Veterinary Surgery31:596-603, 2002

The Effect of Cranial Cruciate Ligament Insufficiency on CaudalCruciate Ligament Morphology: An Experimental Study in Dogs

TERRI A. ZACHOS, DVM, Diplomate ACVM, STEVEN P. ARNOCZKY, DVM, Diplomate ACVS,MICHAEL LAVAGNINO, MS, MSE, and SCOTT TASHMAN, PhD

Objectives—To investigate the effect of cranial cruciate ligament (CrCL) insufficiency on morphol-ogy of the canine caudal cruciate ligament (CdCL).Study Design—In vivo experimental study.Animals—Five adult foxhounds.Methods—Two years after CrCL transection, the histologic appearance of CdCLs from CrCL-deficient and unoperated contralateral control (CrCL-intact) stifle joints were evaluated using lightand transmission electron microscopy.Results—CdCLs from CrCL-deficient joints had extracellular matrix changes, characterized bychondroid metaplasia and disruption of cell architecture. Percent of small-diameter fibrils in CdCLsfrom CrCL-deficient joints was significantly greater (P �.05) than that in CdCLs from CrCL-intactjoints. Collagen fibril density in CdCLs from CrCL-deficient joints (41.09 � 5.39%) tended to be lessthan that in CdCLs from CrCL-intact joints (52.96 � 6.92%); however, this difference was notsignificant (P � .056). Mean eccentricity (ratio of minor to major diameters) of collagen fibrils wassignificantly (P � .0001) lower for CdCLs from CrCL-deficient joints (0.85 � 0.016) whencompared with that for CdCLs from CrCL-intact joints (0.87 � 0.015).Conclusions—Significant alterations were found in the morphology of CdCLs from CrCL-deficientjoints. These changes may be associated with repetitive microtrauma to the CdCL secondary toinstability or enzymatic degradation in the hostile synovial environment of an unstable joint.Clinical Relevance—Regardless of the cause, the switch to a predominantly small-diameter collagenfibril profile may reflect compromised material properties of the CdCL. This should be taken intoaccount when considering surgical techniques that rely on the CdCL to stabilize CrCL-deficientstifles.© Copyright 2002 by The American College of Veterinary Surgeons

JOINT INSTABILITY secondary to cranial cruciateligament (CrCL) insufficiency in dogs has been

used as a model for the study of degenerative jointdisease.1-7 Whereas most studies have focused onalterations in the morphologic and biochemical char-acter of articular cartilage3,6,8-10 and menisci,7,11-13

little attention has been given to potential changes inthe caudal cruciate ligaments (CdCL) of these joints.

Previous kinematic studies have demonstrated that inCrCL-deficient dogs, there is a sudden cranial sublux-ation of the tibia (relative to the femur) at paw-strike.7,14 This is followed by a caudal translation andreduction of this tibial subluxation after lift-off. Be-cause the CdCL is the limiting structure to caudalsubluxation, it is likely that this sudden caudal reduc-tion of the tibia places a repetitive stress on the CdCL.

From the Laboratory for Comparative Orthopaedic Research, College of Veterinary Medicine, Michigan State University, East Lansing,and the Bone and Joint Center, Henry Ford Hospital, Detroit, MI.

Supported by the Laboratory for Comparative Orthopaedic Research and in part by National Institutes of Health Grant No. AR43860.Address reprint requests to Steven P. Arnoczky, DVM, Diplomate ACVS, Director, Laboratory for Comparative Orthopaedic Research,

College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824-1314.© Copyright 2002 by The American College of Veterinary Surgeons0161-3499/02/3106-0013$35.00/0doi:10.1053/jvet.2002.34659

596

Microtrauma, secondary to repetitive stress, hasbeen implicated in the cause of overuse injuries inbones15-25 and tendons.26-33 The response of thesetissues to repetitive trauma has made them more proneto mechanical failure.20-24,29,30 The response of liga-ments to chronic repetitive stress, however, has notbeen investigated. It was the purpose of this study,therefore, to identify morphologic changes in theCdCL after this chronic repetitive stress in CrCL-deficient canine stifle joints. It was our hypothesis thatchronic repetitive stress to the CdCL in CrCL-insuffi-cient joints would result in significant alterations in themorphology of the CdCL.

MATERIALS AND METHODS

Caudal cruciate ligaments from 5 adult foxhounds wereused in this study. The dogs had been subjects of a previousinvestigation7 during which they underwent unilateral (rightstifle joint) transection of the CrCL. Two years postopera-tively, the CdCLs were dissected from both CrCL-deficientjoints and unoperated contralateral (CrCL-intact) joints andwere placed in 10% neutral buffered formalin.

Two 3-mm-thick sections were taken from the middleportion of the CdCLs from each of the CrCL-deficient andCrCL-intact joints. The sections were cut perpendicular tothe long axis of the ligament. One section was processed forroutine histology; the other was processed for transmissionelectron microscopy (TEM). Each section was cut intothirds, resulting in 2 abaxial sections and 1 axial section.Sections processed for routine histology were embedded inparaffin, and serial 5-�m-thick sections were cut. Thesections were stained with hematoxylin and eosin or tolu-idine blue and examined using light microscopy. Cellular-ity, vascularity, and extracellular matrix organization of theCdCLs from CrCL-deficient and CrCL-intact joints werecompared.

For specimens processed for TEM, fixed tissue wasrinsed in 0.1 M phosphate buffer, then placed in 1% osmiumtetroxide in 0.1 M phosphate buffer for 3 hours, dehydratedin graded ethanol solutions (30%, 50%, 65%, 75%, 95%,100%), and transferred to propylene oxide. Specimens werethen infiltrated with an Epon-type resin (Poly/Bed 812;Araldite, and dodenyl succinic anhydride [DDSA] in ratiosof 5:4:12 [Polysciences, Inc, Warrington, PA]). The resinwas infiltrated in 3 steps (50%, 75%, and 100% resin:propylene oxide). These three infiltration processes wereperformed for 12 hours each. The specimens were thenhardened at 60°C for 48 hours.

Thin sections from the central core of the CdCL werestained with aqueous uranyl acetate and lead citrate and thenexamined with a Philips 301 TEM electron microscope

(Philips Electronic Instrument Co, Roselle, IL). Three fieldsper section were photographed, avoiding areas containingartifacts. Photographs were taken at an original magnifica-tion of �25,000. Micrographs were printed at a finalmagnification of �68,400.

The micrographs were digitized, and quantitative assess-ment was performed using an acquisition and analysissoftware package (Scion Image; Scion Corporation, Fred-erick, MD). Five hundred fibrils from each field wereanalyzed. Major and minor collagen fibril diameters, colla-gen fibril density, and eccentricity were determined forCdCLs from both CrCL-deficient and unoperated CrCL-intact joints. Measurements were made using micrographsfrom the axial sections of the ligaments, as described above.The minor collagen fibril diameter was the value interpretedas the actual fibril diameter in all measurements (Fig 1).34-36

Using the minor fibril diameter as the actual diameter isnecessary because of the cylindrical shape of collagenfibrils. In a structure with this morphology, the minordiameter will be a true measurement of the cross-sectionaldiameter of the structure, even when the possibility of aslightly nonorthogonal section is taken into account.

Collagen fibril density was determined as the ratio of thearea of the fibrils in a standardized area to the totalarea.34,36,37 Eccentricity, a measure of collagen fibril align-ment, was determined by calculating the ratio of the minorfibril diameter to the major fibril diameter.34

The numbers of small-diameter and large-diameter col-lagen fibrils were determined for CdCLs from CrCL-intactand CrCL-deficient joints. Small-diameter collagen fibrilswere defined as those with a minor fibril diameter of �100nm; large-diameter collagen fibrils were defined as thosewith a minor fibril diameter of greater than or equal 100nm.38

Mean values for diameters of collagen fibrils, collagenfibril density, fibril eccentricity, and percent small and largediameter fibrils from CrCL-deficient joints were comparedwith these values for fibrils in CdCLs from CrCL-intact

Fig 1. Schematic demonstrating methods of determiningmajor and minor collagen fibril diameters. Collagen fibrileccentricity is calculated as the ratio of the minor fibrildiameter to the major diameter and is a measure of collagenfibril alignment.34-36

597ZACHOS ET AL

joints using a paired 2-sample Student t test. The eccentric-ity and collagen fibril density for each ligament werecompared between individuals using an analysis of variance(ANOVA) and Tukey’s post hoc test. The mean percentageof large-diameter collagen fibrils in CdCLs from CrCL-deficient joints was compared with the mean percentage oflarge-diameter collagen fibrils in CdCLs from CrCL-intactjoints using a paired 2-sample Student t test. The meanpercentage of small-diameter collagen fibrils in CdCLs fromCrCL-deficient joints was compared with the mean percent-age of small-diameter collagen fibrils in CdCLs fromCrCL-intact joints using a paired 2-sample Student t test. P�.05 was considered significant in all statistical analyses.

RESULTS

Histology

CdCLs from CrCL-intact joints had a uniform popu-lation of round to ovoid nuclei surrounded by a homo-geneous extracellular matrix (Fig 2A). The structuralorganization of the extracellular matrix appeared normalwith well-delineated fascicular membranes. No evidenceof abnormal vascularity, increased cellularity, or disor-ganization of the extracellular matrix was noted.

CdCLs from CrCL-deficient joints had evidence ofextracellular matrix disorganization when compared withthe extracellular matrix in CdCLs from CrCL-intactjoints (Fig 2A). Specifically, this was characterized bydegenerative changes such as chondroid metaplasia, hy-aline degeneration, disruption of cell architecture, cyto-plasmic distortion, loss of cell structure, and effacementof discernible cell borders. There was also a loss offascicular membrane integrity (Fig 2B). Whereas therewas variation in the extent of these changes, none of thesections of CdCLs from CrCL-deficient joints had histo-logic appearances that completely resembled those ofCdCLs from CrCL-intact joints. In sections from 4 of the5 CdCLs from CrCL-deficient joints, �90% of the cellshad nuclear and cytoplasmic distortion and disruption ofdiscernible cell borders. In the fifth, these changes werenoted in �75% of the cells.

In addition, chondroid metaplasia was noted onsections from 3 of the 5 CdCLs from CrCL-deficientjoints, comprising less than 10% of the section in thefirst ligament, approximately 25% of the section in thesecond ligament, and �75% of the section in the thirdligament.

Collagen Fibril Diameter

Relative size distributions of collagen fibrils frompaired CdCLs from CrCL-intact and CrCL-deficient

joints are shown in Fig 3. Collagen fibril diameterdistributions for CdCLs from CrCL-intact joints ex-hibited a typical bimodal pattern of large- and small-diameter collagen fibrils (Fig 4A). Large-diameterfibrils were defined as those greater than or equal to100 nm in diameter, whereas small-diameter fiberswere defined as those �100 nm in diameter.38 InCdCLs from CrCL-intact joints, 50.45 � 16.25% offibrils measured were large-diameter fibrils, whereas49.55 � 16.25% of fibrils were small-diameter fibrils.

CdCLs from CrCL-deficient joints had a predomi-nantly small-diameter collagen fibril population (Fig

Fig 2. Photomicrographs of transverse sections of caudalcruciate ligaments (CdCL). (A) CdCL from a CrCL-intactjoint. Note the clearly visible interfascicular septae (arrows)and the presence of cells throughout the section. (B) CdCLfrom a CrCL-deficient joint. The interfascicular septae areabsent, and there is a general lack of organization to the matrix(characterized by hyaline degeneration and loss of cell struc-ture). (Toluidine blue, original magnification �200.)

598 EFFECT OF CrCL INSUFFICIENCY ON THE CdCL

4B). In CdCLs from CrCL-deficient joints, 27.26 �17.28% of fibrils measured were large-diameter fibrils,whereas 72.74 � 17.28% were small-diameter fibrils(Table 1). The percent of small-diameter fibrils inCdCLs from CrCL-deficient joints was significantlygreater (P �.05) than the percent of small-diameterfibrils found in CdCLs from CrCL-intact joints.

The mean collagen fibril diameter in CdCLs fromCrCL-intact joints was 106.50 � 22.09 nm (range, 20.01to 233.03 nm), whereas the mean collagen fibril diameterof CdCLs from CrCL-insufficient joints was 78.56 �18.49 nm (range, 16.04 to 211.32 nm). The mean fibrildiameter was significantly smaller (P �.05) in CdCLsfrom CrCL-deficient joints when compared with that inCdCLs from CrCL-intact joints (Table 2).

Collagen Fibril Density

The mean collagen fibril density in CdCLs fromCrCL-intact joints was 52.96 � 6.92%. The meancollagen fibril density in CdCLs from CrCL-deficient

joints was 41.09 � 5.39%. The difference betweenvalues for CdCLs from CrCL-intact and CrCL-defi-cient joints approached but did not reach statisticalsignificance (P � .056).

Collagen Fibril Eccentricity

Eccentricity varied significantly (P � .0001) betweenCdCLs (Table 3). In 3 of the 5 dogs, the eccentricityvalue was smaller for CdCLs from CrCL-deficient jointswhen compared with that for CdCLs from CrCL-intactjoints. The mean eccentricity of collagen fibrils in CdCLsfrom CrCL-intact joints was 0.87 � 0.015. The meaneccentricity of collagen fibrils in CdCLs from CrCL-deficient joints was 0.85 � 0.016. When all meaneccentricity values were compared, the mean eccentricity

Fig 4. Photoelectronmicrographs of transverse sections ofcanine caudal cruciate ligaments (original magnification�68,400). (A) Ligament from CrCL-intact joint. (B) Ligamentfrom CrCL-deficient joint. Bar � 100 nm.

Fig 3. Collagen fibril diameter distributions for CdCLs fromCrCL-intact joints and CrCL-deficient joints.

599ZACHOS ET AL

of collagen fibrils in CdCLs from CrCL-deficient jointswas significantly smaller (P �.0001) than that for CdCLsfrom CrCL-intact joints.

DISCUSSION

The pathologic changes to the various structures ofthe stifle joint as a sequel to CrCL insufficiency havebeen well documented.1-14 Periarticular osteophyteformation,1,2 meniscal degeneration,7,11-13 and carti-lage degradation3,6,8-10 are known to be predictableconsequences of the joint instability caused by CrCLinsufficiency. The results of the current study havedemonstrated that CrCL insufficiency also results insignificant alterations in the morphology of the CdCL.This was manifested by degenerative changes in thehistologic appearance of the CdCL as well as a changein the collagen fibril diameter pattern.

This and other studies have shown that in normalcruciate ligaments there is a bimodal pattern of large-and small-diameter collagen fibrils.37-41 After CrCLtransection, this pattern appears to change to one ofpredominantly small-diameter fibrils. Increases in thepercentage of small-diameter collagen fibrils in liga-ments have been associated with both degenerative

and reparative processes.42-45 Enzymatic degradationof collagen fibrils has been shown to produce in-creased numbers of small-diameter collagen fibrils inligaments exposed to collagenase in vitro.42 Otherstudies have suggested that ultrastructural changes inthe human anterior cruciate ligament after injury maybe because of enzymatic activity within the synovialenvironment of unstable joints.43-45

Increases in the relative number of small-diametercollagen fibrils have also been documented in liga-ments undergoing repair.35,37,38 Several studies havedemonstrated that injured ligaments are repaired bythe proliferation of type III (small-diameter) collagenfibrils.35,46-48 It is possible that repetitive injury to theCdCL in CrCL-deficient joints may incite a chronicreparative response in the CdCL, resulting in anincrease in small-diameter collagen fibril deposition.

Experimental and clinical studies have demon-strated hypertrophy of the medial collateral ligament(MCL) in animals secondary to CrCL insufficiency.49,

50 The MCL is a secondary restraint (after the CrCL)to the cranial translation of the tibia on the femur inCrCL-deficient stifles.51 This hypertrophy is likely theresult of the repetitive trauma of recurring cranialtibial thrust and the ensuing reparative response of theligament. Kinematic studies on CrCL-deficient stiflesfrom which the CdCLs used in the current study wereobtained had repeated cranial subluxation and caudalreduction of the tibia on weight bearing.7 During thestance phase of gait, increased activity of the ham-string muscles places a caudally directed force on thetibia.52 Because the CdCL is the primary check againstcaudal translation of the tibia,51 it is likely that in aCrCL-deficient stifle the repeated, sudden caudal re-duction of the tibia places a repetitive stress on theCdCL. Whereas it was not possible to accuratelymeasure the cross-sectional areas of the CdCLs eval-

Table 1. Percent Large (�100 nm) and Small (�100 nm) DiameterCollagen Fibrils From CdCLs From CrCL-Intact and CrCL-Deficient

Stifle Joints

Dog

Percent of Total No. of Collagen Fibrils Measured

Left (CrCL Intact) Right (CrCL Deficient)

Large Small Large Small

T 39.10 60.90 23.60 76.40U 42.23 57.77 0.00 100.00V 54.17 45.83 28.80 71.20W 39.40 60.60 40.65 59.35X 77.36 22.64 43.27 56.73

Table 2. Mean Collagen Fibril Diameters of CdCLs From CrCL-Intactand CrCL-Deficient Stifle Joints

Dog

Mean Fibril Diameter (nm) � SD

Left (CrCL Intact) Right (CrCL Deficient)*

T 91.66 � 42.24 75.63 � 29.40U 94.94 � 36.37 48.91 � 8.86V 109.56 � 35.48 80.28 � 31.22W 92.49 � 31.30 93.55 � 35.25X 143.82 � 47.03 94.43 � 41.53

* Significantly different from values from left (unoperated, CrCL-intact)CdCLs (P � .05).

Table 3. Mean Eccentricities of CdCLs From CrCL-Intact and CrCL-Deficient Stifle Joints

Dog

Mean Eccentricity � SD

Left (CrCL Intact) Right (CrCL Deficient)*

T 0.89 � 0.060 0.84 � 0.073U 0.86 � 0.068 0.87 � 0.062V 0.88 � 0.065 0.86 � 0.073W 0.86 � 0.070 0.83 � 0.079X 0.85 � 0.078 0.86 � 0.079

* Significantly different from values from left (unoperated CrCL-intact)CdCLs (P � .001).

600 EFFECT OF CrCL INSUFFICIENCY ON THE CdCL

uated in this study, the increase in the relative numberof small-diameter fibrils could represent a reparativeresponse of the ligament in response to chronic,repetitive stress. Arthrotomy alone does not alter thecollagen diameter profile of canine CdCLs.53

The increase in small-diameter collagen fibrils seen inhealing ligaments has been related to a decrease in thetensile properties of these tissues.39-41,54 Small-diametercollagen fibrils have been associated with disorganizedscar tissue deposition in healing ligaments.38,55 In thecurrent study, eccentricity measurements were used as anindication of collagen alignment.34 The results suggestedthat the fibrils of the CdCLs from CrCL-deficient jointswere significantly more eccentric (less aligned) thanthose in ligaments from CrCL-intact joints. In addition,the collagen fibril density in CdCLs from CrCL-deficientjoints was less, albeit not significantly (P �.056), thanthat of CdCLs from CrCL-intact joints. A decrease incollagen fibril density has been reported in the scar tissueof healing ligaments (when compared with normal liga-ments).56 This scar tissue was also found to have inferiormaterial properties when compared with normal liga-ments.56 These ultrastructural findings appear to correlatewith histologic findings of extracellular matrix disorga-nization observed in these tissues, as determined in thepresent study, as well as by others.55 Based on thesefindings, it is possible that the increase in the proportionof small-diameter collagen fibrils observed in the CdCLsof CrCL-insufficient stifles in the current study couldreflect a compromise in the material properties of thisligament. Further study is warranted to precisely deter-mine the effect of these small-diameter collagen fibers onthe material properties of the CdCL.

The CdCLs in this study were examined 2 yearsafter CrCL transection. Although the exact timelinefor the onset of the observed changes in the morphol-ogy of the CdCLs could not be determined, it ispossible that the change to a predominantly small-diameter collagen fibril profile could have occurredwithin weeks of the initial CrCL transection. Alter-ations in collagen diameter profile of ligaments andtendons have occurred within 4 weeks to 6months.37,57,58 Further studies are required to deter-mine the temporal progression of the morphologicchanges observed in this study.

In our study, the CdCL from the contralateral stiflejoint served as a control in each dog. Although concernshave been raised about the use of contralateral limbs ascontrol subjects,59-61 we believe that a comparison ofcollagen fibril diameters between the two CdCLs from

each dog is a valid one in the model studied here.Significant interanimal variation in collagen fibril diam-eter has been documented.39,40 In 1 report, using internalcontrols (ie, comparing values from one limb to thosefrom the contralateral limb of the same animal), rela-tively few measurements were required from each liga-ment to demonstrate a statistically significant but subtleeffect of a given treatment.39

Further, after unilateral transaction of the CrCL indogs, whereas force was initially transferred to thecontralateral limb, vertical ground reaction force andpeak vertical impulse force equilibrated approximately10 months postoperatively.62 In light of historical datadocumenting the marked significance of interindi-vidual variation in collagen fibril diameter measure-ments, and because the study reported here involvedthe examination of ligaments 24 months after transec-tion of the CrCL (a time interval long after reportedequilibration of vertical ground reaction forces be-tween limbs postoperatively), we believe that the useof the CdCL from the contralateral limb as a controlwas justified in our study.

Although the clinical significance of the morphologicchanges observed in the CdCL of CrCL-insufficientjoints is, at present, unknown, the results of the currentstudy may have implications in the surgical managementof CrCL insufficiency in dogs. A technique for treatingCrCL insufficiency, the tibial plateau leveling osteotomy(TPLO), proposes to alter the angle of the tibial plateau inan attempt to change cranial tibial thrust to caudal tibialthrust.63,64 By doing this, the tibia is forced caudallyinstead of cranially during weight bearing.65 The intactCdCL limits this caudal translation of the tibia and thusbecomes the prime stabilizer of the stifle during weightbearing.65 Recently, it has been shown that after theTPLO procedure the CdCL is subjected to significantincreases in tensile load during weight bearing.65 ACdCL already compromised by the morphologic changesobserved secondary to CrCL insufficiency could be atrisk for further injury from the repetitive loading of theligament likely to occur after the TPLO procedure. Basedon the results of the current study, further investigation ofthe long-term effect of the TPLO procedure on themorphologic and material character of the CdCL iswarranted.

ACKNOWLEDGMENT

The authors thank Ralph Common, Donna Craft, andWilliam Anderst for their technical assistance.

601ZACHOS ET AL

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