Revascularization Process of The

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The American Journal of Sports

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DOI: 10.1177/0363546511398039 2011 39: 1478 originally published online March 10, 2011Am J Sports Med

Aikaterini Ntoulia, Frederica Papadopoulou, Stavros Ristanis, Maria Argyropoulou and Anastasios D. GeorgoulisReconstruction

Contrast-Enhanced Magnetic Resonance Imaging 6 and 12 Months After Anterior Cruciate Ligament Bone Autograft Evaluated byPatellar TendonRevascularization Process of the Bone

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Revascularization Process of theBonePatellar TendonBone AutograftEvaluated by Contrast-Enhanced MagneticResonance Imaging 6 and 12 Months AfterAnterior Cruciate Ligament Reconstruction

Aikaterini Ntoulia,* MD, Frederica Papadopoulou,* MD, Stavros Ristanis,y MD,Maria Argyropoulou,* MD, and Anastasios D. Georgoulis,yz MDInvestigation performed at the Department of Radiology, University Hospital of Ioannina, and theOrthopaedic Sports Medicine Center, Department of Orthopaedic Surgery, University ofIoannina, Ioannina, Greece

Background: Contrast-enhanced magnetic resonance imaging (MRI) studies conducted on animal models have shown that theobserved signal intensity changes are related to the degree of graft vascularity and its biomechanical properties.

Purpose: To evaluate by contrast-enhanced MRI the revascularization process at 3 distinct sites discerned in relation to the sur-rounding microenvironment along the course of bonepatellar tendonbone (BPTB) autograft in uncomplicated human anteriorcruciate ligament (ACL)reconstructed knees.

Study Design: Case series; Level of evidence, 4.

Methods: Thirty-two male patients were assessed with a 3-dimensional fast field echo/short tau inversion recovery (FFE/STIR)MRI sequence at the third postoperative day and at time intervals of 6 and 12 months after surgery. Signal-to-noise quotient(SNQ) was calculated for 3 specific graft sites (intra-articular site, intraosseous tibial tunnel site, and intraosseous juxta screwsite) before and after gadolinium administration. Comparisons of the enhancement index (EI: SNQafter/SNQbefore gadolinium)were performed independently for each graft site and time interval.

Results: Three days postoperatively, insufficient vascularization was noticed at the 3 sites. Six and 12 months after surgery, theenhancement index was significantly increased in all 3 sites (P\ .001). The intra-articular site, 6 months postoperatively, achievedsatisfactory contrast medium uptake (enhancement index .1), with significantly higher enhancement index values compared withthe other 2 sites (P\ .001). Twelve months after surgery, only the intraosseously enclosed sites displayed an increase of theenhancement index, although nonsignificant (P = .09 and P = .07, respectively).

Conclusion: Revascularization of the graft occurs gradually along its length, with the intra-articular site being the first and thefaster part to complete this phase, while both the intraosseous sites are still in progress throughout the first postoperativeyear. Revascularization is an important link at the intrinsic healing chain of the ACL graft. The surrounding microenvironmentdoes seem to play a significant role in this process, and the differences in its composition along the graft course are reflectedat the revascularization progress of the corresponding sites.

Keywords: anterior cruciate ligament reconstruction; graft revascularization; contrast-enhanced MRI; BPTB; enhancement index

After anterior cruciate ligament (ACL) arthroscopic recon-struction, the tendon autograft used to replace the injuredligament undergoes a biological healing process consistingof 4 successive phases: initial necrosis, revascularization,cellular repopulation, and remodeling.4,7,11,18,19,50 Revas-cularization contributes significantly to this process byproviding the nutrient elements to sustain graft viabilityand by facilitating the subsequent remodeling phases tobe conducted.4,18,41,50 The uneventful transition of thesehistomorphological alterations will eventually lead to graftligamentization.4,19,30,31,41,46 As a neoligament, the tendon

zAddress correspondence to Anastasios D. Georgoulis, MD, Ortho-paedic Sports Medicine Center, PO Box 1042, Ioannina 45110, Greece(e-mail: [email protected]).

*Department of Radiology, University Hospital of Ioannina, Ioannina,Greece.

yDepartment of Orthopaedic Surgery, Orthopaedic Sports MedicineCenter, University of Ioannina, Ioannina, Greece.

The authors declared that they have no conflicts of interest in theauthorship and publication of this contribution.

The American Journal of Sports Medicine, Vol. 39, No. 7DOI: 10.1177/0363546511398039 2011 The Author(s)

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autograft will achieve its incorporation to the new environ-ment and therefore its functional adaptation to its newrole.4,9,18,31,56 The earlier this process is completed, thesooner the ACL substitute graft will meet its new loadingdemands, allowing the patient to resume earlier his orher sport activities.4,19,25

In this healing process, the microenvironment sur-rounding the graft plays an important role because it pro-vides the necessary elements for its healing.4,46

According to the composition of this environment, 3distinct sites can be discerned along the graft course:(1) the intra-articular site, (2) the site of the graft thatis located inside the osseous tunnels, and (3) the site ofthe graft that is adjacent to the fixation material insidethe osseous tunnels. The first graft site interacts circum-ferentially with the surrounding synovial environment.The site that is entirely enclosed in the osseous tunnelsinteracts extensively with the cancellous bone, while thegraft site that is located between the fixation materialand the osseous tunnel wall interacts partially withboth of them. Given the prominent differences in the com-position of these surrounding spaces, questions can beraised concerning whether each of the aforementionedgraft sites could be differently affected in terms ofrevascularization due to different nutrient elementssupply.

Several histological studies have been conducted indissimilar in vivo models documenting the revasculariza-tion process of the graft with variable results. On thescope of imaging, MRI with the use of gadolinium-diethylenetriamine pentacetic acid (Gd-DTPA) contrastagent has been used as a valuable tool to provide noninva-sively important information regarding the vascular sta-tus of the graft tissue. It is well established thatenhancement of a tissue after contrast agent administra-tion results in increase of its signal intensity.8,27,36,37,51,52

Weiler et al,52 by means of contrast-enhanced MRI ina sheep model, demonstrated that the signal intensitychanges observed in the graft tissue reflect its vascularityand biomechanical properties. Additionally, evaluation ofquantitative imaging parameters, such as signal-to-noisequotient (SNQ), was correlated with the degree of vascu-larity as it was histologically confirmed. However, inthis animal-based study, only the intra-articular part ofthe graft was examined.

To our knowledge, no previous studies have been con-ducted on human patients to compare noninvasively thedegree of vascularity in the aforementioned distinct graftsites that can be discerned along its course in relation tothe surrounding microenvironment. Therefore, the aim ofthe present study was to quantitatively evaluate, by meansof contrast-enhanced MRI, the signal intensity changes ofthe graft over time that correspond to its revascularization.We hypothesized that the differences in the surroundingmicroenvironment will be reflected in the vascularizationprocess.

MATERIALS AND METHODS

Patients

From June 2007 until September 2009, 215 patients under-went arthroscopically assisted ACL reconstruction by thesame orthopaedic surgeon (A.D.G.). In total, 144 bonepatellar tendonbone (BPTB) autografts were used in 137male and 7 female patients, while 71 quadrupled ham-string tendon (semitendinosus/gracilis) autografts wereused in 56 male and 15 female patients.

Given the objective of the study, examination of a single,although multiparametric, variable, as the vasculature is,requires the establishment of conscientious and explicitpatient selection criteria to maintain the homogeneity ofthe population sample and ensure the validity of theresults. Therefore, for the present study, we recruitedmale patients who had undergone ACL reconstructionwith a BPTB autograft. Women were excluded becausehormonal differences have been related with modified vas-culature responsiveness during revascularization.10,26,55

Patients with multiligament injuries, revision operationof the ACL ligament, as well as serious chondral lesions(Outerbridge classification III or IV), were also excludedfrom this study because the severity of the concomitantinjuries may negatively affect the overall final outcome.Moreover, smokers15,32 and overweight22,48 patients werealso excluded, given that their associated morbidities arerelated to endothelial dysfunction that impairs vesselsmicrocirculation. Furthermore, patients with serious aller-gic reaction to pharmaceutical (previous administration ofcontrast agent or other medication), environmental (severereactions to insects venom), or dietary allergens andrequired medical treatment were also excluded from thisserial study for safety reasons.2,16 Finally, because of socio-economic reasons, patients living more than 1 hour awayfrom the hospital where the present study was conductedwere also excluded.

According to the above criteria, 32 patients were finallyincluded in the study. The mean age of the selectedpatients was 25.6 years (range, 22-32 years), and meanbody mass index (BMI) was 23.7. Informed consent wasobtained from all patients enrolled in this study, in accor-dance with the institutional review board policies of ourUniversity Hospital.

Surgical Reconstruction With a BPTB Graft

The BPTB graft was taken from the medial third of thepatellar tendon.20,34 We did not harvest more than a thirdof the total tendon width. The diameter of the harvestedgraft was 9 to 10 mm. First, the tibial tunnel was drilled.The hole in the tibial plateau was placed approximately5 mm anterior and medial to the anatomic center of thenatural ACL attachment, so that the posterolateral partof the tunnel circumference was located at the ACL attach-ment anatomic center.12 The drilling of the femoral tunnelwas performed arthroscopically through the anteromedialapproach, with the knee joint in 120 of flexion and atReferences 4, 7, 11, 18, 19, 30, 31, 41, 46, 50, 56.

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about the 10-oclock position (for a right knee) or at the 2-oclock position (for a left knee).

The placement of the graft in the femoral tunnel wasperformed with the cortical side of the bone plug close tothe over-the-top position. In the tibia, we twisted the graft90, so as to recreate the anatomic spiral of ACL fibers.20,34

Fixation of the graft was performed in all patients with thesame bioabsorbable interference screws, consisting of poly-lactic acid (PLA) and hydroxylapatite (HA) (BioRCI-HA,Smith & Nephew Endoscopy, Andover, Massachusetts),on both femur and tibia (size, 7 3 20 mm and 9 3 20 mm,respectively). Interference screws were inserted at the can-cellous side of the bone plugs. After the fixation to thefemur, maximal tension was performed manually by pullingthe graft from its tibial edge and pushing the tibia posteri-orly. Holding the knee in 30 of flexion23 and holding thegraft tensioned as we described, we proceeded to the fixationon the tibia with the second interference screw (Figure 1).Finally, the graft was inspected both in full flexion andfull extension so as to exclude graft impingement at theintercondylar roof, the lateral condyle, and the posteriorcruciate ligament. A notchplasty was not performed in anyof our cases.

Clinical Evaluation

All ACL-reconstructed patients followed the same carefullycontrolled rehabilitation protocol. They were allowed toreturn to sports-related activities at 6 months after sur-gery, provided that they had regained full functional

strength and stability. Strength at that time was deter-mined with the BIODEX (System-3, Biodex Corp, Shirley,New York) isokinetic dynamometer, revealing acceptablesymmetry in quadriceps and hamstring strength.45 Clini-cal examination was performed by the same experiencedorthopaedic surgeon at the same day that the MRI exami-nation was performed and included several clinical testsfor objective and subjective evaluation of knee joint stabil-ity. Specifically, the subjective examination included theLachman, anterior drawer, and pivot-shift tests, as wellas International Knee Documentation Committee (IKDC),Tegner, and Lysholm activity scores. In addition, anteriortibial translation was evaluated using the KT-1000 kneearthrometer (MEDmetric Corp, San Diego, California).49

The measurements were performed with the applicationat the tibia of 134-N posterior-anterior external force, aswell as maximum manual posterior-anterior external forceuntil heel clearance.

MRI Evaluation

Each patient underwent static MRI of the knee joint at 3time intervals: third postoperative day and 6 and 12months after surgery. The examination at the third postop-erative day was performed after the removal of the drain-age tubes. Clinical examination and routine laboratoryprofile of the patients, including total white blood cell(WBC) count, C-reactive protein, erythrocyte sedimenta-tion rate (ESR), were performed, and results were normalwith no evidence of surgery-related infection. All the

Figure 1. Tibial (A) and femoral (B) tunnel placement in a left knee.The tibial tunnel was drilled in the center of the ACL footprint.The femoral tunnel was drilled at the 10-oclock position of the left knee (or the 2-oclock position corresponding to the right knee)according to the classification system of the scientific committee of the European Society of Sports Traumatology, Knee Surgeryand Arthroscopy.

1480 Ntoulia et al The American Journal of Sports Medicine


examinations were performed with a 1.5-T imager (Gyro-scan ACS NT, Philips, Medical System Best, Best, the Neth-erlands) and a circular receiver-transmission extremity coil.The knee was positioned in full extension, was comfortableto the patient, and was immobilized with foam padding. Aninfusion line was placed in the patients antecubital veinbefore the examination, so that the patient could remaintotally immobile during the examination. The unenhancedexamination protocol included sequences in all 3 planes toobtain an overview of the graft and joint structures. Theimplementation of a 3-dimensional spoiled gradient echosequence with fat suppression (3-D FFE/STIR) in an obliquesagittal plane along the long axis of the graft, before and 1minute after gadolinium (Gd-DTPA) contrast administra-tion (0.1 mmol/kg body weight), was used for the analysisof signal intensity, which was performed with the computersoftware ImageJ (version 1.43, National Institutes ofHealth, Bethesda, Maryland). The detailed parameters ofthe MRI protocol are outlined in Table 1.

Data Analysis

Two radiologists with experience in musculoskeletal MRIevaluated blindly all MRI examinations, with disagree-ments resolved by consensus. The image that showed thefull extent of the graft from its femoral tunnel exit throughthe joint space up to the tibial tunnel exit was selected forthe analysis. Three sites of the graft were distinguishedaccording to the surrounding microenvironment, andthey were evaluated independently: (1) the intra-articularpart of the graft located centrally within the joint (intra-articular site), (2) the intraosseous part of the graftsurrounded by the osseous tibial tunnel wall (intraosseoustibial tunnel site), and finally (3) the part of the graftlocated at the interface between the osseous tibial tunnelwall and the biodegradable interference screw (intraoss-eous juxta screw site) (Figure 2).

The signal intensity (SI) was calculated at the 3 aforemen-tioned graft sites using an 8-mm2 elliptical region of interest(ROI). The SNQ was then calculated at each of the 3 sites bydividing the SI of the graft by the noise of the image back-ground on the images obtained before contrast agent

administration29,43,52: SNQbefore = SIspecific site of the graft Onoise background (Figure 3). Similarly, on the imagesobtained after the administration of gadolinium, ROIs wereplaced at identical positions by the computer software, andthe measurements were repeated exactly the same way:SNQafter =SIspecific site of the graft O noise background (Figure4). Noise was defined as the standard deviation of the signalin a 250-mm2 ROI placed outside the patient within a 1-cmdistance from the tibial tubercle, at the lower part of theimage29,42 (Figures 3 and 4).

Each measurement was repeated 3 times for the intra-observer variation analysis as well as the calculation ofthe mean values. By dividing the SNQ mean values afterand before contrast administration, a new ratio wasderived to provide direct information about revasculariza-tion. This ratio was named the enhancement index (EI):EI = SNQafterO SNQbefore. Calculation of the enhancementindex was made independently for each graft site and timeinterval. Enhancement index values above 1 were consid-ered as satisfactory revascularization of any graft site,while values equal or less than 1 indicated insufficientrevascularization (Table 2).

Comparison of the enhancement index values was per-formed using analysis of variance (ANOVA), and P values\.05 were considered significant. Tukey post hoc testswere used for comparison between mean values. Poweranalysis was conducted for every comparison we made.Effect sizes .0.8 were considered large according toCohen.13 Finally, k correlation coefficient with 95% confi-dence interval (CI) was used to measure the overall intraob-server and interobserver agreement regarding the ROIplacement. The agreement was classified as poor (k\ 0.4),moderate (k = 0.4-0.8), or excellent (k 0.8). The statisticalanalysis was performed with the Statistica v.8 program(StatSoft, Tulsa, Oklahoma).

RESULTS

Clinical Evaluation

All ACL-reconstructed patients were satisfied with the out-come of the surgery and resumed their preinjury level of

TABLE 1MR Imaging Protocol Series of Different Sequences Performed in Orthogonal Planes to Obtain an Overview of

All Knee Joint Structures and to Evaluate Graft Integritya

Parameters Sequence Plane TR (ms) TE FA NSA Thickness/Gap (mm) FOV/RFOV

PDW/TSE/SPIR Axial 3455 15 90 2 4.00/0.4 180/80PDW/TSE Coronal 3092 16 90 2 3.3/0.33 180/90STIR/long TE Coronal 1798 55 90 3 3.3/0.33 180/90PDW/TSE Sagittal 2711 16 90 2 3.3/0.33 180/90T2W/TSE/SPIR Sagittal 2600 70 90 2 3.3/0.33 180/90T1W/TSE Oblique axial 500 17 90 3 3.0/0.3 180/903D/FFE/SPIR Oblique sagittal 32 5.1 25 2 2.0 180/80

aPDW, proton density weighted; TSE, turbo spin echo; SPIR, spectral presaturation by inversion recovery; STIR, short tau inversionrecovery; TE, echo time; T2W, T2 weighted; T1W, T1 weighted; 3D/FFE, 3-dimensional fast field echo; TR, repetition time; FA, flip angle;NSA, number of sample averages; FOV, field of view; RFOV, rectangular field of view.



sports participation. Negative Lachman, anterior drawer,and pivot-shift tests indicated that the knee joint stabilitywas regained clinically for all the patients. For the BPTBgraftreconstructed patients at 6 months postoperativetime interval, the median Lysholm score was 93 (range,83-100) and the Tegner score was 6 (range, 4-8) at thetime of examination, while for patients at 12 months aftersurgery, the median Lysholm score was 98 (range, 93-100)and the Tegner score was 7 (range, 6-8). The KT-1000arthrometer results revealed that the mean differencebetween the anterior tibial translation of the reconstructedand intact sides at 6 months was 1.5 mm (range, 0-3 mm)for the 134-N test and 1.9 mm (range, 1-3 mm) for the max-imum manual test, respectively. The KT-1000 arthrometerresults at 12 months were 1.2 mm (range, 0-2 mm) for the134-N test and 1.5 mm (range, 1-3 mm) for the maximummanual test.

MRI Evaluation

Calculation of the enhancement index at the 3 graft siteson the third postoperative day MRI examination showedmean values of 0.95 6 0.03, 0.93 6 0.02, and 0.90 6 0.02for the intra-articular, intraosseous tibial tunnel, andintraosseous juxta screw sites, respectively. According tothe biomedical meaning of the enhancement index, no sig-nificant vascularization can be assumed at this time. How-ever, 6 months after surgery, the enhancement index meanvalues were 1.52 6 0.14, 1.36 6 0.16, and 1.29 6 0.15,respectively. All these values were found to be significantlyincreased above the threshold value of 1, demonstrating

satisfactory revascularization. Similarly, 12 months aftersurgery, the enhancement index mean values were 1.53 60.15, 1.45 6 0.13, and 1.38 6 0.15, respectively, indicatingalso satisfactory revascularization. Post hoc analysis withTukey tests revealed significant differences in the enhance-ment index values of the 3 graft sites at the 3 time intervalson respective MRI examinations.

Intra-articular Site. Three days after surgery, the intra-articular site of the graft demonstrated insufficientenhancement index (0.95 6 0.03). However, 6 months aftersurgery, this site of the graft achieved satisfactory contrastmedium uptake, displaying at the same time significantlyhigher enhancement index values compared with the other2 sites (P\ .001). Nevertheless, by 12 months, no furthersignificant increase of the enhancement index was notedcompared to the 6 months (P = .1).

Intraosseous Tibial Tunnel Site. Similarly, 3 days aftersurgery, the graft site that was enclosed inside the tibialtunnel displayed enhancement index values inferior tothe threshold value (0.93 6 0.02). On the other hand, 6and 12 months after surgery, the enhancement indexwas clearly increased as judged by the effect size,although this change was not statistically significant (P= .09). This indicates that revascularization was still inprogress.

Figure 2. Graft sites. Three sites can be discerned along thegraft course according to the composition of the surroundingmicroenvironment: the intra-articular site, the intraosseoustibial tunnel site, and the intraosseous juxta screw site.

Figure 3. Before contrast agent administration. A sagittalsection through the reconstructed knee obtained beforeintravenous administration of gadolinium. Three regions ofinterest (ROIs), seen as numbered circles, were assigned atthe midportion of every graft site (ROI 1, 2, and 3). Calcula-tion of the SNQ values was performed independently foreach graft site and every time interval. The noise of the imagebackground was measured with the assignment of a ROI (4)at the lower part of the image and within a distance of 1 cmfrom the tibial tubercle.



Intraosseous Juxta Screw Site. Finally, at the thirdpostoperative day, the intraosseous juxta screw siteexhibited enhancement index values lower than 1 as theother 2 sites did. Six and 12 months after surgery, increaseof the enhancement index values was noted, although non-significant as well (P = .07).

Moreover, comparison of the enhancement index valuesbetween the 2 intraosseous graft sites showed that the sitethat was entirely surrounded by the cancellous bone achievedhigher values than the juxta screw site during the wholeobservation period. This finding was supported by the largeeffect sizes, although significant differences were also notdetected. The above findings are summarized in Figure 5.

Power analysis revealed that all effect sizes of our com-parisons were .0.8. This enabled us to confidently regardas sufficient the population sample for detecting correctlythe true experimental outcome (lack or presence of differ-ences) between graft sites at every time interval. Theintraobserver and interobserver agreement, as assessedby the k coefficient, was excellent in every case (0.92: CI,0.9-0.94 and 0.9: CI, 0.89-0.91, respectively).

DISCUSSION

The successful outcome of ACL reconstruction requires theincorporation of the tendon autograft into its new environ-ment.4,30,56 This process will take the intrinsic metamorphosis

of a tendinous tissue into a neoligament with the uneventfultransition through 4 distinct phases: avascular necrosis, revas-cularization, cellular repopulation, and finally remodeling andreorganization of the collagen fibers.|| The phase at which thegraft tissue recovers its vascular supply plays the key role inthis process by acting as a prerequisite for the other phasesto be conducted and ensuring the long-term viability of thegraft. Therefore, it is important to elucidate how this phaseproceeds over time along the graft course.

The present study is an MRI-based quantitative analy-sis of the enhancement that the BPTB autograft achievesafter contrast medium administration corresponding toits vascularity at the 3 specific sites examined along itslength. These 3 sites are namely the intra-articular sitelocated centrally within the joint, the intraosseous siteenclosed in the osseous tibial tunnel, and the site locatedat the interface between the osseous tibial tunnel walland the fixation material. Our hypothesis was that becausethe microenvironment of the graft is different at these 3sites, the graft tissue would also be differently affectedduring its revascularization process.

Our results show that, initially, the absence of function-ing vasculature due to necrosis after graft harvestingresults in absence of contrast agent uptake and thereforeinsufficient enhancement of the graft tissue with valuesof the corresponding index below 1 in all cases. This imag-ing finding is in agreement with the results from previoushistological studies that reported extensive ischemic necro-sis taking place throughout the graft substance initiallyafter its harvesting.28,47

Six and 12 months after surgery, however, satisfactoryenhancement was noticed in all graft sites, indicating ade-quate perfusion of the harvested tissue. Interestingly, theintra-articular site was found to be the first graft sitethat reached maximum enhancement index values at 6months without any significant alteration noted at 12months, indicating the completion of the revascularizationprocess for this site by this time period. On the other hand,the site of the graft that was entirely enclosed by the

Figure 4. After contrast agent administration. The same sag-ittal section is depicted after intravenous administration ofgadolinium. Uptake of the contrast medium can be seen asincreased signal intensity within the vessels of the poplitealregion. Identical ROIs were assigned at the exact same posi-tions of the 3 graft sites by the software program, and calcu-lations of the SNQ values were similarly repeated.

TABLE 2Enhancement Index Numerical and Biomedical Definitiona

Enhancement Index EI SNQafterSNQbefore

SNQafter . SNQbefore, EI . 1 Satisfactory revascularizationSNQafter SNQbefore, EI 1 Insufficient revascularization

aEnhancement index ratio provides directly interpretativeinformation regarding the presence, or not, of revascularization.Uptake of the contrast medium will result in the SNQ valuesobtained after its administration being greater than before, andtherefore, their resultant ratio will be greater or at least equalto the numeric value of 1. This signifies satisfactory revasculariza-tion for the specific graft site. On the contrary, enhancement indexvalues lower than 1 mean insufficient revascularization. There-fore, 1 is the threshold value for demonstrating revascularization.

||References 4, 7, 11, 18, 19, 28, 30, 31, 41, 46, 50.



osseous tibial tunnel wall, as well as the site that partiallyinteracted with the interference screw, showed a slowerprogression of revascularization process. Both these sitesat 6 months, despite the fact that they exhibited satisfac-tory enhancement, had indices that were significantly infe-rior compared with the intra-articular site (P \ .001,respectively). By 12 months, the increase of the enhance-ment index was clearly noted in both these sites, suggest-ing that graft revascularization was still in progressduring all this period. However, mean enhancement indexvalues were inferior to those of the intra-articular site,although no significant differences were observed at thattime. Nonsignificant differences as well were found whenmean enhancement index values of the intraosseous tibialtunnel and intraosseous juxta screw sites between 6 and 12months time interval were compared (P = .07 and .09,respectively), although the increasing trend of their meanvalues during this time period is supported by large effectsizes (.0.8), as a power analysis revealed. The above-mentioned differences in the enhancement index valuesreflect the differences in the revascularization progressat the different graft sites, supporting our initial hypothe-sis that the microenvironment conditions existing aroundthe graft would affect its revascularization process.

The rapid completion of revascularization at the intra-articular site could be explained by the fact that its freesurface facilitates the provision of the nutrient elementsdirectly from synovial fluid in the initial postoperativeperiod.3,4,14,17,21,37,54 Several studies have established therole of diffusion as an important instantaneous pathwaybetween interstitial synovial fluid and synovial lining neo-vessels for the delivery of the nutrients.14,54 Later, micro-vessels and neovessels originating from the infrapatellarfat pad and the posterior synovial tissues embrace circum-ferentially its surface to form a rich vascular envelop that

contributes with numerous intraligamentous branches toperfusion of this graft site.2,3,5,19,41,44,47 On the otherhand, the slower revascularization progress of both intra-osseous graft sites could be explained by the fact thatnutrient elements are provided mainly by endosteal vesselbranches developing from the adjacent cancellous bone.6,28

Specifically, the graft site entirely located in the osseoustunnel and which interacted perimetrically with the can-cellous bone demonstrated higher enhancement index val-ues during the whole examination period, compared withthe juxta screw site, which interacted partially with thecancellous bone. And despite the fact that we did not noticestatistical significant differences in this case, these find-ings seem to have remarkable clinical significance.

Contrary to what was previously suggested based on theresults from animal studies,11 that the femoral and tibialtunnels contribute predominantly to early vasculature for-mation at the corresponding graft sites, our findings showthat the intra-articular site was the first graft site to com-plete this crucial phase. These differences could be attributedto substantial differences in the precision of the surgical tech-nique used and the overall success of the reconstructed sur-gery at the knees of experimental animal models that mayaffect the final healing outcome.7,11,50 Results from previoushistological studies conducted on human ACL specimens,however, are in accordance with our findings. Falconieroet al19 studied the maturation characteristics of BPTB auto-grafts at the midportion and noted maximum vascularresponse of the graft tissue 6 months after surgery, whileat 12 months, the degree of graft vascularity closely resem-bled that of normal ACL. Abe et al,1 analyzing biopsy speci-mens obtained at the time of second-look arthroscopy inhuman ACL-reconstructed knees, reported that revasculari-zation of the graft occurs during the early postoperativeperiod with no differences detected between 24 and 52 weeksafter surgery. Similarly, Rougraff and Shelbourne41 obtainedarthroscopic biopsy samples from a limited area of the intra-articular portion of BPTB autografts in uninjured ACL-reconstructed knees and revealed that although this processwas not entirely equivalent with what was observed in ani-mal models, revascularization of the graft does take place,ensuring the grafts long-term survival.

Among noninvasive procedures, MRI has been widelyused for the postoperative evaluation of ACL graft integ-rity.33,39,40,53 Several MRI studies were performed todescribe the longitudinal imaging appearance of the ACLgraft substitute. Many of these studies were performedwithout the administration of contrast agent, and there-fore, the serial changes noted on the graft were character-ized on a visual scoring basis, reflecting indirectly itsoverall healing course.24,35,38 However, without the objec-tive measurements of contrast medium uptake, the tempo-ral changes of signal intensity could only potentially beattributed to vascular supply.

The use of a paramagnetic contrast agent such as Gd-DTPA can improve the diagnostic sensitivity and specific-ity by direct visualization of tissue vascularity.27 It isknown that enhancement of a tissue after contrast agentadministration results in an increase of signal inten-sity.8,27,36,51,52 Therefore, contrast-enhanced MRI can

Figure 5. Changes over time of the enhancement indexmean values at the 3 examined graft sites. The intra-articularsite is the first to complete this phase, while for both the other2 intraosseously enclosed sites, the revascularization pro-cess is still in progress throughout the first postoperativeyear.



noninvasively monitor the serial signal intensity changesnoted during the revascularization process of the ACLgraft. On the basis of these changes before and after theadministration of contrast agent, we calculated theenhancement index, which reflects this time-dependentbiological phenomenon and most importantly gives directlyquantitative information about the revascularizationdegree. Muramatsu et al36 compared the revascularizationprogress in autografts and allografts in humans, establish-ing a fundamental methodological basis for quantitativeanalysis by means of contrast-enhanced MRI. Weileret al,52 similarly in a sheep model, demonstrated that thesignal intensity changes observed in the graft tissue reflectits vascularity. However, in this animal-based study, onlythe intra-articular part of the graft was examined. In ourstudy, we assessed graft revascularization in 3 specificsites identified along its length. To our knowledge, this isthe first study to evaluate the outcome of the revasculari-zation process in association with the microenvironmentsurrounding each site of the graft. More interestingly,with the use of the enhancement index as a quantitativeparameter, this can be performed in a comprehensiveand reproducible way, which provides directly comparableresults for every patient at each time interval.

Limitations of our study might be considered in that wehave not included in our study the corresponding sites ofthe graft that are located inside the femoral tunnel. How-ever, because the surgical technique ensures the sameintraosseous environment for both tibial and femoral tun-nels, we can presume that the microenvironment condi-tions are identical, and therefore, the revascularization ofthe graft in the tibial tunnel itself can be provided asa model for the interaction between cancellous bone andfixation material with the graft. Imaging of the intrafe-moral graft portion and calculation of its signal intensitychanges require a different MRI scan design in an obliquecoronal plane. Future studies in this field would providevaluable information regarding the revascularization rateof this graft portion as compared with the intra-articularand intratibial parts.

Another limitation of this study is the lack of correlationbetween our findings and histological or biomechanicalanalysis of graft material properties. However, the designof our study included uncomplicated reconstructed kneesof human patients, and thus, any kind of interventionalprocedure was inappropriate.

As far as the future prospective of the present study isconcerned, new hypotheses in the field of several in vivoclinical applications can be developed and tested, benefit-ing from the level of quantitative information that can beacquired noninvasively by contrast-enhanced MRI. Admin-istration of various blood products, such as platelet-richplasma and bone morphogenic proteins and examinationof their acceleration effect on the revascularization rateas well as the effect that different fixation materials (metalscrews or biodegradable screws) and surgical techniquesmight have on this process, can be accurately demon-strated with this imaging modality.

To conclude, in an uncomplicated knee joint havingundergone ACL reconstruction, the graft gradually achieves

revascularization, which is clearly and objectively demon-strated by contrast-enhanced MRI. This process occurs inassociation with the surrounding microenvironment, withthe intra-articular site of the graft being the first fullyrevascularized site even 6 months after surgery, while theintraosseous sites were still in progress throughout the firstpostoperative year. Enhancement index is an objectiveimaging parameter to directly assess this phenomenonwith contrast-enhanced MRI, which can be used as a valu-able noninvasive tool to evaluate the healing process ofthe ACL graft.

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