An In Vivo Injury Model of Posterolateral Knee Instability · eral knee as the human posterolateral...

Posterolateral rotatory instability of the knee is a difficultproblem to diagnose and treat. It was thought to be a rareinjury in the past, but it is becoming increasingly recog-nized that the incidence of posterolateral knee injurieshas been underestimated and underreported.† It has alsobecome clear that untreated or unrecognized posterolat-eral knee injuries are a frequent source of both ACL and

PCL reconstruction graft failures.11,18,20,23,28 In light of thisunderstanding, there has been an increasing interest inthe anatomy, biomechanics, and diagnosis of posterolateralknee injuries during the past decade.8,9,11,16-21,25-28

The natural history of posterolateral knee injuries has notbeen well defined. Kannus14 reported that untreated grade 3posterolateral knee injuries resulted in residual instabilitywith functional limitations and the eventual development ofmeniscal tears and osteoarthritis, suggesting that the long-term consequences can be serious. A recent study has foundthat the rabbit knee has similar attachment sites andcourses for the important main stabilizers of the posterolat-eral knee as the human posterolateral knee.6 A follow-upstudy demonstrated that the fibular collateral ligament andpopliteus tendon in the rabbit knee did not heal when surgi-cally sectioned and that there was a significant amount of

An In Vivo Injury Model of PosterolateralKnee InstabilityRobert F. LaPrade,*† MD, PhD, Fred A. Wentorf,† MS, Erik J. Olson,‡ DVM,and Cathy S. Carlson,‡ DVM, PhDFrom the †University of Minnesota, Department of Orthopaedic Surgery, Minneapolis,Minnesota, and the ‡University of Minnesota, Department of Veterinary Population Medicine,St. Paul, Minnesota

Background: Posterolateral knee instability is a difficult clinical problem to diagnose and treat. To best evaluate its natural historyand the effects of interventional treatments, it is important to develop an in vivo model in an attempt to validate human cadavericand clinical observational studies.

Purpose: To develop an in vivo model of posterolateral knee instability in the rabbit and to determine the natural history ofuntreated posterolateral knee injuries at 6 months postoperatively.

Study Design: Controlled laboratory study.

Methods: The fibular collateral ligament and popliteus tendon were surgically transected in 1 knee in each of 10 skeletally matureNew Zealand White rabbits, with the contralateral knee serving as a control knee. At 6 months postoperatively, the rabbits wereeuthanized, the knees were removed, and biomechanical testing of knee joint stability was performed. After biomechanical test-ing, histological sections of each proximal tibia and distal femur were evaluated, and each proximal tibia was graded using anosteoarthritis grading scheme.

Results: Biomechanical testing revealed a statistically significant difference in amount of rotation in the operated knees com-pared with the unoperated control knees to varus moments at 30°, 60°, and 90° and to external rotation torques at 30° and 60°.Morphological analysis revealed no significant difference in lesions of osteoarthritis in the medial compartment of the operatedknees compared with the unoperated knees.

Conclusions: Although the presence of posterolateral knee instability was validated 6 months after surgery in this model, theauthors did not identify significant differences in lesions of osteoarthritis in the operated knees compared with the unoperatedcontrol knees.

Clinical Significance: This study confirms that posterolateral knee instability can be created in an animal model. It also recapit-ulates the clinical observation in humans that untreated posterolateral knee injuries do not heal. Similar to animal models ofmedial collateral ligament sectioning, further studies to validate a posterolateral instability knee model may lead to interventionalstudies to determine the best way to treat this complex knee instability pattern.

Keywords: animal model; fibular collateral ligament; osteoarthritis; popliteus tendon; posterolateral knee; rabbits

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*Address correspondence to Robert F. LaPrade, MD, Department ofOrthopaedic Surgery, University of Minnesota, 2450 Riverside AvenueSouth, R200, Minneapolis, MN 55454 (e-mail: [email protected]).

†References 1, 7, 12, 13, 15-17, 21, 23, 28.No potential conflict of interest declared.

The American Journal of Sports Medicine, Vol. 34, No. 8DOI: 10.1177/0363546506286785© 2006 American Orthopaedic Society for Sports Medicine

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1314 LaPrade et al The American Journal of Sports Medicine

increased varus motion in the rabbit knee at 3 months afterinjury.22 Complete histological evaluations were not performedin that study because the gross lesions seen at necropsywere minimal. We hypothesized that extending the studyperiod from 3 to 6 months would allow sufficient time forosteoarthritis development.22 Therefore, the goal of thisstudy was to determine if posterolateral knee instability per-sisted over the longer term (6 months after injury) and if itled to the development of osteoarthritis.

MATERIALS AND METHODS

Approval for this study was obtained from the University ofMinnesota Institutional Animal Care and Use Committee.Ten skeletally mature, adult, New Zealand White rabbitswith a mean weight of 4.7 kg (range, 3.7-5.3 kg) were used.An a priori power calculation based on preliminary biome-chanical data indicated that 10 animals per treatment groupwould be sufficient to identify significant treatment effects.

One knee was operated on in each rabbit, alternatingbetween right and left knees with each surgery, while thecontralateral knee served as an unoperated control. The rab-bits were anesthetized with 3.5 mL of an intramuscularinjection of 80% ketamine (30 mg/kg), 8% xylazine (6 mg/kg),and 12% acepromazine (0.90 mg/kg) for these surgeries.

After the operated knee was shaved, a 4-cm longitudinalincision was made 1 cm lateral and parallel to the patellartendon to expose the posterolateral structures. Immediatelyon entering the subcutaneous tissues, the biceps femoriswas exposed. The distal aspect of the biceps femoris muscu-lar attachment was taken down off the tibia and retractedproximally to expose the fibular collateral ligament.

An incision was then made in the fascia anterior andposterior to the fibular collateral ligament, and a curvedK-wire was inserted medial (deep) to the fibular collateralligament. Several small longitudinal incisions were madein the ligament to weaken it sufficiently to allow it to beruptured with lateral traction. Brisk lateral tractionresulted in a midsubstance mop-end tear of the fibularcollateral ligament.22 The ends of the fibular collateral lig-ament were left in situ.

In a similar fashion, the popliteus tendon was exposedby a small vertical incision in the posterolateral joint cap-sule deep to the fibular collateral ligament. A K-wire wasplaced under the popliteus tendon near its femoral attach-ment, while several small longitudinal incisions weremade in the tendon. The K-wire was then pulled upwardand lateral to rupture the tendon, and the ruptured endswere left in situ.

After hemostasis was obtained with a Bovie coagulator,the biceps fascia was reapproximated with 5-0 absorbablesutures. The skin was then closed with a 5-0 subcuticularabsorbable suture. The rabbits were given intramuscular(IM) injections of buprenorphine (0.05 mg/kg) for analgesicrelief. Enrofloxacin (5 mg/kg IM) was also given immediatelyafter surgery and continued for 3 days postoperatively forprophylaxis against infection. Acetaminophen (1 to 2 mg/mL)was provided for each animal in the drinking water for3 days postoperatively to provide further pain control.

All rabbits were allowed unrestricted cage activity overthe course of the study. They were checked daily for infec-tion, extent of use of the operated leg, and for any signs ofdiscomfort.

All rabbits were euthanized 6 months after the surgicalprocedure, and both hindlimbs were disarticulated at thehip joint. To prepare the knees for biomechanical testing,the skin, biceps femoris, and gastrocnemius muscles wereremoved without disruption of the deeper structures. Theproximal epiphysis of the proximal femur and the distalepiphysis of the distal tibia were then removed. The shaftsof the femur and tibia were injected with polymethylmethacrylate, and a threaded 2-mm K-wire was insertedinto the intramedullary cavity of the proximal end of thefemur and the distal end of the tibia for attachment to thetesting fixture.

Biomechanical Limits of Motion Testing

Biomechanical testing of motion limits for varus-valgusmoments and internal-external torques was done on allknees using a custom fixture attached to a materials test-ing machine (MTS Systems Corp, Eden Prairie, Minn).The custom fixture included a 50-N Interface load cell(Interface, Scottsdale, Ariz) to accurately measure appliedloads and a base plate to fix the knee at a variety of flexionangles. A cyclic application of varus-valgus moments from0.3 N!m of varus to 0.3 N!m of valgus was applied at a rateof 1 deg/s to the tibia at 30°, 60°, and 90° of knee flexion.Displacement of the tibia with respect to the femur wasrecorded using a linear variable displacement transducer(LVDT). The testing cycles were performed 5 times at eachflexion angle, and the results were averaged. Similar test-ing was performed with application of 0.1-N!m internal and0.1-N!m external rotation torques at a rate of 1 deg/s to thetibia. Rotation of the tibia with respect to the femur wasmeasured using a potentiometer (Maurey Instrument Corp,Chicago, Ill).

Osteoarthritis Grading

India Ink Staining. Each knee joint was disarticulated,and the articular surfaces of each proximal tibia and distalfemur were covered with India ink using a small paintbrush, allowed to set for 2 minutes, and rinsed with salineuntil all excess ink was removed (approximately 30 seconds).A semiquantitative grading scheme was devised and usedto assess the relative amount of India ink retention in thefemoral condyles and tibial plateaus. Each anatomical site(femoral condyle or tibial plateau) was divided into quad-rants, and the percentage of the articular surface area con-taining retained ink stain was estimated for each quadrantand assigned a subjective score. The greater the surfacearea affected (with retained ink/black) and the more quad-rants affected, the higher the score (Table 1).

Histological Evaluation. After the femora and tibiaewere evaluated grossly, they were fixed in 10% neutral-buffered formalin. Each proximal tibia was bisected in acoronal plane using a diamond saw (Allied High Tech





Vol. 34, No. 8, 2006 In Vivo Injury Model of Posterolateral Knee Instability 1315

Products Inc, Rancho Dominguez, Calif), and each distalfemur was bisected in a coronal plane that included thetrochlear groove and the central weightbearing region ofthe femoral condyles. Specimens were decalcified in 10%ethylenediamine tetraacetic acid (EDTA), processed rou-tinely, and embedded in paraffin. Sections from each sitewere cut at 6 µm and stained with hematoxylin and eosinas well as toluidine blue stains.

All sections were randomized and relabeled with a codenumber to ensure that they were graded and measuredwithout knowledge of treatment. Sections from all sites(tibial plateaus, femoral condyles, and femoral trochlea)were examined to subjectively evaluate the location andseverity of articular cartilage and bone lesions. In alljoints, the lesions were most severe in the medial tibialplateau; therefore, histological grading was confined tothis site. All medial tibial plateau sections were evaluatedusing a previously described, semiquantitative gradingscheme (Table 2).2-5,10 The following parameters were eval-uated: articular cartilage structure, loss of toluidine bluestaining, and bone cysts using a 2 " objective; maximumnumber of tidemark lines, chondrified vessels, blood ves-sels crossing the tidemark, chondrocyte clones, and tide-mark clefts using a 20 " objective.

In addition, the area and thickness of the articular carti-lage, calcified cartilage, and subchondral bone of the medialtibial plateau were evaluated using the Osteomeasure BoneMorphometry System (Osteometrics, Decatur, Ga).The innerand outer perimeters of articular cartilage (from surface tomost superficial tidemark), calcified cartilage (from mostsuperficial tidemark to calcified cartilage-bone junction),and subchondral bone (from calcified cartilage-bone junctionto marrow space) were traced in a centrally defined area of3.5 " 3.5 mm that included two thirds to three fourths of thewidth of the plateau and excluded the extreme axial andabaxial areas (peripheral margins).10 Articular cartilage,calcified cartilage, and subchondral bone areas and widthswere calculated from the perimeter measurements.

Histological sections of the medial tibial plateau also wereevaluated for the presence of abaxial osteophytes (periarticu-lar osteophytes of the medial tibial plateau), and the areas

of all osteophytes were measured using Spot Advancedmorphometry software (Diagnostic Instruments, SterlingHeights, Mich) and a 2 " objective. The outer margin (at theoutermost edge of the calcified cartilage) of each osteophytewas hand traced with a computer mouse starting at the artic-ular to fibrocartilage transition, distally along the tibia, andending at the lower margin of the osteophyte or to a maximaldepth of 8960 µm (the depth of 2 grid squares) for those casesin which the osteophyte formed a thin margin parallel to theshaft of the tibia distally. A straight line was then drawn toconnect this point with the tracing start point (Figure 1).

Statistical Analyses

Motion differences (varus and valgus opening; internaland external rotation) between the operated and controlknees were evaluated using a paired, 2-tailed t test.Morphological grades and measurements were summa-rized using principal components (factor) analysis, and theresulting factors from operated and unoperated controlknees, as well as the individual continuous variables, werecompared using paired, 2-tailed t tests. Statistical signifi-cance was defined as P < .05. The nonparametric Wilcoxonsigned ranked test was used to compare ranked data,including India ink scores, articular cartilage structurescores, and loss of toluidine blue staining scores.

The following 7 variables were included in the factoranalyses: articular cartilage structure score, loss of tolui-dine blue staining score, articular cartilage area and width,subchondral bone area and width, and calcified cartilagewidth. The remaining variables were excluded from the fac-tor analyses because they were not present in any of thesections evaluated (chondrocyte clones, tidemark clefts, andbone cysts), they did not correlate well with any of the fac-tors (maximum number of tidemark lines, chondrified ves-sels, and abaxial osteophyte area), or because of redundancyof the data (ie, high degree of correlation with other vari-ables used in the analysis; these included calcified cartilagearea, total cartilage area and width, and the inner andouter perimeters).

TABLE 1Morphologic Grading Scale for India Ink Staining in Rabbit Tibial Plateaus and Femoral Condyles

Gross Morphologic Parameter:India Ink Staining Score Feature

Grade 0 Articular cartilage smooth and intact (no ink retained in any of the 4 quadrants)

Grades 1-4 Retained ink affecting #20% of the articular cartilage surface area and involving 1 quadrant,2 quadrants, 3 quadrants, and 4 quadrants, respectively

Grades 5-8 Retained ink affecting >20% but #50% of the articular cartilage surface area and involving 1 quadrant,2 quadrants, 3 quadrants, and 4 quadrants, respectively

Grades 9-12 Retained ink affecting >50% but <100% of the articular cartilage surface area and involving1 quadrant, 2 quadrants, 3 quadrants, and 4 quadrants, respectively

Grades 13-16 Retained ink affecting 100% of the articular cartilage surface area and involving 1 quadrant,2 quadrants, 3 quadrants, and 4 quadrants, respectively






RESULTS

All rabbits did well perioperatively and postoperatively,exhibiting little evidence of pain or discomfort. Within2 weeks postoperatively, all rabbits were observed to havenormal cage activity, with no evidence of lameness.

At necropsy, superficial dissection of the skin and under-lying biceps femoris revealed no evidence of healing ofeither the fibular collateral ligament or popliteus tendonin the operated knees. Minimal scar tissue formation waspresent in the area of the transected structures. Grossly, nomeniscal tears were observed in any of the rabbits. Onerabbit had severe bilateral osteoarthritis of the knee joints(characterized by marked India ink retention of thefemoral condyles and tibial plateaus bilaterally, with themedial compartment being the most severe) that wasobserved at necropsy; it was interpreted to be preexisting

and not related to the surgical treatment. Morphologicaland biomechanical data from this animal were excludedfrom the statistical analyses, although it was noted thatthe control knee was stable and the operated knee wasunstable (demonstrated an increase in both varus dis-placement and external rotation at all tested knee flexionangles).

Biomechanical analysis identified several differences inthe limits of knee motion when comparing the operated andthe control limbs (Table 3). Testing revealed a significantdifference in the amount of varus displacement at 30° (P <.05), 60° (P < .01), and 90° (P < .01) for the operated kneescompared with the unoperated control knees (Figure 2). Asignificant increase in external rotation was seen at both30° (P < .01) and 60° (P < .01) for the operated knees com-pared with the unoperated control knees (Figure 2). Therewas no significant difference in the amount of external

TABLE 2Histological Grading Scale for Rabbit Tibial Plateaus

Histologic Parameter Feature

Articular cartilage structure

Grade 0 Articular cartilage smooth and intact

Grades 1-4 Minimal/mild superficial fibrillation (less than one tenth of the articular cartilage thickness) involving<20%, 20%-50%, >50% but <100%, and 100% of the plateau, respectively

Grades 5-8 Fibrillation and/or clefts and/or loss of cartilage affecting the superficial one third or less of thearticular cartilage thickness involving <20%, 20%-50%, >50% but <100%, and 100%of the plateau, respectively

Grades 9-12 Fibrillation and/or clefts and/or loss of cartilage affecting more than one third, but less than twothirds, of the articular cartilage thickness involving <20%, 20%-50%, >50% but <100%, and100% of the plateau, respectively

Grades 13-16 Fibrillation and/or clefts and/or loss of cartilage affecting more than two thirds and up to the fullthickness of the articular cartilage involving <20%, 20%-50%, >50% but <100%, and 100% of the plateau, respectively

Grades 17-20 Fibrillation and/or clefts and/or loss of cartilage extending into calcified cartilage and/or subchondral bone involving <20%, 20%-50%, >50% but <100%, and 100% of the plateau, respectively

Loss of toluidine blue staining

Grade 0 None

Grades 1-4 Loss of staining affecting #20% of the articular cartilage depth and involving <20%, 20%-50%, >50% but <100%, and 100% of the plateau, respectively

Grades 5-8 Loss of staining affecting >20% but <50% of the articular cartilage depth and involving <20%,20%-50%, >50% but <100%, and 100% of the plateau, respectively

Grades 9-12 Loss of staining affecting >50% but <100% of the articular cartilage depth and involving<20%, 20%-50%, >50% but <100%, and 100% of the plateau, respectively

Grades 13-16 Full-thickness loss of staining involving <20%, 20%-50%, >50% but <100%, and 100% of the plateau,respectively

Chondrocyte clones (a cluster Countedof 6 or more chondrocytes)

Abaxial osteophytes, Countedmaximum number of tidemarks,vessels crossing the tidemark,tidemark clefts, chondrified vessels, and bone cysts






rotation between the operated and unoperated controlknees at 90° of knee flexion. There were no significant dif-ferences in internal rotation motion or valgus displacementbetween the operated and unoperated control knees at anyflexion angle.

The mean India ink scores from the medial compartmentof the knee (tibial plateau and femoral condyle) were higherthan those from the lateral compartment (Table 4); how-ever, only one of the comparisons was significant (medial vslateral tibial plateau of the operated knees; P < .01), with atrend toward significance in the medial versus lateralfemoral condyles of the operated knees (P = .06). In allcases, except for the lateral tibial plateau (where the scoreswere equal), the India ink scores from the operated kneeswere higher than those from the unoperated knees; how-ever, none of the comparisons reached statistical signifi-cance (Table 4, Figures 3 A and B).

Histology Findings

As indicated previously, the medial tibial plateau was themost severely affected site and was the only site in whichhistological grading/measuring was performed. In bothtreatment groups, the histological lesions were relativelymild (eg, the highest articular cartilage structure scorewas 14, and the overall mean score was 7.3) (Figures 3 Cand D).

The factor analysis resulted in 3 factors that explained86% of the variation in the data (Table 5). Factor 1 wasweighted by articular cartilage width and area and calcifiedcartilage width, and explained 32% of the variation in thedata. Factor 2 was composed of subchondral bone width andarea and calcified cartilage width, and explained 28% of thevariation in the data. Factor 3 was composed of articularcartilage structure score, loss of toluidine blue stainingscore, and articular cartilage area (because this parameterhad a loading score of less than 0.50, it is not shown inTable 5), and explained 26% of the variation in the data.

None of the factors were significantly affected bytreatment, although there was a trend for factor 1 to behigher in the operated knees as compared with the unop-erated knees (P = .08). Similarly, none of the individualvariables/scores were significant, although there was atrend toward higher mean articular cartilage area in theoperated knees compared to the unoperated control knees(P = .06) (Table 5). Although the mean articular cartilagestructure scores and the scores involving loss of toluidineblue staining were higher in the operated knees comparedwith the unoperated knees, neither difference was statisti-cally significant (Table 5, Figure 4).

The presence of medial abaxial osteophytes in the unoper-ated control knees was nearly identical to that in the operatedknees (8/9 knees, 88.9% and 9/9 knees, 100%, respec-tively). The mean area (±SD) of the abaxial osteophyteswas somewhat smaller in the unoperated rabbit knees (4.68 ±2.50 mm2) than in the operated knees (5.08 ± 2.28 mm2), butthis difference was not statistically significant.

DISCUSSION

Although the overall functional implications of anuntreated posterolateral corner injury can be signifi-cant,11,12,14,18,20,21,23 the natural history of posterolateralknee injuries has not been well described or followed inthe past. Patients with untreated posterolateral cornerinjuries have been found to have residual instability aswell as functional limitations and, frequently, a varusthrust gait pattern.12-14,16,21,23 The long-term sequelae ofchronic posterolateral instability have been described to bemeniscal tears and degenerative arthritis over time.14 Inaddition, some of the more significant instability patternsseen with both ACL and PCL injuries have been found tobe in association with combined posterolateral cornerinjuries.7,11,18,20,24 Several biomechanical and clinical stud-ies have demonstrated that untreated or unrecognized pos-terolateral knee injuries can place significant stress oncruciate ligament reconstructions, which can potentiallylead to the grafts stretching out and failing over time.11,18,20

There have been multiple previous articles published onin vivo animal models to characterize the healing patternof the medial collateral ligament complex of the knee.29-31

This information has led to an improved understanding ofthis ligament’s repair process and has helped clinicians tovalidate the appropriate method of treatment for humanmedial collateral ligament injuries. These articles onmedial collateral ligament injuries in animals have beentranslated to human clinical practice to the point where wenow treat isolated, and some combined, medial collateralligament injuries nonoperatively, and with early motion,compared with the previous techniques of operative repairand/or cast immobilization.

The present study focused on the development of ananimal model for posterolateral knee instability. Our studyconfirms that untreated grade 3 posterolateral kneeinjuries in the rabbit remain unhealed 6 months aftersurgery and recapitulates the clinical observation thatuntreated posterolateral knee injuries in humans do not

Figure 1. Toluidine blue section of the medial tibial plateaufrom an unoperated rabbit knee. The black tracing demon-strates the measurement scheme of the abaxial (peripheral)osteophyte. Bar = 1000 µm.






heal and should be treated with early repair or reconstruc-tion to maximize functional outcomes.1,12,14 We believe thatthe rabbit model will prove useful in the development ofinterventional studies to treat isolated and combined pos-terolateral knee injuries, similar to the findings in medialcollateral ligament animal models. We hypothesize thatthere was no significant increase in external rotation at 90°of knee flexion because the cruciate ligaments act to con-strain external rotation at higher knee flexion angles.8,9 Inhumans, the average increase of external rotation at 90° ofknee flexion in isolated posterolateral structure sectioningstudies is approximately 5°, which is much less than theaverage increase of 13° to 17° of increased external rotationat 30° of flexion.8,9 If rabbits are similar to humans,increases in external rotation at 90° of knee flexion wouldbe more difficult to identify than at 30° of knee flexion; alarger group of experimental animals may be required.

We theorized that an in vivo model of posterolateral insta-bility of the knee was necessary to determine the outcome ofuntreated posterolateral corner knee injuries on residualstability, knee function, and osteoarthritis of the knee. It wasalso theorized that an animal model was necessary to test

TABLE 3A Comparison of the Limits of Motion for Operated (Posterolateral Instability)

and Control (Unoperated) Rabbit Kneesa

Rotation (mean ± SD), deg

Applied Load Flexion Angle, deg Operated Control P

Varus (0.3 N!m) 30 5.1 ± 2.1 2.7 ± 1.8 <.0560 18.3 ± 3.8 9.2 ± 3.4 <.0190 22 ± 4.6 8.1 ± 3.1 <.01

Valgus (0.3 N!m) 30 3.4 ± 1.6 3.8 ± 1.3 NS60 7.8 ± 2.7 7.2 ± 3.2 NS90 7.1 ± 2.5 7.6 ± 2.7 NS

Internal rotation (0.1 N!m) 30 3.2 ± 1.8 2.9 ± 2.1 NS60 5.1 ± 1.5 5.0 ± 1.9 NS90 5.6 ± 1.8 5.9 ± 1.9 NS

External rotation (0.1 N!m) 30 6.5 ± 2.3 3.7 ± 1.8 <.0160 10.4 ± 2.1 5.3 ± 2.0 <.0190 9.2 ± 2.2 7.6 ± 2.3 NS

aNS, not significant.

0

5

10

15

20

25

30

Knee Flexion Angles

Var

us (d

egre

es)

Operated Control

30° 60° 90°

P<0.05

P<0.01

P<0.01

A

0

5

10

15

20

25

30

35

Knee Flexion Angles

90˚30˚ 60˚

Ext

erna

l Rot

atio

n (d

egre

es)

Operated Control

P<0.01

P<0.01

B

Figure 2. (A) Varus angulation created by a 0.3-N·m varusmoment applied at the knee at 30°, 60°, and 90° of flexion inoperated (posterolateral instability) and control (unoperated)rabbit knees. (B) External rotation of the tibia with respect tothe femur created by a 0.1-N·m external rotation torqueapplied at the knee at 30°, 60°, and 90° of flexion in operatedand control rabbit knees.

TABLE 4India Ink Scores for Operated (Posterolateral

Instability) and Control (Unoperated) Rabbit Knees

India Ink ScoreAnatomical Site Treatment Group (mean ± SD)a

Medial tibial plateau Unoperated 3.22 ± 2.86Medial tibial plateau Operated 5.00 ± 2.40Lateral tibial plateau Unoperated 1.78 ± 1.72Lateral tibial plateau Operated 1.78 ± 1.56Medial femoral condyle Unoperated 3.22 ± 2.68Medial femoral condyle Operated 4.33 ± 3.28Lateral femoral condyle Unoperated 1.56 ± 0.88Lateral femoral condyle Operated 2.67 ± 1.58

aNone of the comparisons between treatment groups within ananatomical site were statistically significant.






the previous results of biomechanical studies on the effectsof posterolateral knee injuries on cruciate ligament grafts inthe multiple ligament injured knee.11,18,20 After completion

of a rabbit study on posterolateral knee anatomy revealedthat there were enough similarities in its bony and liga-mentous architecture to proceed with an in vivo testing

Figure 3. Proximal tibia; rabbit. Gross (A and B) and histologic (C and D) appearances of the proximal articular surface of thetibia of an unoperated rabbit knee (A and C) and the most severely affected operated rabbit knee (B and D). In the unoperatedknee (A), the articular cartilage is intact and smooth, with minimal India ink retention in the medial tibial plateau (*). In the oper-ated knee (B), there is moderate surface fibrillation (*) in the articular cartilage and moderate retention of India ink stain (arrow) inthe medial tibial plateau. Histologically, the articular cartilage of the medial tibial plateau of the unoperated knee (C) is smoothand intact. In the corresponding site of the operated knee (D), there is surface fibrillation and clefts (arrow) and a moderatedecrease in the staining of the proteoglycan matrix with toluidine blue. Toluidine blue stain, bar = 500 µm.

TABLE 5Results of the Factor Analysis and Values for Individual Histological Grades and Measurements Comprising

the Factors for Operated (Posterolateral Instability) and Control (Unoperated) Rabbit Kneesa

Individual Variables

Factors Treatment Groupc

% of Variance Factor Factor Control OperatedFactor No. Explained P Composition Loadingb (n = 9 knees) (n = 9 knees) P

1 32 .081 AC width/thickness, µm 0.93 0.46 ± 0.14 0.53 ± 0.12 .267AC area, mm2 0.83 1.78 ± 0.55 2.40 ± 0.69 .062CC width/thickness, µm –0.74 0.10 ± 0.03 0.09 ± 0.02 .531

2 28 .665 SCB width/thickness, µm 0.90 0.57 ± 0.09 0.60 ± 0.11 .623SCB area, mm2 0.89 3.10 ± 0.46 3.05 ± 0.47 .834CC width/thickness, µm 0.51 0.10 ± 0.03 0.09 ± 0.02 .531

3 26 .420 AC structure, score 0.92 5.89 ± 3.89 8.78 ± 3.34 .188Loss of toluidine blue 0.92 5.22 ± 3.53 5.78 ± 3.63 .734

staining, score

aAC, articular cartilage; CC, calcified cartilage; SCB, subchondral bone. None of the factors or individual variables comprising the factorswere significantly different between operated and unoperated controls.

bOnly those variables having a factor loading value of greater than the absolute value of 0.50 are shown.cValues are presented as mean ± SD.






method,6 a pilot study was performed that verified that agrade 3 posterolateral knee injury pattern could be createdin the rabbit model.22 Although our short-term results at3 months revealed that there was significant residual lax-ity, lesions of osteoarthritis were minimal and were notquantified. For this reason, we extended the present studyto 6 months. The current study confirms the human clini-cal observation and confirms our hypothesis from our pre-vious rabbit posterolateral knee instability model thatgrade 3 posterolateral knee injuries do not heal and maylead to the development of osteoarthritis with time.

The India ink grading scheme used in this study wasdeveloped in our laboratory. Staining joint tissues withIndia ink at the time of necropsy is done routinely andallows a global assessment of lesions of osteoarthritis withinthe joint. We attempted to standardize the interpretation ofIndia ink staining patterns by devising a semiquantitativegrading scheme that allowed assignment of a staining scoreto each site based on clear criteria. The semiquantitativedata obtained using this scheme are complementary to thehistological data but provide different information than doesa midcoronal histological section. Although India ink stain-ing indicates the topographical distribution of the lesions, itprovides no information on severity. Conversely, the histo-logical scores provide a more detailed assessment of lesionseverity but are highly limited to a specific location withinthe joint.

The histological grading scheme was initially developed toevaluate a broad range of osteoarthritis severities, from veryminimal changes to severe changes. In the present study,where all of the lesions were mild, the scheme has some lim-itations. In the animals of the present study, several param-eters included in the scheme were not present in the sections(eg, bone cysts, which tend to be found only in very severe dis-ease). Thus, these parameters were excluded from the analy-sis because they did not occur. Another category of variablesdid not correlate well with any of the other variablesand would have remained individual (factor weighted by asingle variable), rather than being grouped with correlated

variables (factor weighted by multiple variables). Theseincluded maximum number of tidemark lines, number ofchondrified vessels, and abaxial osteophyte area. The osteo-phyte area measurements represented continuous data andwere easily evaluated individually. The number of tidemarklines was high in virtually all cases and did not appear toprovide useful information. Similarly, the number of chondri-fied vessels was so low that this variable provided no addi-tional useful information. A third category of variablesincluded those that were so closely correlated with othervariables that they became redundant and were thus elimi-nated. An example of this was calcified cartilage area, whichprovided essentially the same information as calcified carti-lage width.

We anticipated that there would be more severe lesionsof osteoarthritis in the medial compartment of the oper-ated knees than in the unoperated knees because of theadditional stress placed on the medial compartment sec-ondary to the lateral compartment gapping that occurswith varus instability. Although the role of isolated pos-terolateral knee instability on the amount of increasedforce seen on the articular cartilage has not been biome-chanically studied, it has been demonstrated that there isa significant increase in force seen on the medial compart-ment of cadaveric knees with combined posterior PCL andposterolateral knee injuries compared to isolated PCLinjuries.24 In addition, it has been observed arthroscopi-cally that many patients with chronic posterolateral kneeinjuries develop significant chondromalacia of the antero-medial aspects of the medial femoral condyle and medialtibial plateau, which is more severe in patients with avarus thrust gait pattern.16 Supporting this observation isthe trend toward increased width and area of articular car-tilage in the operated knees in the present study, sugges-tive of matrix edema that likely precedes fibrillation.

An a priori power study indicated that 10 animals pertreatment group would be sufficient to identify significanttreatment effects; however, this was based on preliminarybiomechanical data because we did not have histologicaldata at the time. Clearly, this number was not sufficientto obtain significant differences in osteoarthritis severity.Using the histological data from the present study, we havedetermined that a sample size of 25 animals would berequired to demonstrate histological differences in theseverity of osteoarthritis. Increasing the postsurgical inter-val beyond 6 months would also be expected to increasethe severity of lesions of osteoarthritis present at necropsybut would significantly increase the costs of the study.Laboratory rabbits housed in cages are not very active (loco-motive/physical exercise), and this fact may influence theamount of weightbearing on the joints and the developmentand progression of osteoarthritis. The severe, bilateralosteoarthritis that was present in 1 of the 10 rabbits used inthis study almost certainly predated the surgical procedure.This case was rather unusual in its severity and may havebeen identified by means of preoperative radiographywhich, unfortunately, was not performed. In general, it isassumed that preexisting degenerative changes are as likelyin the control joints as in the operative joints and that theywill not mask the effects of the treatment.

0

2

4

6

8

10

12

14

Operated Control

Treatment Group

Mea

n S

core

Figure 4. Articular cartilage structure scores for the medialtibial plateau in the operated (posterolateral instability) versusthe control (unoperated) rabbit knees 6 months after thesurgery. Error bars represent SDs.






Acquiring preoperative/presurgical baseline radiographsof the rabbit knees would aid in the interpretation oflesions of osteoarthritis, could be used to screen for severepreexisting lesions, and may have allowed visualizationof preexisting osteophytes. The fact that control and oper-ated knees had similar numbers and sizes of osteophytessuggests that they may have been preexisting; however,because preoperative radiographs were not taken, thissupposition could not be confirmed.

In conclusion, we confirmed that the fibular collateral liga-ment and popliteus tendon in the rabbit knee do not healwhen sectioned, and there is a significant amount of increasedresidual posterolateral instability of the rabbit knee at 6months after injury. We believe this demonstrates that in vivomodels of posterolateral knee injuries will potentially allow forverification of in vitro cadaveric studies of posterolateral kneeinjuries and the development of models of combined postero-lateral and cruciate ligament injuries of the knee.

ACKNOWLEDGMENT

The Orthopaedic Research and Education Foundation(R.F.L.), the Sports Medicine Research Fund (MMF-5021) ofthe Minnesota Medical Foundation (R.F.L.), and NationalInstitutes of Health Grant RR14099 (C.S.C.). The authorsacknowledge the assistance of Conrad Lindquist for hisassistance on the biomechanical testing portion of thisstudy. The authors also thank Bruce Lindgren for statisticalassistance and Anne Undersander and Josh Parker for tech-nical assistance.

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An In Vivo Injury Model of Posterolateral Knee Instability · eral knee as the human posterolateral...

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