the effects of immobilization on the characteristics of articular cartilage: current concepts and...

Osteoarthritis and Cartilage (2002) 10, 408–419© 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved. 1063–4584/02/$35.00/0doi:10.1053/joca.2002.0529, available online at http://www.idealibrary.com on

The effects of immobilization on the characteristics of articular cartilage:current concepts and future directionsB. Vanwanseele, E. Lucchinetti and E. StussiLaboratory for Biomechanics, Swiss Federal Institute of Technology Zurich (ETHZ), CH-8952 Schlieren,Switzerland

Summary

Objective: The purpose of this paper is to review current data and concepts concerning the effect of immobilization on articular cartilage inanimal models. We also evaluate the methods to measure articular cartilage changes in humans.

Methods: Studies looking at the effects of immobilization on morphological, biochemical, and biomechanical characteristics of articularcartilage are reviewed.

Results: Articular cartilage changes in immobilized animals include altered proteoglycan synthesis, as well as thinning and softening of thetissue. The overall thickness of articular cartilage in the knee decreases up to 9% after 11 weeks of immobilization and the deformation rateunder test load increases up to 42%. Quantitative data about changes in human articular cartilage following immobilization are not available.This is mainly due to the lack of an accurate, reproducible, and non-invasive method to characterize articular cartilage.

Discussion: An understanding of the alterations in articular cartilage following short and long term immobilization in humans is essential forthe optimization of rehabilitation programs. Refined imaging techniques combined with state-of-the-art visualization tools could allow thesystematical monitoring of articular cartilage morphology changes in immobilized humans. © 2002 OsteoArthritis Research SocietyInternational. Published by Elsevier Science Ltd. All rights reserved.

Key words: Immobilization, Articular cartilage, Morphological, Biochemical, Mechanical.

Introduction

Joint pain and loss of mobility are among the most commoncauses of impairment in middle-aged and elderly people. Inmany instances, articular cartilage degeneration and con-comitant alterations in other joint tissues, cause pain and adecreased range of motion. An understanding of thedegeneration process in articular cartilage, and the poten-tial for restoring its properties depend to a large extent onan appreciation of the biological behavior and the respon-siveness of articular cartilage to injury and immobilization.

Mechanical loading influences the development, main-tenance, and aging of skeletal tissues including articularcartilage. Specifically, intermittent hydrostatic pressure isthought to maintain cartilage. Shear stresses, prolongedstatic loading or absence of loading encourage cartilagedestruction and ossification1. An understanding of the rela-tionships between joint use/disuse and joint degenerationrepresents a critical step in the process of developingstrategies to prevent and treat joint diseases such asosteoarthritis (OA).

In this review, we focus our attention on the problemsresulting from joint immobilization and discuss the ability ofarticular cartilage to recover following remobilization. In thefollowing section, we review the effects of reduced joint

408

motion on articular cartilage morphology, and the subse-quent section 3 focuses on experiments aimed at quantify-ing biochemical changes in articular cartilage afterimmobilization. The effects of reduced joint motion on themechanical behavior of articular cartilage are then dis-cussed. In the final section we present novel techniquesthat can be applied non-invasively to monitor early changesin articular cartilage in humans.

Received 13 August 2001; accepted 8 January 2002.Address correspondence to: Vanwanseele Benedicte, Labora-

tory for Biomechanics, Swiss Federal Institute of Technology(ETH), Wagistrasse 4, CH–8652 Schlieren, Switzerland. Tel: +41 16336211; Fax: +41 1 633 11 24; E-mail: [email protected]

Morphological changes

STRUCTURAL CHARACTERISTICS OF ARTICULAR CARTILAGE

The structure of articular cartilage changes with depthfrom the joint surface2,3. Although these variations arecontinuous, articular cartilage has been divided into fourdistinct zones or layers referred to as the superficial tan-gential zone, the middle or transitional zone, the deep orradial zone, and the calcified zone (Fig. 1)4. The superficialzone is thin and exhibits the largest amount of collagen andthe lowest amount of proteoglycans (a more detaileddescription is given in ‘Biochemical changes’). The colla-gen fibers in the superficial zone are oriented parallel to thejoint surface and the chondrocytes appear flattened. Themiddle zone is oriented in a cross pattern with a transitionfrom horizontal to vertical cell and collagen orientation. Thecollagen fibers in the deep zone are oriented vertically. Thefibrils emerge from the underlying calcified cartilage wherethey are anchored. The calcified cartilage represents atransition zone between the articular cartilage and theunderlying subchondral bone (Fig. 1)5.

Osteoarthritis and Cartilage Vol. 10, No. 5 409

Collagen fibrils and proteoglycans are the structuralcomponents of hyaline cartilage supporting the internalmechanical stresses that result from loads applied to thearticular surface. The general orientation of the surfacecollagen fibrils was first shown by pricking the surface,which resulted in a split-line pattern (Fig. 2)6,7. The orien-tation of fibrils in the superficial collagen matrix was foundto be approximately coincident with the sliding direction ofthe joint. Based on X-ray diffraction studies8, polarized lightmicroscopy9, and electron microscopy studies9, the fibersseem to be aligned to the split line pattern. Benninghoffhowever, postulated that the fibrils would originate at theosteochondral junction and run radially towards the sur-face. There, these fibrils bend over tangential to the surfaceand, finally continue to the articular margin7. These obser-vations were later supported by investigators using scan-ning electron microscopy10,11, and a multiple-plan freezefracture technique12, but are still object of controversy.Studies by Broom13, suggest that the fibrils are not continu-ous over their entire length but only continue for a shortdistance along the articular surface. The superficial tangen-tial layer, which is near the articular surface, consists ofsheets of tightly woven collagen fibrils14. This regionaccounts for the highest concentration of collagen. Thefibers in the middle zone, on the other hand, appearrandomly oriented and homogenously dispersed. In thedeep zone, the fibers come together to form larger, radially

oriented fiber bundles. These bundles enter the calcifiedzone, crossing the tidemark, to form an interlockingnetwork that anchors the tissue to the bony substrate(Fig. 1)3,15.

The morphological development of articular cartilage isinfluenced by its adaptation to functional demands ofabsorbing and redistributing compressive forces. The tide-mark represents the interface between the hyaline cartilageand the calcified cartilage (Fig. 1). Macro-morphologicalparameters, such as tissue volume, thickness, and jointsurface areas can be used to characterize the differentia-tion and functional adaptation of the cartilaginous tissue tothe mechanical stresses. Detailed data on the quantitativemorphology of articular cartilage are important input par-ameters for computer models of diarthrodial joints. Thesemodels allow the prediction of load transmission injoints16,17, and the simulation of adaptational processes inthe tissue18. In orthopedics and skeletal radiology, reliabledetermination of cartilage thickness is useful for the stagingof joint disease and for the evaluation of pharmacological orsurgical chondroprotective treatments.

Superficial tangential(10–20%)

Middle (40–60%)

Deep (30%)

Calcified cartilage

Articular surface

Tidemark

Fig. 1. Representation of collagen fibril ultrastructure throughoutthe depth of the cartilage depicting the distinct idealized zones

(reproduced with permission from Mow et al., 1974)4.

Fig. 2. Diagrammatic representation of a split line pattern of thefemoral condyles (reproduced with permission from Hultkranz,

1898)6.

MORPHOLOGICAL CHANGES DUE TO IMMOBILIZATION

Most investigations examining the consequences of jointimmobilization were performed using large laboratory ani-mals (dogs). The studied animals were immobilized bymeans of a cast (non-rigid) or an external fixator (rigid) fora given time period. The control animals were free to movewithin their cages. Both groups were sacrificed at the endof the prescribed study period and the joints wereevaluated19–23. On gross examination, the articular carti-lage appeared smooth and continuous with no signs offibrillation. Jurvelin et al. measured articular cartilage at 20different locations across the femoral and the tibial jointsurfaces22. Large thickness variations were demonstratedin control dogs. In these animals, the thickest cartilage waslocated at the central areas of the medial condyle of thefemur and tibia. The thinnest cartilage was found in theposterior part of the lateral condyle of the femur. After11 weeks of splinting the right leg, the overall thickness ofthe articular cartilage in femur, tibia, and patella wasdecreased by 9% (Table I)22. The immobilization signifi-cantly decreased the thickness of the hyaline cartilage atthe summit of the medial condyle (20%) and at the patellarsurface (19%) of the femur21. After a remobilization periodof 50 weeks, no significant difference was seen in compari-son to age-matched controls. No changes were seen in themedial condyle of the tibia or in the patella. The cartilagethickness of the summit of the femoral lateral condyleor the central point of the intermediate part of the tibiallateral condyle showed no changes after immobilization(Table I)20.

O’Connor confirmed that the thickness of articular carti-lage is determined, at least in part, by mechanical factorsassociated with joint loading and movement24. Sixty 100-day-old male rats with open epiphyses, were randomlydivided in three equal sized groups (caged controls, hindlimb unweighted, and hind limb unweighted with cast immo-bilization). Articular cartilage thickness was measured onhistological sections stained with toluidine blue, whichprovided excellent color discrimination between bone andcalcified cartilage. A distinct basophilic line marked thelocation of the tidemark and was used to discriminatebetween calcified and non-calcified cartilage. After 28 daysof joint unweighting by tail suspension in rats, the authors

410 B. Vanwanseele et al.: Effects of immobilization on articular cartilage

did not find any changes in the total articular cartilagethickness (Table I). However, after discriminating betweencalcified and non-calcified cartilage, an increase up to 41%in thickness was seen in the calcified layer, whereas thethickness of the non-calcified layer decreased. In theanterior part of the femur however, no changes wereobserved. After 28 days of unweighting with casted immo-bilization, there were no changes in the calcified and in thenon-calcified layer (Table I). This tidemark advancementwas nearly twice as high in the unweighting group, while inthe immobile unweighting group no changes wereobserved. Both groups were osteopenic as compared tothe control group. There was no difference between bothgroups in mean epiphyseal mineralized tissue area. Vascu-lar invasion, resulting in contact between epiphyseal mar-row spaces and the tidemark, occurred in 80% of theanimals in the unweighting and immobilization group. Thisstudy shows that joint immobilization, and unweighting of amobile joint do not have the same effects and that biologi-cal adaptation may be regulated in different ways.Unweighting a mobile joint accelerates advancement of thetidemark, while motion restriction activates resorption atthe chondroosseus interface. The tidemark response tounweighting is consistent with the predictions of Carter’s

theory, according to which the reduction of cyclic hydro-static loading causes a reduced inhibition of the naturaltendency of the subchondral ossification front to advancetowards the joint surface25.

Leroux et al. immobilized 10 skeletal mature dogs with acast during 4 weeks26. The thickness of articular cartilage,calcified cartilage, and subchondral bone was measuredfrom scanned and digitized images of stained sections asthe average of 5, 10 and 10 equidistant measurements.They did not found changes in the cartilage, calcifiedcartilage, or subchondral bone thickness on the medialtibia. These results are consistent with previous findingsfrom Haapala et al., at this part of the knee joint21.

Table IMorphological changes due to immobilization

Authors Study time Parameter Measurement points Results

Haapala et al.21 11 weeks, rigid Mean thickness Med femur–Summit Decrease of 20%–Anterior Decrease of 19%–Patellar No changesMed tibia No changesPatella No changes

Haapala et al.20 11 weeks, rigid Mean thickness Lat femur No changesJurvelin et al.22 11 weeks, rigid Mean thickness Femur Decrease of 13%

Med tibia Decrease of 6%Lat tibia Decrease of 4%Patella Decrease of 7%

O’Connor24 28 days, unweighting Total thickness Tibia post No changesTibia ant No changesFemur post No changesFemur ant No changes

Calcified thickness Tibia post Increase of 41%Tibia ant Increase of 41%Femur post Increase of 26%Femur ant No changes

Uncalcified thickness Tibia post Decrease of 7–13%Tibia ant Decrease of 7–13%Femur post DecreaseFemur ant No changes

28 days, non-rigid Total thickness Tibia post No changesTibia ant Decrease of 10%Femur post No changesFemur ant Decrease of 15–22%

Calcified thickness Tibia post No changesTibia ant No changesFemur post No changesFemur ant No changes

Uncalcified thickness Tibia post No changesTibia ant No changesFemur post No changesFemur ant No changes

Leroux et al.26 4 weeks, non-rigid Cartilage thickness Medial tibia No changesCalcified thickness Medial tibia No changesSubchondral bone thickness Medial tibia No changes

Lat=lateral, Med=medial, Post=posterior, Ant=anterior.

Biochemical changes

COMPOSITION OF NORMAL ARTICULAR CARTILAGE

Articular cartilage is a specialized connective tissue witha large amount of extracellular matrix. This matrix is com-posed of a dense network of collagen fibers (10–30% bywet weight), large aggregating and non-aggregating pro-teoglycans (3–10% by wet weight). The remaining 60–80%


is water, inorganic salts, and small amounts of other matrixproteins, glycoproteins and lipids27.

The collagen fiber network, mainly consisting of type IIcollagen (with small amounts of collagen type V, VI, IX, andXI), provides the shape and serves as confinement to theentrapped proteoglycans, which cause the tissue to swellbecause of the large osmotic pressure they generate27.This enables articular cartilage to withstand the compres-sive stresses associated with load bearing. Cartilageproteoglycans (PG) are large protein-polysaccharide mol-ecules that exist either as monomers or as aggregates28.The monomers form exists of an approximately 200 nm-long protein core to which about 150 glycosaminoglycan(GAG) chains and both O-linked and N-linked oligosaccha-rides are covalently attached29. The glycosaminoglycansare relatively short chains of repeating disaccharide units ofsulphated hexosamines. In cartilage, the most importantproteoglycan molecule is aggrecan, which consists ofnumerous PG monomers non-covalently bond tohyaluronic acid to form aggregates with a molecular weightof up to 2×108 Daltons (D) and a length of approximately2 �m. Besides aggrecan, several small non-aggregatingproteoglycans, such as decorin and biglycan, are alsopresent. Unlike collagen, the percentage of proteoglycansis lowest near the articular surface and increases withdepth. Finally, other glycoproteins such as fibronectin,anchorin, and cartilage oligomeric matrix protein (COMP)are far less abundant, but also play a role in cartilagebiology27.

Distributed within the matrix is a sparse population ofcells (with a density of less than 10% of the tissue’svolume), the chondrocytes, which are responsible for thesynthesis and the maintenance of the matrix components.The turnover of the cartilage matrix is regulated by thechondrocytes, which are capable of synthesizing a varietyof proteolytic enzymes, such as matrix metalloproteases(MMPs)30–33. It is well known that in OA both aggrecanand collagen are degraded34–39. The proteolytic cascadeinvolves collagenases (interstitial collagenase or MMP-1,neutrophil collagenase or MMP-8, and collagenase-3 orMMP-13), gelatinases (MMP-2 and MMP-9), and strome-lysins (in particular stromelysin-1 or MMP-3)27. Tissueinhibitors of metalloproteases (TIMPs) inhibit the cataboliceffects of MMPs. It is believed that the ratio MMP-to-TIMPis tightly regulated by the chondrocytes themselves towarrant tissue homeostasis.

the trunk in 90° flexion. Only after 8 weeks was an increasein water content observed, but confined to the tibial plateau.Leroux et al. did not find any changes in water content after4 weeks of cast-immobilization in the medial tibia26. Thisresult is consistent with the findings of Muller et al.40, wherechanges appear only after 8 weeks.

Behrens’ group also reported a 6.4% decrease in thetotal solid component after 6 weeks of casting. In theexternally fixed joints, the difference was almost 30%(Table II)19. Looking closer at this solid component, adecrease of the proteoglycan content at almost all jointlocations was observed in the casted and in the externalfixator group (Table II)19. The proteoglycan content in theother joint locations showed a clear decreasing trend.When no joint movement was allowed the loss of hexuronicacid was more dramatic than in the case of limited kneemotion (casting)19. The values returned to normal after1 week of cast removal, but in some areas of the externalfixator joints, hexuronic acid levels were still significantlydepressed after 1 week of recovery. Allowing a smallmovement during the immobilization was already sufficientto permit a faster recovery. These findings were confirmedby Haapala et al., who found a decrease in total uronic acidconcentration of up to 32% in patella, tibia and femur after11 weeks of rigid immobilization (Table II)41. The sameinvestigators observed that the glycosaminoglycan concen-tration of the articular cartilage was slightly decreased, butnot significantly reduced, at the lateral condyle of femur andtibia20. In this study, 29-weeks-old female dogs were immo-bilized for 11 weeks in 90° flexion with a fiberglass cast, tiedto the body to prevent loading. Earlier microspectrophoto-metric and polarized light microscopy results from the sameexperiment showed, however, that a significant decrease inthe GAG concentration took place in the medial femoraland tibial condyle (20–23%) but the recovery in these sitesafter 50 weeks of remobilization was not complete21. Theseresults are in contrast to Behrens et al., who found adecrease in the medial as well as in the lateral condyle ofthe tibia and femur after a shorter immobilization period anda full recovery19.

After a short (4 weeks) period of immobilization, nochanges in sulfated GAG concentration at the medial tibiawere found by Leroux and associates26. Unfortunately, adirect comparison of these studies is not possible becausethe authors used animals of different age and differentbreeds. Additionally, the immobilization time was different.

Haapala et al. found a decrease in the hyaluronancontent after 11 weeks rigid immobilization at the tibialcondyle and the patellar surface of the femur (Table II)41.The proportion of hyaluronan to total uronic acid remainedunchanged because of a concurrent decrease in aggrecan.In contrast to these results, Muller and associates showedthat the hyaluronan content was maintained at controlvalues after 8 weeks of joint disuse40. The results of thecentrifugation studies of the non-dissociatively extractedproteoglycans indicated a decrease in the amount ofaggregates. However, the loss of aggregates is primarilyassociated with a decrease of the slow-sedimenting ones.

Some authors studied the metabolism of cartilage follow-ing immobilization and measured the rate of PG synthesisin explant cultures. Behrens et al. showed that the proteo-glycan synthesis decreased by 48% in the external fixationgroup and by 34% in the casted group as compared tocontrols (Table II)19. Only in the external fixation group asignificant difference was measured. These results agreewith the results of Palmoski42, who observed a 41% reduc-tion in synthesis (on a wet-weight basis) in pooled femoral

EFFECTS OF IMMOBILIZATION ON THE BIOCHEMICAL

COMPOSITION OF ARTICULAR CARTILAGE

The effects of 6 weeks of immobilization on the bio-chemical characteristics of articular cartilage were thor-oughly investigated for the first time by Behrens et al.19.Adult dogs (2–3 years old) were immobilized by rigidexternal fixation or by casting, allowing limited knee motionbut normal transarticular forces, as generated by themuscles. Water content increased at 20 different locationsacross the patella, the femur, and the tibia (Table II)19.These findings are partly in agreement with the results ofSetton and co-workers23, where an increase in hydration ofarticular cartilage at the posterior site of the tibial plateauafter 8 weeks of joint disuse (non-rigid immobilization) wasfound (Table II). However, Setton’s group did not reportchanges in the femur. Muller et al. also did not find changesin water content in femur after 4 and 8 weeks non-rigidimmobilization (Table II)40. In this study, the right limb of 16skeletal mature female dogs was bounded and strapped to


condyle and patellar cartilage from a single 6 weeks casteddog. The external fixator group also showed an increase ina low-molecular weight proteoglycan (as measured bychromatography) that was present only in very smallamounts in normal cartilage and in only slightly higherquantities in the casted animals (Table II)19. The rate ofproteoglycan synthesis and aggregation is very sensitive tothe physico-chemical environment (i.e., hydration, pH,osmotic pressure, etc.) of the chondrocytes43–46. Conse-quently, it seems reasonable to postulate that chondrocytesmay respond to changes induced by unloading and disuse,and remodel their surrounding extracellular matrix. Markersof cartilage and synovium metabolism (MMPs and TIMPs)

were measured by Grumbles and collaborators47 and byHaapala and co-workers48 in an attempt to detect proteo-lytic events associated with joint immobilization. Grumbleset al. found an elevation of neutral metalloprotease(MMP-2) after 4 weeks of non-rigid immobilization in adultmongrel dogs. Concurrently, a striking fall in TIMP levelswas displayed47. A return of TIMP and MMP-2 towardscontrol values was observed 2 weeks after removal of thesling. Haapala et al. monitored the concentration ofmarkers of cartilage and synovium metabolism in the kneejoint synovial fluid of young beagles subjected to 11 weeksof rigid immobilization48. The joint lavage fluid levels ofinterleukin 1�, TIMP-1 and the concentration of chondroitin

Table IIResults of several studies on the effect of immobilization on the biochemical characteristics of articular cartilage


Behrens et al.19 6 weeks, rigid Dry weight Overall DecreasePG synthesis (explant culture) Overall Decrease (−48%)Low mol. weight PG Overall IncreaseNewly synthesized aggregates Overall No changesHexuronic acid Med Femur Tendency to decrease

Lat Femur DecreaseMed Tibia DecreaseLat Tibia DecreasePatella Decrease

6 weeks, non rigid Dry weight Overall Tendency to decreasePG synthesis (explant culture) Overall Tendency to decrease (−34%)Low mol. weight PG Overall No changesNewly synthesized aggregates Overall DecreaseHexuronic acid Med Femur Tendency to decrease

Lat Femur Tendency to decreaseMed Tibia Tendency to decreaseLat Tibia Tendency to decreasePatella Decrease

Setton et al.23 4 weeks, non-rigid PG: collagen ration Distal Femur No changesWater content Femur No changes

Tibia No changes8 weeks, non-rigid PG: collagen ratio Distal Femur No changes

Water content Femur No changesTibia Decrease

Muller et al.40 4 weeks, non-rigid Water content Tibia* No changesFemur** No changes

PG content Tibia* DecreaseFemur** Decrease

Collagen content Tibia* No changesFemur** No changes

8 weeks, non-rigid Water content Tibia* DecreaseFemur** No changes

PG content Tibia* No changesFemur** No changes

Collagen content Tibia* No changesFemur** No changes

Haapala et al.41 11 weeks, rigid PG content Patella, tibia and femur DecreaseUronic acid content Patella, tibia and femur Decrease

Haapala et al.21 11 weeks, rigid PG content Med femur Decrease (−30 to −44%)Med tibia Decrease (−29%)

Haapala et al.20 11 weeks, rigid PG content Lat Tibia and Femur No changesGrumbles et al.47 28 days, non-rigid MMP-2 Femur Increase

TIMP Femur IncreaseHaapala et al.48 11 weeks, rigid Chondroitin sulphate Synovial fluid Decrease

MMP-3 Synovial fluid No changesTIMP-1 Synovial fluid Decrease

Leroux et al.26 4 weeks, non-rigid Water content Medial tibia No changesPG content Medial tibia No changes

PG=proteoglycan, GAG=glycosaminoglycan, MMP=matrix metalloproteases, TIMP=tissue inhibitors of metalloproteases,Prox=proximal, Post=posterior, Ant=anterior, Lat=lateral, mol=molecular, *=full thickness, **=surface zone.


sulfate were decreased. In contrast to Grumbles’ data,MMP-3 was not affected by the immobilization period. Jointremobilization during 50 weeks restored the decreasedconcentrations of markers to control levels.

The other solid part component of articular cartilage,namely the collagen, appeared to be very resistant toreduced joint loading, as shown in several studies20,21,23.No changes in concentration could be detected (Table II).However, immobilization reduced the amount of collagencross-links after an 11 week period of rigid immobilization inthe femur of dogs. Remobilization restored cross-link levelsto normal21. The ability of articular cartilage to sustainrepeatedly applied compressive stresses over a lifetime ofnormal joint function is attributed to the tightly wovencollagen network and the high concentration of proteogly-cans49. It has been suggested that once the integrity of thisnetwork is lost, the cells might be exposed to abnormallyhigh stresses50. The observation of a decreased numberof collagen cross-links might therefore result in altered bio-mechanical properties and, ultimately, lead to an abnormalbiological response of the residing chondrocytes.

EFFECT OF IMMOBILIZATION ON BIOMECHANICAL PARAMETERS

The effect of 11 weeks’ immobilization on the stiffness ofarticular cartilage in the canine knee was investigated byJurvelin and associates22. They characterized the mechan-ical properties in 20 different locations using the in situindentation method (Fig. 3). In an indentation test a punchwith a certain radius (a) is pressed under constant load (P)normally to the articular surface and the deformation (�) ismeasured at specific time points during the test55.

From the deformation at a given time point (t0), theelastic modulus (E(t0)) is calculated as follows:

K is a geometrical scaling function (using the value ofthickness at the measuring point) and � the Poisson’s ratio.In cartilage, the instant elastic deformation after applicationof the constant load is followed by a time-dependent creepdeformation. From the obtained load-displacement curves,they calculated the instantaneous elastic modulusE(t0=0s), and the modulus 15 s after load applicationE(t0=15s). The retardation time spectrum, which character-izes the time-dependent deformation in viscoelasticmaterial, was calculated using the 2 s and 15 s shearcompliance (shear compliance=1/shear modulus), accord-ing to the method described by Parsons and Black56. Theretardation spectrum is then used to assess the rate of fluidflow.

Splinting of the knee joint caused an overall softening ofthe articular cartilage22. The value of the instantaneouselastic modulus decreased by 17%, and the 15-s elasticmodulus was reduced by 25% compared with the controldogs (Table III). After 11 weeks of immobilization, thechanges in E(t0=0s) were found at four indentation points,three on the femur and one on the tibia. Six indentationpoints on the femur and three on the tibia showed signifi-cant changes in the 15-s elastic modulus. The rate of fluidflow under compression, used as a measure for the tissuepermeability, increased in the cartilage of the immobilizedleg. As reported in earlier studies all biomechanical par-ameters showed large variability depending on their ana-tomical location22,57. After splinting, softening was moreremarkable in non-contact areas of the joints studied22.This classification in contact vs non-contact areas wasestimated radiographically and is purely indicative.

In contrast to these results, the instant shear modulusG(t0=0s) of cartilage on compressive loading measured bymeans of the in situ indentation test did not change after arigid immobilization during 11 weeks in female Beagle dogs(Table III)20. In this study only the summit of the lateral

Articularcartilage

Rigiddie

Load

Loading pad withspherical punch

Subchondralbone

B C

A

σo

Time

Com

pres

sive

load

B

C

A

ε∞

Time

Cre

ep d

efor

mat

ion

Copiousfluid

exudation

Equilibriumdeformation

No exudation

Fig. 3. Setup of an unconfined indentation method and the typical biphasic behavior of articular cartilage. When compressive stress (�0) isapplied to the tissue (a), fluid exudes, resulting in compaction of the matrix (b). At equilibrium (c), there is no fluid flow; the equilibrium

compressive strain of the solid matrix is given by �0 (adapted from Armstrong et al., 1980 with permission)55.

Biomechanical changes

DEFORMATION OF NORMAL ARTICULAR CARTILAGE UPON

APPLICATION OF LOAD

Articular cartilage is a viscoelastic material, with twodistinct phases: a solid phase (the organic solid extracellu-lar matrix) and a movable fluid phase (the interstitial waterwith the inorganic salts dissolved in it)14. It can be consid-ered as a fluid-filled, porous-permeable medium. Bio-mechanically, articular cartilage absorbs and distributesload and provides a smooth, lubricated surface that facili-tates movements with little friction between the articulatingsurfaces. Biomechanical properties of articular cartilageshow distinct topographical variations in human andanimal joints22.

The compressive viscoelastic behavior of articular carti-lage is due primarily to the flow of the interstitial fluid and tothe intrinsic viscoelasticity of the matrix51. Articularcartilage also exhibits viscoelastic behavior in tension52,which is attributable to both the internal friction associatedwith polymer motion and the flow of the interstitial fluid. Intension, the tissue is strongly anisotropic (being stifferand stronger for specimens harvested in the directionparallel to the split line pattern than for those harvestedperpendicular to this pattern)53. Also, cartilage is inhomo-geneous53. These anisotropic and inhomogeneous charac-teristics are believed to be due to the varying collagen andproteoglycan structural organizations of the joint surfaceand the layering structural arrangements found within thetissue54.


condyle of the femur and the central point of the intermedi-ate part of the lateral condyle of the tibia were mechanicallytested. The instant modulus E(t0=0s) and instant shearmodulus

were calculated to indicate the tissue stiffness according toHayes et al.58. These results are similar to data gathered atapproximately the same indentation points in a study pre-viously published by the same investigators (Table III)59.Another remarkable point is that in this previous study, twopoints very close to each other (i.e. central and marginalintermediate points on the lateral tibia (TLIc and TLIm)),can largely differ in terms of E(t0=0s). In the control dogsthis parameter is already much larger at TLIm than at TLIc.The effect of immobilization on the E(t0=0s) as well as onthe E(t0=15s) is much more pronounced in TLIc than TLIm.Because of this important site-dependency, it is extremelydifficult to compare results.

The equilibrium shear modulus measured 1 h after loadapplication i.e. G(t0=3600s) decreased in the lateral femurby 28% and in the lateral tibia by 25% (Table III)20. In thisstudy, the instantaneous modulus did not change but theequilibrium modulus was reduced, i.e., the articular carti-lage matrix alone was softer. After 11 weeks, the dogs wereallowed cage activity for 50 weeks. The equilibrium shearmodulus recovered to control levels in the tibia but was stillreduced by 15% in the femur. These observations showthat the effects of immobilization are not totally reversible.

Leroux and co-workers calculated the equilibrium shearmodulus from linear regression of the equilibrium shearstress as a function of the applied shear strains over thefour step-wise applied strain increments (E0=0.005, 0.01,0.02, and 0.03)24. The resulting torque was monitored for1200 s, followed by a 600 s recovery period upon removal

of the strains. They observed even a bigger reduction ofapproximately 70% for the equilibrium shear modulus in themedial tibia following 4 weeks of joint immobilization (TableIII). They found an equal decrease for the dynamic shearmodulus, which was calculated by applying an oscillatorydisplacement, corresponding a shear strain with an ampli-tude of 0.01 and an angular frequency from 0.1 rad/sto 100 rad/s. Calculations assume a linear viscoelastic be-havior. In contrast, the equilibrium elastic modulus didnot change.

Setton et al. reported no significant changes in compres-sive properties of the articular cartilage after 4 or 8 weeksof joint disuse (Table I)23. The non-rigid immobilization wasused as a model for joint disuse, eliminating weight-bearingforces across the joint, while allowing for some limited limbmotion. The indentation tests were performed at two siteson the medial tibial plateau, corresponding to the anteriorand posterior sites of contact to the femoral condyle. The insitu indentation protocol and biphasic theoretical analysiswere used to numerically determine the permeability of thearticular cartilage, and the elastic properties of the matrix(aggregate modulus corresponding to the equilibriummodulus and Poisson’s ratio). In contrast to Jurvelin etal.22, no changes were found. However, looking at theequivalent positions (tibia medial anterior and posterior) inthis study, no changes were observed in this latter study.The period during which the animals were immobilized wasalso shorter in this study. Setton et al. used female dogs ofolder age and the immobilization method was different23.The same group studied the effect of non-rigid immobiliz-ation on the tensile elastic modulus of the femur23. Teststrips of articular cartilage were placed in the jaws of thetesting apparatus and straightened under a tare load of0.02 N. Series of successive tensile stress-relaxationexperiments was then performed as described23. A signifi-cant increase of the elastic modulus after 8 weeks ofimmobilization was found in the distal groove of the femur,

Table IIIResults of investigations looking at the effect of immobilization on the biomechanical properties of articular

cartilage


Jurvelin et al.22 11 weeks, rigid Instant EM Femur, tibia and patella Decrease (−17%)15-second EM Decrease (−25%)

Haapala et al.20 11 weeks, rigid ESM Summit femur lat Decrease (−30%)Summit tibial lat Decrease

ISM Summit femur lat No changesSummit tibial lat No changes

Setton et al.23 4 weeks, non-rigid Compressive EM Medial tibia post No changesMedial tibia ant No changes

Tensile EM Lat femur ant Tendency to increaseLat femur post Tendency to increaseDistal groove femur Tendency to increaseProx groove femur Tendency to increase

8 weeks, non-rigid Compressive EM Medial tibia post No changesMedial tibia ant No changes

Tensile EM Lat femur ant Tendency to increaseLat femur post Tendency to increaseDistal groove femur IncreaseProx groove femur Tendency to increase

Leroux et al.26 4 weeks, non-rigid EM Medial tibia Trend to decreaseESM Medial tibia Decrease (−75%)Dynamic SM Medial tibia Decrease (−71%)

EM=elastic modulus, ESM=equilibrium shear modulus, ISM=instant shear modulus, Prox=proximal,Post=posterior, Ant=anterior, Lat=lateral.


where the femur is in contact with the patella (Table III).There was already a clear tendency to this result after4 weeks in all measured points. A period of 3 weeksof remobilization after 4 or 8 weeks of immobilization didnot restore the value for the tensile modulus measuredin control cartilage.

Indeed, there is a lot of discrepancy between resultsfrom different studies. This discrepancy may be related todifferences in the protocol used for biomechanical testing,the type and duration of immobilization, and finally the ageand breed of the animals used. Inherent to the indentationmethod, used in most biomechanical investigations, is thesite-dependency, which renders a direct comparisonbetween results from different studies very difficult. Also,the technique is invasive since it requires the opening of thejoint to expose the articular surfaces. Additionally, in theanimal studies discussed, the gathered data were fitted todifferent models. Jurvelin et al. assumed that articularcartilage can be modeled as an elastic layer with finitethickness bonded to a rigid half-space60. In contrast, Settonet al. applied the biphasic theory and calculated thepermeability, aggregate modulus, shear modulus, andPoisson’s ratio23. One of the reasons for discrepancybetween Leroux’s results26 and the previous results20,23

may be that torsional shear, used in this study, directlymeasures the average response of the full-thicknesssample, while compressive indentation testing is stronglyinfluenced by the properties of the superficial articularcartilage and the Poisson’s ratio needed for the theoreticalsolution.

Although it is an invasive technique, indentation hasbeen recently used to assess biomechanical properties ofarticular cartilage in humans61,62. Other non-invasivemethods to asses human articular cartilage, albeit not froma biomechanical point of view, will be presented in the nextsection.

The technique is fast, cheap, and non-invasive. The prob-lems of ultrasound are the lack of reproducibility and itsinsufficient accuracy68,69. Additionally, this method is onlyapplicable to superficially located joint surfaces, and theposition of the images relative to the joint surface is hard todefine. Computer tomography (CT) arthrography is inva-sive, requiring a contrast agent to be injected into the jointcavity70,71. Its routine use in clinical medicine is thereforeproblematic and the technique cannot be employed inhealthy volunteers. Diagnostic arthroscopy is consideredthe gold standard for evaluating surface alterations. Theexamination can provide information on cartilage mechan-ical properties if an arthroscopic indentation instrument isused61,62. Again, this method is invasive, which restricts itsroutine clinical use. Additionally, it is very difficult to definethe measuring location in an accurate and reproducibleway, which is essential for cross-sectional as well aslongitudinal studies.

Magnetic resonance imaging (MRI) is presently the mostaccurate imaging modality to evaluate the articular carti-lage, due to the high soft tissue contrast and the oppor-tunity to evaluate the total joint surface (Fig. 4). MRI uses astrong static and high frequency spatially inhomogeneousmagnetic fields to obtain sectional images. In MRI, thetissue contrast can be substantially modulated by choosingdifferent types of pulse sequences, and by changing thespecific parameters of the sequences (repetition time, echotime, flip angles, etc). For the analysis of cartilage macro-morphology, the bone-cartilage interface and the articularsurface need to be delineated accurately72,73. In particular,the spatial resolution must be sufficient to permit quantita-tive measurements. Therefore, a high resolution fat sup-pression gradient sequence is used to visualize thearticular cartilage with a high contrast to the surroundingtissues (Fig. 4)74–76. Fat-suppression is achieved by apply-ing a pre-pulse, preventing the fat-bound protons fromcreating a signal during the subsequent data acquisition77.However, sequences with selective excitation of only thewater-bound protons have been introduced recently, inwhich fat-signal elimination can be obtained with muchshorter acquisition times78–80. Quantitative evaluationrequires some image-processing steps including the seg-mentation of the cartilage, 3D analysis (3D reconstructionand 3D thickness computations), and potentially 3Dregistration.

Knee-joint cartilage volume measurements did not devi-ate more than 5–10% on average from data collected fromanatomical sections73, and from CT arthrography81–83. Thethickness distribution calculated out of the MRI showed nodifferences compared to histological sections84, A modeultrasound74,81 and stereophotogrammetry85. The repro-ducibility has been studied in healthy volunteers andpatients, by repeating measurements after joint reposition-ing and reshimming of the magnet86–88. The reproducibilityof cartilage volume and thickness measurement are rela-tively high, ranging between 1.5% and 3.8%88. The carti-lage volume and thickness results agreed within 4%between the fat-suppression and the water excitationsequence with slightly higher values for the water excita-tion79. Another potentially valuable image sequence forquantitative cartilage imaging is driven equilibrium Fouriertransform imaging (DEFT)89. It generates contrast betweencartilage and joint fluid by enhancing the signal from thejoint fluid, rather than by suppressing the cartilage. This is apromising technique but the accuracy and precision ofquantitative measurement remains to be established. Bio-chemical information of the cartilage can be obtained by

Monitoring cartilage changes in humans

Animal studies showed that immobilization result in sev-eral major changes of articular cartilage. Mechanical, bio-chemical, and morphological characteristics are alteredand do not always totally recover upon remobilization of thejoint. Due to the lack of an accurate, reproducible, andnon-invasive method to characterize articular cartilage,quantitative data about changes in human articular carti-lage after a given period of immobilization are not yetavailable. However, novel techniques will allow themeasurement of morphological parameters in the nearfuture.

Conventional radiography can provide indirect informa-tion about cartilage destruction from signs such as ‘jointspace’ narrowing, subchondral sclerosis or cysts, or thepresence of osteophytes63,64. However, it is a two-dimensional technique that is sensitive to artefacts result-ing from malpositioning65. Another disadvantage ofradiography is the unability to differentiate between femoraland tibial cartilage loss. Radiography is less sensitive tolocal than to general cartilage lesions, and cannot demon-strate the pattern of cartilage destruction throughout thejoint surface. Due to these limitations, measurements of thejoint space width in radiographs are not ideal for reliablyevaluating cartilage thickness and surface alterations. Anattempt to use ultrasound for determination of articularcartilage thickness has been made by McCune et al., Aisenet al., Castriota-Scanderbeg et al., and Martino et al.66–69.


using the negatively charged contrast agent gadoliniumdiethylene triamine pentaacetic acid (Gd(DTPA)2−). Thiscontrast agent distributes in cartilage in inverse relation tothe negatively charged GAG concentration. Using thispremise, the spatial distribution of GAG can be easilyobserved in T1-weighted and T1-calculated MRI studies inintact human knee joints90, with good histological corre-lation. Furthermore, in vivo clinical images of T1 in pres-ence of (Gd(DTPA)2−) correlated well with the validated exvivo results after total knee replacement surgery90. It couldbe demonstrated that degenerated cartilage had a higherGd(DTPA)2− uptake than normal cartilage91. ConsequentlyGd(DTPA)2− enhanced MRI gives us the opportunity to getbiochemical and structural information from articular carti-lage. However, it necessitates the injection of contrastagents, which is invasive and risky.

Several studies showed the power of MRI to detectedearly degenerative changes in articular cartilage91–94.Thut et al. used six different image sequences and aself-developed score system which takes into accountthe signal abnormality, cartilage thickness change, andsurface irregularity92. Early degenerative changes weredistinguished in thicker cartilage regions of rabbit knees,whereas some changes were still undetectable at locationswhere the cartilage is very thin. This demonstrates not onlythe power of MRI to detect early degenerative cartilagechanges but also the importance of image resolution. Calvoet al. showed that focal increases in cartilage thickness isone of the earliest measurable changes in osteoarthritisand precedes subchondral bone remodeling93. Four weeksafter partial medial meniscectomy in the left knee of rabbits,the femoral articular cartilage was already significantlyincreased. Tibial cartilage showed a significant increaseafter 6 weeks, while no changes were detected in themicroradiographs of the subchondral bone even 10 weeksafter inducing OA. Measuring the cartilage thickness vari-ations with MRI can be used to follow the course of OA andto evaluate potential beneficial effects of novel therapies.

Another parameter derived from MR images is the T2map94. This map is generated, on a pixel by pixel basis, byextrapolating from the signals obtained at two differentecho times. Significant T2 differences are observed

between healthy volunteers and osteoarthritic patients. TheMRI technique for T2 mapping complements the high-resolution cartilage thickness and volume measurementtechnique. Vanwanseele et al. showed recently that carti-lage thickness decreases after spinal cord injury95. Theyused MRI to monitor systematically morphological changesin articular cartilage. Preliminary results showed that after6 months of immobilization, there is already a clear tend-ency to thinning in medial tibia. Twelve months after thespinal cord injury the decrease in cartilage thickness waseven bigger.

Fig. 4. Methods for quantitative 3D analysis of cartilage morphology from MR imaging. Panel (a) Coronal MR image of the human kneeobtained with a fat-suppressed gradient echo sequence; segmentation of medial tibial cartilage with a B-spline Snake algorithm. Panel (b)

3D volume reconstruction of the medial and lateral tibial cartilage.

Summary

Articular cartilage alterations following immobilizationhave been thoroughly studied in laboratory animals. Theseinvestigations demonstrated that stress deprivation altersthe morphological, biochemical and biomechanical charac-teristics of articular cartilage. Data about changes in humanarticular cartilage after immobilization are not yet available.In healthy volunteers and in patients suffering from severeOA, cartilage thickness and volume could be measured byMRI using a special pulse sequences combined with thestate-of-the-art visualization tools. This technique allowedalso first assessments of how physical activity could influ-ence articular cartilage morphology. Measuring parameterssuch as tissue volume and mean or maximal cartilagethickness give some insight into the influence of unloadingand/or restricted motion on the articular cartilage in patientsimmobilized for a certain period. Such data can provideimportant information on the effects of immobilization onhuman articular cartilage and on the time course of thesechanges. This knowledge will help physicians and thera-pists in the planning and optimization of rehabilitationprograms after surgical procedures or prolonged motionrestriction or immobilization.

Acknowledgments

Support for this project was provided by the Swiss Para-plegic Foundation, Basle, Switzerland.


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the effects of immobilization on the characteristics of articular cartilage: current concepts and...

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