Molecular biology and biomechanics of normal and healing ligaments--a review* 1

Osteoarthritis and Cartilage (1999) 7, 130–140? 1999 OsteoArthritis Research Society 1063–4584/99/010130+11 $12.00/0Article No. joca.1998.0168, available online at http://www.idealibrary.com on

Molecular biology and biomechanics of normal and healingligaments—a review

B C B. F, D A. H N G. SThe McCaig Centre for Joint Injury and Arthritis Research, University of Calgary, Calgary, Alberta,

Canada T2N 4N1

Summary

Objective: In this review article, we discuss current data and concepts concerning the molecular biology andbiomechanics of both normal and healing ligaments in a rabbit model.

Method: Data is presented from light microscopy, transmission electron microscopy, molecular biology (RT-PCR),and biomechanical measurements (laxity, stress at failure, modulus, and static creep) of normal, pregnant and healingrabbit medial collateral ligaments.

Results: ‘Flaws’ in scar matrix, smaller-than-normal diameter collagen fibrils, and failure of collagen cross-linkmaturation may be particularly important deficiencies which appear to be related to ligament scar weakness andperhaps to scar creep. The mechanical behaviours of both normal and healing ligaments are altered by relative statesof joint motion and normal ligaments are a#ected by systemic hormones (particularly during pregnancy).

Discussion: Molecular analysis of ligaments and ligament scars, combined with ongoing morphological andbiomechanical studies of ligament structure and function, will ultimately reveal which factors can be manipulatedclinically to optimize the restoration of normal ligament properties after ligament injuries. Further studies on themechanisms of ligament healing, genetic markers of repair, and gender-specific di#erences in ligament repair responsesare required.

Key words: Ligament, Scar, Healing, Collagen.

Introduction

AS NOTED in the previous article, ligaments andtendons have very interesting biomechanical prop-erties which are highly adaptive for the complexfunctions that each of these structures must serve.These properties are the collective product of all ofthe cells within that structure during its normalgrowth and development; ultimately producing anadult tissue which can perform complex biome-chanical roles while maintaining some ability tochange, if necessary, during aging [1, 2]. In fact,whilst ligaments were once thought to be almostinert, it has now been recognized that they areresponsive to a number of local and systemicfactors which influence their cells. Changes inloading (e.g., during exercise or immobilization)are particularly well described local stimuli tochanges in ligament and tendon cell behaviours

130

[3–11] during growth [12, 13] but also in adulthood[14]. Hormone variations, on the other hand, haveonly recently been recognized as a stimulus forligament cells to change, producing surprisingalterations in ligament properties during preg-nancy [15] as well as important implications to thegenesis of ligament injuries in females [2, 16].Injury to a ligament induces a dramatic change inits structure and in its biology, creating a situ-ation in which ligament functions need to restoredby a healing process during which both cellularcomposition and loading are altered, and in whichhormone variations may still be influential.

In this article, we review briefly the cell andmatrix changes in a healing ligament versus anormal ligament in a rabbit model. For more com-prehensive sources of information to ligament biol-ogy and biomechanics, the reader is referred toseveral excellent reviews on these topics [17–22].

Sources of Support: Alberta Heritage Foundation forMedical Research, Medical Research Council of Canada, TheArthritis Society of Canada.

Address correspondence to: Dr C. Frank, Department ofSurgery, 3330 Hospital Drive N.W., Calgary, Alberta, CanadaT2N 4N1. Tel.: (403) 220-6881; Fax: (403) 283-7742; E-mail:[email protected]

Normal ligament structure

Normal skeletal ligaments are defined as densebands of connective tissue which stabilize jointsand guide joint motion. They have the gross

Osteoarthritis and Cartilage Vol. 7 No. 1131

appearance of being made up of a collection ofdensely packed fibres which span a joint and thenbecome anchored to the bone at either end (Fig. 1).On closer inspection, surfaces of ligaments oftenhave a membrane on them (an ‘epiligament’ forextraarticular ligaments or synovium on intra-articular ligaments) which can obscure theirfibrous detail. As this membrane is stripped away,the fibrous architecture of the ligament becomeseven more apparent in some ligaments, with ahierarchical organization into groups of fibresknown as ‘bundles’. It is di$cult to separate thesebundles, suggesting that they are interconnectedin some ways. With joint movement there is clearlysome interaction between fibres as they maintainthe gross appearance of a single structure whilesome of them can be seen to tighten or loosendepending on the bone positions and the forcesthat are applied.

Under a microscope, ligaments are appreciatedto be much more complex. The epiligament can beseen to extremely cellular and vascular, also con-

taining some sensory and proprioceptive nerves.The ligament itself is much less vascular, but doeshave some organized blood supply and some inner-vation. The physiological impact and implicationsof these nerves and vessels are just beginning to bestudied in detail [23–25].

The most obvious histological feature of liga-ments is that they contain relatively few cellsscattered among a longitudinally aligned fibrousmatrix. These cells are mainly spindle shapedfibroblasts or fibrocytes which are generallyaligned with the matrix. Within vascular profiles,some endothelial cells can also be seen. Fibrebundles are appreciated along with a wavy appear-ance to the collagen fibres, known as its ‘crimp’.Other components of this fibrous matrix can beenhanced with other stains or immunohistochemi-cal techniques (e.g., minor amounts of elastin,proteoglycans, actin, etc). These histological fea-tures of normal ligaments, including their verycomplex insertions into bones have been describedin detail elsewhere [26–28].

Ultrastructural studies have revealed that liga-ment fibroblasts have long cell processes withsome apparent gap junctions between cells. Thevast majority of collagen fibres are aligned alongthe long axis of the ligament with a few smallfibres appearing to ‘cross-connect’ in some way.Three dimensional orientations of fibres and truefibre connections along the lengths of ligaments,however, remain obscure. Transmission electronmicroscopy has shown that collagen fibrils withineach collagen fibre exist in a variety of sizes fromabout 60 nm to about 4000 nm in diameter [29], andthat elastin, proteoglycans and minor collagens(e.g., type VI collagen) are found within the inter-fibrillar spaces in some very interesting distribu-tions [30]. Some of these components have beenshown to change quantitatively during growth,maturation and aging and vary as a function ofanatomic location along the length of the liga-ments as well [31]. The functional role of each ofthese components is currently being studied, withthe tensile load-carrying collagens (types I and III)receiving the greatest focus and what are thoughtto be elements which contribute to the viscoelasticproperties of ligaments (water, proteoglycans, elas-tin, minor collagens) also beginning to receivesome attention.

FIG. 1. Gross appearance of a normal rabbit medialcollateral ligament (MCL).

Comparing normal ligaments to healingligaments

It must first be made very clear that it has longbeen recognized that there are di#erences in heal-ing characteristics between di#erent ligaments,

132 Frank et al.: Ligament biology and biomechanics

making it di$cult to provide a review of a ‘typicalhealing ligament’. In fact, the anterior cruciateligament (ACL) of the knee appears to have par-ticularly poor healing capacity, for reasons whichare being defined [32–34]. The medial collateralligament (MCL) of the knee has been studied morethoroughly, since it does have a more consistenthealing capacity that can be evaluated [35]. Inparticular, it can provide insights into how heal-ing ligaments di#er from normal ligaments, evenunder what are assumed to be ‘reasonable healingcircumstances’ (in an extraarticular compartmentwhich is vascular and cellular, and in whichstresses are low).

Even though the rabbit MCL heals under whatmay be called ‘better circumstances’, it still healswith ‘scar tissue’ [36]. The new tissue appears to beformed by similar cellular and matrix processes tothose described in wound healing [36]. This anal-ogy invokes potential applications of a great dealof other information to the study of ligaments, butit does not eliminate the need to understandspecific processes and products in injured liga-ments, since the biomechanical properties of theseproducts define their functions.

Healing MCLs undergo many changes duringthe healing process. These changes are driven bycellular processes can be classified into at leastthree gross, overlapping, sequential stages: bleed-ing and inflammation (days to weeks), cell prolifer-ation with matrix production (weeks to months),and matrix remodelling (months to years). Ratherthan describing these changes in detail, a few keyfactors will be emphasized [37].

Scar tissue in a healing ligament is formed bythe cells which proliferate in the ligament gap.These cells are a mixture of fibroblasts, inflamma-tory cells and vascular cells [38]. The matrix thatthey produce is initially very disorganized, how-ever it organizes over time. That reorganization ispart of the remodelling process. Remodelling doesnot progress to the point of recreating a normalligament and it appears that the scar remainsdi#erent from normal ligaments in some importantways. First, it appears that scars contain a numberof ‘flaws’ [39]. While the number and sizes of theseflaws (fat cells, inflammatory foci, blood vessels,loose collagen and disorganized collagen) de-creases over time, it doesn’t appear that they areever completely removed (Fig. 2). Secondly, while

it appears that there are some changes in thequantities of various key matrix components overtime, with some of them recovering to withinnormal ligament quantities [40], other matrix com-ponents likely do not recover as well. It wouldappear that there are chronic abnormalities inproteoglycan types [41] collagen types and colla-gen cross-links [42] in MCL scars. Thirdly, itappears that collagen fibrils in scars are smaller indiameter than normal fibrils for several years fol-lowing the injury [43]. While some small increasesin fibril size do occur during the second year ofremodelling [44], they remain abnormally small(Fig. 3) for reasons which have not yet beendefined.

Healing ligaments exhibit changes in their bio-mechanical properties over time as a result of theproliferation and remodelling of matrix notedabove. Given the highly organized heterogeneity oftendons and ligaments, it is not surprising that theresponse to injury is unable to replicate or restorethe original structural organization. The conse-quence is an inability to match structural ormechanial behaviour even two years after injury.In general, there are improvements in load-deformation properties over the first few months ofhealing, however the properties of a normal unin-jured ligament are likely never restored [45]. Heal-ing ligaments appear to permanently remainweaker and less sti# than normal MCLs. Thesedeficiencies are influenced by the size of the gapbetween ligament ends [35] and by joint motion[46, 47]. Decreasing the size of the gap betweenligament ends and some joint motion both appearto enhance scar ultimate strength and sti#ness tosome extent. In a material sense, however, thesetreatments do not appear to enhance scar qualityby any appreciable amount. While there are someimprovements in MCL scar material strength andsti#ness over the first 3 months of healing, scarsnever regain more than about 30–40% of normalMCL properties [37, 45] for reasons which remainunclear. Fiber malalignment [39, 48], failing colla-gen cross-link maturation [42], and the failure ofscar collagen fibrils to mature into normal sizefibrils [43] have all been noted as potential causesof scar weakness, however, which is the mostimportant has remained obscure.

Recently, a specific association has been madebetween fracture mechanics and the strength ofthe healing rabbit MCL [39]. In fracture mech-anics, flaws in a material are recognized as beingthe cause of fracture under tensile loading. Cracks


FIG. 2. Histologic appearances of midsubstance ‘flaws’ within rabbit MCL scars, showing di#erent types of defectswithin the new matrix: (A) a blood vessels, (B) fat cells, (C) loose collagen, (D) disorganized collagen, (E) inflammatorysite with little matrix, (F) a combination of all. All Haematoxylin and Eosin stain #125 magnification (Fig. 2 from

Shrive et al. [41], with permission).

propagate from flaws perpendicular to the direc-tion of applied tension with sharp thin voids beingthe most dangerous. When the stress intensity atthe tip of the void reaches the fracture toughnessof the material, the crack propagates. Fracture ispredicted from the critical flaw, typically the larg-est void perpendicular to the applied stress. The‘flaws’ in a healing ligament are described above.Over time from injury the number and size of theseflaws decreases. Also the collagen fibers align withthe applied tension, so the fracture toughnessincreases. Strong associations were found betweenthe number and size of flaws, and the stress atfailure of the tissue. The number of flaws presentwas also expected to a#ect the overall sti#ness ofthe healing ligament, with more flaws providingless sti#ness [39]. Again, a strong association wasfound between the area of the cross-section thatwas ‘flaw’ and the elastic modulus of the material.(Fig. 4).

Soft tissues however, are not subject to highstresses for the majority of their working life.

Recent evidence [49, 50] has suggested that thestrain levels induced in the ACL by various pas-sive and active movements in both humans andanimals may remain within the toe region ofa typical stress–strain curve. The tissues aresubject to repeated low loads. The response of aligament under these loading conditions in theviscoelastic one of creep. Creep is the increase instrain over time when the material is subject toconstant (or repeated) stress. We have begunto analyze the response of MCLs and MCL scarsto repeated low loads in vitro: scar creepsconsiderably more than normal ligament [51]. Itappears that MCL scars subjected to cyclicand static loads which are only a fraction oftheir failure loads respond by creeping more thantwice as much as a normal MCL (Fig. 5). Thenormal MCL was surprisingly found to creepless than would have been anticipated fromprevious load relaxation tests [52], we suspect, dueto fibre recruitment. Thus, we have speculatedthat normal ligaments may be highly adapted to


resist creep; a property which scars may neverobtain.

Analysis of normal and healing ligaments usingthe tools of molecular biology has been the subjectof investigation in recent years. Such investiga-tions have been hindered by a lack of e#ectivemethods to study very hypocellular tissues, as wellas a relative paucity of appropriate reagents toinvestigate tissues from many of the species com-monly used to investigate the mechanical proper-ties of the tissues (i.e., rabbits, goats, sheep etc.compared to rodents). Recently, we have developeda method which reproducibly yields intact RNAfrom ligaments, tendons, cartilage, meniscus andother dense connective tissues [53]. This methodol-ogy has greatly facilitated the study of normal and

healing ligaments at the molecular level. Thesecond advance in this area has been the devel-opment of rabbit-specific reagents to investi-gate this mechanically and biochemically wellcharacterized model. As summarized in Table I,rabbit-specific primers for reverse transcription-polymerase chain reaction (RT-PCR) analysis ofa number of molecules relevant to connective tis-sues have been developed, validated and used inthis model system. These include matrix mole-cules, proteinases and inhibitors, growth factorsand receptors, hormone receptors, inflammation-related molecules and house-keeping genes. Whilethe methods to date have been applied to a semi-quantitative analysis of RNA from normal andhealing ligaments, some very interesting findingshave been obtained.

With regard to ligaments specifically, the mol-ecules identified in Table I with * have beendetected in RNA extracted from normal rabbitMCL. Analysis of some of these molecules from3 week MCL scar tissue by semiquantitativeRT-PCR has revealed that a number of the tran-scripts are elevated in the scar tissue compared tonormal tissue from age-matched controls [54–56].With regard to matrix molecules, as shown in Fig.6, some of the transcripts for a subset of matrixmolecules are significantly elevated in scar andthis correlates with recent biochemical evidence[41] Thus, there is a correlation between themolecular findings and the biochemical results,indicating that the transcripts are likely trans-lated and that regulation of mRNA synthesis andstability is very important. Similar analysis ofmRNA for growth factors and growth factor recep-tors have also indicated that some are elevated,while others are not [54]. Thus, this molecularapproach should provide new insights into themechanisms regulating ligament development andmaturation, as well as the response to injury.

Effects of gender on normal ligaments

FIG. 3. Transmission electron microscopic comparison ofcollagen fibril sizes in cross-sections of a normal MCL(A), versus fibrils in an MCL scar (B). Magnification

#30 000.

There are a number of ways in which the biome-chanical properties of joints and joint tissues canbe described. At a clinical level, individual struc-tures cannot be isolated and instead only a collec-tive mechanical behaviour can be described. Thebest example of this phenomenon is the clinicaldefinition of ‘whole joint laxity’; a crude mechan-ical test of the relative sti#ness of tensile loadcarrying structures under low loads when musclesare relaxed and bones are displaced manually fromtheir anatomic resting positions. Relative isolation


FIG. 4. Plots of flaw size and distribution against material properties of 3, 6 and 14 week MCL scars showing strongcorrelations. (A) Stress at failure versus mean area of the largest flaw; (B) Stress at failure versus the fourth root of themean area of the largest flaw; (C) Stress at failure versus the inverse of the square root of the mean of the largest flaw;

(D) Mean elastic modulus versus mean flaw area (Fig. 4 from Shrive et al. [41], with permission).

of structures can be achieved by controlling thedirection of forces and isolating a particular direc-tion of bony displacement, such as one direction oftranslation, or one rotation. The well-knownanterior drawer test of the knee in which ananterior manual force is applied to the tibia in aknee flexed at ninety degrees and the examinerassesses relative tibial displacement, is oneexample of this definition of laxity for the anteriorcruciate ligament. Using this type of gross assess-ment of all of the major ligaments, the laxity of thenormal knee has been clearly noted to varybetween individuals, with some individuals havinghypermobile joints and others less lax joints. Morewomen than men are reported to have joint hyper-mobility [57, 58] and female athletes experiencemore joint injuries than their male counterparts[16]. The basis for the gender di#erences is notdefinitively known, however hormones such asestrogen have been implicated. Interestingly,

receptors for estrogen and progesterone have beendetected in both human and rabbit ligaments byimmunolocalization [59] and by RT-PCR [54]. Inboth species, the receptors have been detected inligaments from females and males. While the dataobtained thus far is consistent with the receptorsbeing of a functional size in both males andfemales, some experiments using ligament tissuefrom male and female rabbits have indicated thatMCL tissue from female rabbits is responsive toestrogen in vitro, while that from males is notresponsive to estrogen in vitro [60]. Thus, theregulation of hormone responsiveness in these tis-sues is apparently not only based on the presenceof hormone receptors.

Ligament laxity has also been reported to bealtered during pregnancy [15]. The increases arereported to occur during the second trimester andpersist post-partum for a number of weeks [15]. Thelaxity of the isolated rabbit MCL, when tested


in vitro, also increases during pregnancy [60]. Thisdefinition of laxity is specific to this model; repre-senting a displacement measurement of very lowload tensile sti#ness of the isolated MCL as thefemur is distracted from its anatomic contactpoint with the tibia. The statistically significantincreases in this specific form of ligament laxityoccur between the second and third week of preg-nancy and are maximal during the fourth week ofpregnancy (Fig. 7). Interestingly, the high loadbehaviour of the MCL is not influenced by preg-nancy and the changes appear to be restricted tochanges in low load behaviour and laxity (Hart,Frank and Shrive, manuscript in preparation). Theextent of the changes in MCL laxity that occurduring pregnancy are very similar irrespective ofwhether the rabbit was in the first pregnancy orwas multiparous. The mechanism(s) responsiblefor these changes in laxity are the subject ofcurrent molecular analysis.

FIG. 5. Static creep curves for a typical 6 week MCL scar and normal MCL tested at 4 MPa showing that the scar creepsnearly twice that of the normal at the same stress level.

-

The studies described thus far have implicated,but not proven a role for the pregnancy-associated

hormone relaxin in the process [15]. Relaxin, amember of the insulin-like growth factor family[61], increases during pregnancy and the levelsdrop dramatically post-partum. The temporallyinduction of relaxin varies from species to species,but in the rabbit serum levels rise at approxi-mately 15 days of gestation and then drop quicklypost-partum [62]. Initially, the role of relaxin wasthought to be primarily related to relaxing thepubic symphysis, but recent findings indicate awider range of roles [61]. Interestingly, relaxin isalso found in milk and pups of mothers withprolonged expression of relaxin in the milk are athigh risk to develop hip dysplasia due to laxcapsular ligaments [62]. In vitro, relaxin alterscollagen synthesis by rabbit articular cartilage[64] and in vivo, it inhibits collagen deposition inmurine models of fibrosis [65]. Recent studieshave investigated the e#ects of porcine relaxin(from Dr Sherwood, Univ. of Illinois), sincerabbit relaxin is not available, on ligament cellsfrom rabbits. The preliminary studies haveindicated that porcine relaxin can alter the pat-tern of gene expression by these ligament cells,but the alterations are very complex. It remainsto be determined whether rabbit relaxin also


induces such complex alterations in ligament cellsand tissues.

Table IList of molecules for which rabbit-specific primers sets have been developed and validated

Matrix Molecules Proteinases/Inhibitors*Collagen I, II, III *MMP-1 (Collagenase)*Biglycan *MMP-3 (Stromelysin)*Decorin *Urokinase*Lumican *TIMP-1, -2, -3*Versican *PAI-1, -2Aggrecan*Fibromodulin Growth Factors/Receptors

*TGF-â1, -â2 *Insulin ReceptorsInflammatory Molecules *EGF PDGF-â Receptors

*IL-1 *iNOS NGF EGF Receptors*TNF *COX-2 *ET-1

*bFGFHormone Receptors* PDGF-A

*Estrogen *IGF-I, -II*Progesterone

Constitutive (Housekeeping)*Beta-Actin*GAPDH*HPRTCOX-1cNOS

Two independent clones (Tac and Pfu) were isolated and sequenced to verify the products.*Indicates transcripts detected in RNA from normal MCL tissue.

FIG. 6. Comparison of mRNA levels for matrix moleculesbetween normal rabbit MCL tissue and 3 week MCLscar. MRNA levels determined by semi-quantitativeRT-PCR and normalized to housekeeping gene (HPRT)levels. Statistical significance between groups deter-

mined by ANOVA.

FIG. 7. Laxity of MCL in non-pregnant skeletally maturerabbits (12 month old retired breeders) and skeletallymature multiparous rabbits that were 4 weeks pregnant(gestation in the rabbit=31–32 days). Statistical signifi-

cance between groups determined by ANOVA.

-

Analysis of RNA from the MCL of non-pregnant,pregnant and post-partum rabbits has indicatedthat pregnancy induces a set of complex altera-tions in the pattern of gene expression that istissue specific, age-specific (adolescent vs skel-etally mature) and somewhat ligament-specific(Hart et al. manuscript submitted). With regard to

ligaments, changes in mRNA expression in theMCL is di#erent from those of the ACL, and bothare somewhat di#erent from patellar tendon. Thecomplexity of the changes di#er from thosedetected in relaxin-treated cells, indicating thateither the e#ects in vivo have multiple etiologies,or the e#ect of relaxin on cells in vitro does notreflect its e#ects in the in-vivo milieu. In vivo,pregnancy-associated changes in matrix mol-ecules, proteinases and inhibitors, growth factorsand inflammatory molecules have been detected(Hart et al. manuscripts submitted and in prep-aration). As transcripts for some molecules areelevated during pregnancy and others depressed


(Table II), it will be di$cult to establish a simplecorrelation between the changes in mechanicalbehaviour of the ligaments and alterations in geneexpression without more detailed investigationand analysis. These studies are ongoing.

?

As discussed in previous sections, the healingprocess in ligaments is complex. Furthermore,gender-related variables appear to influence nor-mal ligament function, risk to incur ligamentinjury, as well as ligament function during preg-nancy. These findings raise the question ofwhether gender-related variables influence theoutcome of ligament healing. A search of theliterature has revealed a paucity of ‘hard evidence’one way or the other, however there are sugges-tions that gender-related factors may play a roleduring healing. This area is in need of furtherinvestigation at the mechanical, molecular, andbiochemical levels to clarify these potentiallyimportant factors which could influence outcomesin clinical populations. Studies on this topic areongoing.

Conclusions

As key stabilizers and proprioceptive elementsin diarthrodial joints, ligaments potentially servevery important roles in normal joint functioning.Following injury, it is known that the mechanicalbehaviours of ligaments are altered significantly,creating the potential for altered joint kinematics,joint dysfunction, and osteoarthritis. We are onlybeginning to appreciate the relationships betweenthe cell biology and mechanical functioning ofligaments in health and in disease. We are alsoonly beginning to recognize that there may beimportant, gender-specific di#erences in ligamentproperties with relevance to arthritis. Furtherunderstanding of the molecular, cell, and matrixbiology of ligaments, combined with furtherdefinitions of the structure–function relationshipsof ligaments, will eventually lead to improved

treatment and prevention of some forms ofosteoarthritis.

Table IISummary of statistically significant changes in mRNAlevels in the multiparous rabbit MCL during pregnancy

UnchangedElevated Depressed (not significant)

Decorin Biglycan VersicanCollagenase Type I Collagen IL-1PAI-1 Type III Collagen TGF-â1iNOS TNF TIMP-1

bFGF UK

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Molecular biology and biomechanics of normal and healing ligaments--a review* 1

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