significantly r e d u c e d o n the
CONNECTIVE TISSUERESPONSE TO IMMOBILITY:AN ACCELERATEDAGEINGRESPONSE.~
295
TABLE 4. ACID MUCOPOLYSACCHARIDECONTENT OF CONTROL AND IMMOBILIZED PATELLAR AND QUADRICEPSTENDONS Dog
Knee Control
Crude AMP % 0.3 M before Changer NaCI* Fractionation* 2"66
J1
% Changer
0"93 -33.8
0"9 M % 0"75 M % MgCI~ Changer MgCI2* Changer in HAc* 1 "05
-46"2
1 "76 2.45
Immobilized Control
2-10 2"79
Immobilized Control
2.09 2.41
Immobilized Control
1"38 2.55
Immobilized Control
1 "61 2"11
Immobilized Control
1.69 2.40
Immobilized Control
2.12 3"42
Immobilized
2.34
0"76
0.93
0'48
2"60 0"388
0.867 0.155
1-050 0.166
0.528 0.117
-14"3
J4
-20"0 0"52 0"90
-25.1
J6
Ks
K10
-
-- 9-7
-33"9
-14"6 0"41 0"73
-30"1
--35"8
-34"2
--24"9 0.788 0.170
3"0
0"34 0"48
0.84 1 "33
0"555 0"129
- 34"6 0"34 0"33
-23.0
-23"7
--27.0 1 "89 0"324
- 35.9
0,67 0,93
0"71 1"15
-38.0 0"31 0.52
0.66 0,87 -28.4
-31"6
Control SD Mean Values Immobilized SD
-37"8
- 45 "3
0'58 0'93
- 13.5 0'45 0'50
0,56 1 '03
0"47 0"81
-37"5 0"40 0'52
-21.4
-48"6
-11"7
3-9
0"99 0,90
0"36 0.86
-19"9
K9
-
-40.0
- 36"9
0"34 0"64
0"99 1 '26
0.54 0-70 -42"7
K7
0"66 1 "03
-32.0
Immobilized Control J~
0"50 0"65
0"50 -37"1
--25.2 0"384 0-061
* Results expressed in micrograms of uronic acid per gram of fat-free connective tissue. t Immobilized-Control x 100 Control All values were significantly different P < 0"01. e x p e r i m e n t a l side. T h i s f i n d i n g is i n c o n t r a d i c t i o n w i t h p r e v i o u s results o n pooled m a t e r i a l w h e r e a l t h o u g h r e d u c t i o n s were f o u n d i n the 0.75 M MgCI2 f r a c t i o n the c h a n g e s were n o t statistically significant. I n T a b l e 5, t h e A M P c o n t e n t of c o n t r o l a n d i m m o b i l i z e d a n t e r i o r capsule a n d synovial m e m b r a n e is given. Significant r e d u c t i o n i n the A M P c o n t e n t was f o u n d i n the c r u d e m a t e r i a l before f r a c t i o n a t i o n a n d i n the 0"3 M NaC1 a n d the 0.9 M MgC12 i n acetic acid fractions. T h e f r a c t i o n c o n t a i n e d i n the 0.75 M MgC12 i n a q u e o u s s o l u t i o n was n o t significantly different f r o m the c o n t r o l values. T h i s result c o r r e s p o n d s to p r e v i o u s f i n d i n g s w i t h pooled s t r u c t u r e s . T a b l e s 6, 7 a n d 8 c a n n o t b e assessed statistically b e c a u s e of p o o l i n g of tissue f r o m several animals. T h e values for
296
W. H. AKESON, D. AMIEL, D. LAVIOLETTE AND D. SECRIST
T ~ L E 5.
A C I D MUCOPOLYSACCHARIDE CONTENT OF CONTROL AND IMMOBILIZED ANTERIOR CAPSULE AND SYNOVIAL MEMBRANE
Dog
Knee
Crude AMP before Fraetionatione,a~
% Changer
J1 Control
% Changer
SAMPLE LOST 0"40
1.95
J2
0.3 M NaCI *(~
-
32"8
-45
0.9 M MgCI~ in HAc*cbj
0"89
"0
0.75 M % MgCI~*lc~ Changer
0"54 -
37"1
1.31 1 "30
Immobilized Control
0.95 1.33
Immobilized Control
1.12 1.51
Immobilized Control
1.16 1-45
Immobilized Control
1.43 1.72
Immobilized Control
1.24 1.87
Immobilized
1.55
0"40
0"59
0"34
1.50 0.259
0.444 0.139
0.671 0.120
0"373 0.100
-26.9
J6
-32.1 0"19 0.34
-15"8
K7
K9
-
-11"7 -27"9
-10"3 0"26 0"39
-18"1
--31"4 0.299 0"081
+ 6"5 0"49 0"29
0-49 0"72 -32"2
--20"7 1.251 0.200
-- 17"9 0"23 0'46
0-53 0"68 -40-3
--17"1
Control SD Mean Values Immobilized SD
-- 20"3
9"1
0"37 0"59
+10"7 0"31 0.28
0'47 0"60
0-30 0"62 -27"9
K10
+ 7"1
-- 34"5
1"4
+ 2.7 0"38 0"28
0"75 0.59
0.36 0"33 -
-13.5
-26"5
--12.8
--17"4 0"549 0"102
2 5 "9
0"40 0"37
0"45 0"70
0"25 0-55 -- 23 "2
Ks
0"56 0"52
-
Immobilized Control J4
0"22 0.28
% Changer
-- 6"7 0.344 0"088
* Results expressed in micrograms of uronic acid per gram of fat-free connective tissue. t Immobilized-Control × 100. Control (a) All values were significantly different P < 0"01. (b) 0"9 M M g C h values were significantly different P < 0. (c) T h e control and immobilized values were not significantly different.
A M P c o n t e n t o f p o s t e r i o r capsule, c r u c i a t e l i g a m e n t a n d collateral l i g a m e n t s t r u c t u r e s are g i v e n in t h e s e t a b l e s in t h e m a n n e r s i m i l a r to t h e tables p r e v i o u s l y d e s c r i b e d . P o o l i n g was n e c e s s a r y b e c a u s e o f t h e s m a l l a m o u n t s o f tissue available for analysis. T h e results shown suggest that the changes were consistent with those previously noted. The d i r e c t i o n o f c h a n g e s e e m e d t h e s a m e in all cases. T h e n o t a b l e q u e s t i o n arises w i t h r e s p e c t to t h e f r a c t i o n in 0"75 M M g C I 2 in a q u e o u s s o l u t i o n w h e r e in s o m e cases t h e c h a n g e s w e r e g r e a t e r t h a n in others. T h e significance o f this f i n d i n g is u n c e r t a i n at t h e present time.
CONNECTIVE TISSUE RESPONSE TO I M M O B I L I T Y : AN ACCELERATED AGEING RESPONSE~
297
T A B L E 6. A C I D MUCOPOLYSACCHARIDE CONTENT OF CONTROL AND IMMOBILIZED POSTERIOR CAPSULE Dog
Knee
Control
Crude AMP before Fractionation*
% Changet
1.84
J~
0.3 M NaCI #
0"36 - 21 "7
Immobilized Control KT,Ks (pooled) Immobilized Control K%K10 (pooled) Immobilized
% Changet
1.44 1-82 -34.6
--39"0
--36.6
- 28-0 0-36 0-33
-- 9"2
-31"6
% Changer
0"50
0"89 1.21
0-39
0-75 M MgCh ~
- 16"2 0"83 0"98
0"25 0"57
1 "54
% Changet
0"99 - 30" 5
0"25 0"41
1.19 2"43
0.9 M MgCh in HAe#
-- 6-1 0.31 0"55
--31"4 0"83
-- 7"0 0.51
* Results e x p r e s s e d in m i c r o g r a m s of u r o n i c acid p e r g r a m of fat-free c o n n e c t i v e tissue. t Immobilized-Control x 100. Control
T A B L E 7. A C I D MUCOPOLYSACCHARIDE CONTENT OF CONTROL AND IMMOBILIZED CRUCIATE LIGAMENTS Dog
Knee
Control All Dogs Pooled Immobilized
Crude AMP before Fractionation °
% Changet
4"0
0.3 M NaCI #
% Changer
0"89 - 45 "8
2'17
0.9 M MgCh in HAe#
% Changet
2"66 -- 53 "9
0"41
0-75 M MgCh #
% Changet
0"54 - 48" 1
1 "38
- 37 "0 0"34
* Results expressed in m i c r o g r a m s of u r o n i c acid p e r g r a m of fat-free c o n n e c t i v e tissue. t Immobilized-Control x 100. Control
T A B L E 8.
ACID
MUCOPOLYSACCHARIDE CONTENT OF CONTROL AND IMMOBILIZED COLLATERAL LIGAMENTS
Dog
Knee
Control All Dogs Pooled Immobilized
Crude AMP before Fractionationo
% Changer
2"09
0-3 M NaC1#
% Changer
0.57 -13.4
1 "81
0"9 M MgCll in HAc*
1 "8 -33"3
0"38
% Changet
0.75 M MgCla °
0"58 - 1 1 "1
0"96
% Changet
0"0 0-58
* Results e x p r e s s e d in m i c r o g r a m s of u r o n i c acid p e r g r a m of fat-free c o n n e c t i v e tissue. t" I m m o b i l i z e d - C o n t r o l × 100. Control
298
W. H. AKESON, D. AMIEL, D. LAVIOLETTE AND D. SECRIST
TABLE 9.
LIMITING VISCOSITY NUMBERS I/z] OF HYALURONICACID OBTAINED FROM PERIARTICULAR CONNECTIVE TISSUE OF CONTROL AND IMMOBILIZEDKNEES OF DOGS
Animal # 23 24 27 28 29 30 Mean Values
Control 5'39 4.15 2' 90 4" 44 3" 02 2.62 3' 75
Immobilized 2-44 3' 62 2' 70 1 •88 3- 57 3' 21 2' 90
There is no significant difference in the control and immobilized values when paired observations are compared by the T test.
Limiting viscosity numbers The results of viscosity determination are given in Table 9 where they are presented as limiting viscosity number (LVN). The mean value on the experimental side was 23 per cent less than on the control side. However, a significant difference was not observed. A typical plot of the limiting viscosity number for pooled material is given in Fig. 1. ~.©
3'00-
2"50 n-n s nsc 2'00
j/
1.50-
o.65 o.io 0% o.~o o.~5 Concentration ( G m / l O O m I )
FIG. 1.
LVN of hyaluronic acid from control and immobilized canine periarticular connective tissue.
DISCUSSION A superficial similarity between immobility and ageing includes prominent atrophy of bone and muscle in the extremities of older individuals. In addition, certain chemical changes in the gel-fibre ratio have been described in such tissue as skin (Sobel and Marmorston, 1956) (Boas and Foley, 1954) and nucleus pulposus (Hallen, 1958) (Davidson and Small, 1963). However, the nature of the gel-fibre ratio changes is not fully established in human material, particularly in fibrous connective tissue structures and the full
CONNECTIVE TISSUE RESPONSE TO IMMOBILITY: AN ACCELERATED AGEINGRESPONSE?
299
implications of the changes described in the paper as they relate to ageing cannot be appreciated until such studies have been completed. The present study was undertaken to determine whether the connective tissue response to immobility is a uniform response involving all the fibrous connective tissue structures or whether the changes are restricted to strategic sites as Peacock has suggested (Peacock, 1963). The results indicate that the response is indeed a nearly uniform one. There was a uniformly negative finding in so far as total collagen concentration was concerned. No statistically significant changes in collagen concentration were found in any of the structures studied, including the posterior capsule. The results of Peacock which are at variance with these in so far as the structures of the posterior aspect of the knee are concerned are probably best explained as simply an artefact resulting from scar formed secondary to the insertion of the threaded wire in the popliteal region. The tissue sampling in this study was a different one, and attention was paid to including none of the tissue in the immediate vicinity of the threaded wire used for internal fixation. The posterior capsule structures themselves which would be the structures ordinarily involved in a flexion contracture of the knee showed no increase in collagen concentration. All of the structures studied, namely tendon, capsule, collateral ligaments and cruciate ligaments showed changes in the same direction and of the same order of magnitude in so far as total hexosamine and total acid mucopolysaccharide concentration is concerned. The hyaluronic acid and chondroitin-4 and 6-sulfate changes confirm the previous findings on pooled periarticular connective tissue of the canine knee. Dermatan sulfate changes were less uniform. Dermatan sulfate was significantly reduced in patellar and quadriceps tendon but not in the anterior capsule. Statistical analysis was not possible in the case of the ligamentous and posterior capsule structures because pooling was necessary to provide sufficient material for AMP separation. A previous study on pooled periarticular connective tissue showed that dermatan sulfate concentration was reduced, but the change was not statistically significant (Akeson, Amiel and LaViolette, 1967). Presumably, the change in quadriceps and patellar tendon was masked by the pooling procedure. The changes were sufficiently large in the cruciate ligament and posterior capsular material to suggest that they could be statistically significant. The determination of limiting viscosity numbers on a hyaluronic acid fraction showed small changes between the control and experimental sides but these were not statistically significant. These findings suggest that depolymerization of ground substance is probably not an important mechanism in the decrease in AMP concentration observed. Previous studies in which half-time of turnover of hyaluronic acid was determined in this experimental model suggested that accelerated breakdown of hyaluronic acid did not occur. The present findings on limiting viscosity number are consistent with that conclusion. From the available information, the reduced concentration of hyaluronic acid is probably the result of reduced synthesis rather than accelerated breakdown. These findings point to the importance of activity in the maintenance of normal connective tissue homeostasis. They raise questions as to whether certain changes associated with ageing might be secondary to reduced physical stress. The final interpretation of the question of acid mucopolysaccharide and water influence on connective tissue pliability awaits further elucidation of the organization of fibrous connective tissue on a molecular level. In the meantime, considerable work of a descriptive type remains to be done in anticipation of the time when molecular organization is sufficiently understood to relate changes such as those just described to the ageing process as a whole.
300
W. H. AKESON, D. AMIEL, D. LAVIOLETTE AND D. SECRIST REFERENCES
AKESON, W. H. (1961)07" Bone ~ 07oint Surg. 43-A, 1022. AKESON, W. H., AMIEL, D. and LAVIOLETTE, D. (1966) I n Biochimie et Physiologie du Tissue Conjonctlf (Edited by COMTE, P.), pp. 679-689, Societe Ormeco et Imprimerie, Lyon, France. AKESON, W. H., AMmL, D. and LAVIOLETTE, D. (1967) Clin. Orthop. 51, 183. ANTONOPOULOS, C. A., GARDELL, S., SZlRMAI, J. A. and DE TYSSONSK, E. R. (1964) Biochim. biophys. Acta 83, 1. BITTER, T. and MUIR, H. M. (1962) Anal. Biochem. 4, 330. BOAS, N. F. (1953)07. biol. Chem. 204, 553. BoAs, N. F. and FOLEY, J. B. (1954) Proc. Soc. exp. Biol. ivied. 86, 690. BROOKE, J. W. and SLACK, H. G. B. (1959) Ann. rheumat. Dis. 18, 129. DAVlDSON,E. A. and SMALL, W. (1963) Biochim. biophys. Acta 69, 445. DlSCHE, Z. (1947)07. biol. Chem. 167, 189. HALLEN, A. (1958) Acta chem. Scand. 12, 1869. PEACOCK, E. E. (1963) Surg. Forum 14, 440. SLACK,n . G. B. (1954)Biochem. 07. 60, 112. SOBEL, H. and MARMORSTON,J. (1956)07- Gerontol. U , 2. WOESSNER, J. F. (1961) Arch. Biochem. 93, 440. S u m m a r y - - A certain similarity between changes secondary to lack of extremity function and those of ageing has stimulated the present inquiry. The chemical changes occurring in immobility are described and compared with available information on such changes in the ageing process. A change in the gel-fibre ratio was noted to be a uniform process which occurred in all fibrous connective tissue structures of the immobilized canine knee. There was no change in total collagen to support the hypothesis that contractures occur as a result of stimulated collagen synthesis in strategic sites. Acid mucopolysaccharides on the immobilized side were uniformly reduced in terms of concentration of hyaluronic acid and chondroitin-4 and 6-sulfate. Changes in the dermatan sulfate fraction were less consistent. R 6 s u m 6 - - U n e certaine similitude entre les alt6rations consfcutives g u n manque de fonctionnement des extr6mit6s et celles provoqu6es par le vieillissement a conduit la pr6sente recherche. Les modifications chimiques se manifestant en cas d'immobilisation sont d6crites et comp~r6es avec l'information disponible concernant de teUes modifications au cours du processus de vieillissement. On a constat6 qu'une modification de la proportion gel/fibres est un processus g6n6ralis6 se d6roulant dans routes les structures de tissu conjonctif fibreux du genou immobilis6 chez le chien. I1 n'y avait pas de variation du collagbne total pouvant 6tayer l'hypothfise que des contractures seraient la cons6quence d'une stimulation de la synthbse du collagbne g des points strat6giques. Au c6t6 immobilis6, les mucopolysaccharides 6taient g6n6ralement r6duits en ce qui concerne la concentration de l'acide hyaluronique et le sulfate -4 et -6 de chondro/tine. Les variations de la fraction sulfate de dermatan 6taient moins constantes. Z u s a m m e n f a s s u n g - - D i e vorliegende Untersuchung wurde wegen einer gewissen •~hnlichkeit zwischen den Ver~inderungen nach dem Verlust einer Extremit~itenfunktion und den Altersver~inderungen unternommen. Die chemischen ~ n d e r u n g e n bei der Immobilit~t werden beschrieben und mit den vorhandenen Ergebnissen tiber derartige ~ n d e r u n g e n im AltersprozeB verglichen. Die Ver~inderung in dem GelFaser-Verh~iltnis zeigte uniformes Verhalten bei allen Faserstrukturen des Bindegewebes eines stillgelegten Hundeknies. Eine ~ n d e r u n g im Gesamtkollagen war nicht zu sehen, welche die Hypothese gestiJtzt h~itte, dab als Ergebnis angeregter Kollagensynthese an besonders wichtigen Stellen Kontrakturen entstehen. Saure Mukopolysaccharide wie Hyalurons~iure une Chondroitin 4- und 6-sulfat waren auf der stillgelegten Seite in ihrer Konzentration vermindert. Die ,~nderungen in der Dermatansulfatfraktion waren weniger eindeutig.
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