The ‘‘Ligamentization’’ Process In

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DOI: 10.1177/0363546511402662 2011 39: 2476 originally published online April 22, 2011Am J Sports Med

Steven Claes, Peter Verdonk, Ramses Forsyth and Johan BellemansHuman Graft? A Systematic Review of the Literature

The ''Ligamentization'' Process in Anterior Cruciate Ligament Reconstruction: What Happens to the

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The Ligamentization Process inAnterior Cruciate LigamentReconstruction

What Happens to the Human Graft?A Systematic Review of the Literature

Steven Claes,*y MD, Peter Verdonk,z MD, PhD, Ramses Forsyth, MD, PhD,and Johan Bellemans,y MD, PhDInvestigation performed at the Department of Orthopedic Surgery and Traumatology,University Hospitals Leuven, Belgium

Background: Surgical anterior cruciate ligament reconstruction using tendon grafts has become the standard to treat the functionallyunstable anterior cruciate ligamentdeficient knee. Although tendons clearly differ biologically from ligaments, multiple animal studieshave shown that the implanted tendons indeed seem to remodel into a ligamentous anterior cruciate ligamentlike structure.

Purpose: The goal of this study was to systematically review the current literature on the ligamentization process in humananterior cruciate ligament reconstruction.

Study Design: Systematic review.

Methods: A computerized search using relevant search terms was performed in the PubMed, MEDLINE, EMBASE, and CochraneLibrary databases, as well as a manual search of reference lists. Searches were limited to studies examining the healing of theintra-articular portion of the tendon graft based on biopsies of this graft obtained from a living human.

Results: Four studies were determined to be appropriate for systematic review, none of them reaching a level of evidence higherthan 3. All reports considered autografts. Biopsy specimens were evaluated by light or electron microscopy and analyzed for vas-cularization, cellular aspects, and appearance of extracellular matrix. All authors universally agreed that the tendon grafts survivein the intra-articular environment. Based on changes observed in the healing grafts with regard to vascularization, cellularaspects, and properties of the extracellular matrix, different chronologic stages in the ligamentization process were discerned.

Conclusion: The key finding of this systematic review is that a free tendon graft replacing a ruptured human anterior cruciateligament undergoes a series of biologic processes termed ligamentization. The graft seems to remain viable at any time duringthis course. Histologically, the mature grafts may resemble the normal human anterior cruciate ligament, but ultrastructural differ-ences regarding collagen fibril distribution do persist. Different stages of the ligamentization process are described, but no agree-ment exists on their time frame. Problematic direct transmission of animal data to the human situation, the limited number ofreports considering the ligamentization process in humans, and the potential biopsy sampling error attributable to superficial graftbiopsies necessitate further human studies on anterior cruciate ligament graft ligamentization.

Keywords: ACL reconstruction; ligamentization; biology; human knee; systematic review

Injuries to the anterior cruciate ligament (ACL) arevery frequent; in the United States, 250 000 newACL ruptures are estimated to occur each year, makingACL reconstruction one of most common surgical proce-dures in sports medicine.16,30 Rupture of the ACL impairsthe stability of the knee, resulting in difficulties withsporting activities and increased risk of subsequentmeniscal injury and early osteoarthritis.25 It has beenshown extensively that a ruptured ACL will not healspontaneously with nonoperative management.5 Nonaug-mented primary ACL repair (ie, just suturing the tornends of the ligament) has also been proven to be unsuc-cessful, thus making ACL reconstruction using tendon

*Address correspondence to Dr Steven Claes, Department of Ortho-pedic Surgery and Traumatology, University Hospitals Leuven Campus,Pellenberg, Weligerveld 1, B-3212 Pellenberg, Belgium (e-mail:[email protected]).

yDepartment of Orthopedic Surgery and Traumatology, UniversityHospitals Leuven, Belgium.

zDepartment of Orthopedic Surgery and Traumatology, UniversityHospital Ghent, Belgium.

N. Goormaghtigh Institute of Pathology, University Hospital Ghent,Belgium.

The authors declared that they had no conflicts of interest in theirauthorship and publication of this contribution.

The American Journal of Sports Medicine, Vol. 39, No. 11DOI: 10.1177/0363546511402662 2011 The Author(s)

M

2476

Basic Science Review

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grafts the current standard of care for the ACL-deficientknee.17

Both tendons and ligaments are composed of dense con-nective tissue primarily containing types I and III collagen,proteoglycans, and cells. Their precise composition and thearrangement of matrix macromolecules, however, clearlydiffer to provide the specific mechanical properties requiredby each structure to function effectively. For example, com-pared with tendons, ligaments are more metabolicallyactive, contain cells with rounded nuclei, and have higherDNA content, more type III collagen, more proteoglycans,less total collagen, a different amount of nonreducible colla-gen cross-links, and a different distribution of collagen fibrildiameters.14,36 Interestingly, some authors even demon-strated significant differences between different ligaments(eg, ACL versus medial collateral ligament),19 while otherswere able to discern different tendons on their ultrastruc-tural basis (eg, hamstrings versus patellar tendon).18 Mostrecently, Pereira et al32 found gender-specific morphologicand (immuno)histochemical differences between hamstringtendons of women and men.

Despite these (ultra)structural differences between ten-dons and ligaments, tendon grafts have become the stan-dard to replace the ruptured ACL. In 1986, Amiel et al3

demonstrated that a tendon autograft transplanted intothe rabbit knee to replace an excised ACL underwent a pro-cess of ligamentization. They described this phenomenonas the continuous development of a tissue that was origi-nally a patellar tendon into a substance very similar toa normal ACL, thus laying the foundation for currentACL reconstruction techniques. To date, numerous animalstudies in dogs,4,42 goats,12,31 sheep,6,15 rabbits,3 and mon-keys10 have been undertaken to examine the fate of bothautografts and allografts used to replace the ACL. How-ever, the complexity of the human ACL anatomy, surgicaltechniques, postoperative rehabilitation protocols, andtesting conditions impede direct transmission of animaldata to the human situation. Nevertheless, a thoroughunderstanding of the biologic processes occurring inhuman grafts is essential for optimizing ACL reconstruc-tion and maintaining long-term joint health, as these fac-tors directly affect the mechanical properties of thereconstructed ligament. Therefore, the purpose of thisstudy is to systematically review the literature withrespect to the healing of the intra-articular portion of ten-don grafts used in human ACL reconstruction.

METHODS

Search Strategy

We performed a systematic review of the literature to iden-tify all studies concerning the biology of a successfullyreconstructed human ACL. The PubMed, MEDLINE,EMBASE, and Cochrane Library databases were searchedfrom their earliest entry points to April 4, 2010, includingarticles published online as Epub ahead of print. Thecomputerized search was performed using combinationsof the following search terms: anterior cruciate ligament

or ACL reconstruction, human, histolog*, ligamenti-zation, graft, healing, and remodeling.

Selection

Searches were limited to English-language studies examin-ing the healing of the intra-articular portion of the tendongraft based on biopsies of this graft in a living human.Reports exclusively focusing on the biology of graft-tunnelincorporation were excluded. Reports on the biology of syn-thetic grafts used in human ACL reconstruction wereexcluded as well. In addition, each reference list from theidentified articles was manually checked to verify that rele-vant articles were not missed for the current review.

Study Quality Assessment

The methodologic quality of the different studies wasassessed with respect to study design; the presence ofnative tendon and/or ligament controls; adequate reportingof the ACL biopsy site, procedure, and sample size; the uti-lization of independent and/or blinded observers; and thedetailed description of the techniques used to examinethe tissue samples.

Data Extraction

Each study was evaluated for the following variables:study type; method of ACL reconstruction; type and originof grafted tendon; number of ACLs biopsied; technique,site, and size of the biopsy; study method and techniquesused to examine the samples; number of control biopsysamples of native tendon or ligament; interval betweenACL reconstruction and biopsy; and proposed ligamentiza-tion time frames. Relevant data from each included studywere extracted and recorded on multiple worksheets.

RESULTS

Study Identification

The initial computerized search using the aforementionedsearch terms identified around 300 potentially relevantarticles. Review of all available abstracts revealed 30articles that were retrieved for further evaluation. Thesemanuscripts were studied in detail and the reference listswere cross-checked by hand in order not to miss a relevantreport. After this procedure, 24 articles discussing the biol-ogy of the reconstructed ACL based on tissue biopsies ofliving humans could be withheld. Subsequently, 2 articlesreporting on biopsies of a ruptured reconstructed ACLwere excluded from the review,7,33 as were 4 articlesreporting on a single case.11,23,28,46 Finally, 9 studies with-out control biopsies of either native tendon or ligamentwere excluded,2,9,22,24,26,34,39-41 as well as 5 studies not pro-viding a ligamentization timescale.8,20,27,44,45 Therefore, 4studies were determined to be appropriate for systematic

Vol. 39, No. 11, 2011 Ligamentization Process in ACL Reconstruction 2477


review.1,13,35,37 The Quality of Reporting of Meta-analyses(QUOROM)29 flow diagram depicts the number of studiesidentified, included, and excluded as well as the reasonsfor exclusion (Figure 1).

Study Characteristics

Two studies were conducted in the United States,13,35 1study was conducted in Europe,37 and another in Japan.1

The ACL reconstruction procedures were performedbetween 1984 and 2008. The mean number of ACLs biop-sied per study is 31 (range, 21-43), with a total of 124ACL biopsies. With regard to graft origin, none of theincluded studies reports on allografts. Two of the includedstudies consider bonepatellar tendonbone (BPTB) graftsexclusively,1,35 1 study considered hamstring grafts,37 and

the remaining study performed biopsies on both BPTB andhamstring grafts.13 Surgical approaches and graft fixationtechniques greatly differed between reports; Abe et al,1 forexample, performed half of their surgeries by an open tech-nique in the early 1990s, while Sanchez et al37 publishedabout 2 decades later on an arthroscopic technique withthe addition of a preparation rich in growth factors(PRGF) in half of their cases. The main study characteris-tics are summarized in Table 1.

Study Quality

Assessment of the methodologic quality of these studiesrevealed that there were no randomized controlled trials(level of evidence 1). Moreover, no prospective studiescould be retrieved, probably because of the invasive nature

Studies identified using search terms in Pubmed,Cochrane, and Embase

(n = 304)

Studies retrieved for more detailed evaluation(n = 30)

Studies excluded (n = 263)Reason: nonhuman subjects, synthetic grafts, no

biopsies taken, nonreconstructed ACLs

Studies excluded (n = 6)Reason: biopsies from tendon-bone

interface of the graft

Studies concerning human ACL reconstruction with biopsies of the intra-articular

portion of the graft (n = 24)

Studies concerning successfully reconstructedhuman ACLs

(n = 22)

Studies excluded (n = 2)Reason: biopsies of ruptured grafts

Studies excluded (n = 4)Reason: case reports

Potentially appropriate studies for inclusion insystematic review

(n = 18)

Studies excluded (n = 14)Reason: no ligamentization timescale mentioned

no tendon or ligament controls performed

Studies withheld for systematic review(n = 4)

Figure 1. The Quality of Reporting of Meta-analyses (QUOROM) flow diagram, depicting the number of studies identified,included, and excluded, as well as the reasons for exclusion.29

2478 Claes et al The American Journal of Sports Medicine


of the biopsy procedure (level of evidence 2). One reportwas set up as a retrospective comparative study (level ofevidence 3).37 The remaining 3 were case series (level of

evidence 4).1,13,35 Only half of the included reports useda blinded, independent examiner to study the tissuesamples.13,37

TABLE 1Primary Study Characteristicsa

Rougraff et al (1993)35 Abe et al (1993)1 Falconiero et al (1998)13 Sanchez et al (2010)37

Study qualityLevel of evidence 4 4 4 3

Primary surgery:ACL reconstructionGraft origin Autograft Autograft Autograft AutograftGraft type BPTB BPTB Hamstrings (n = 8) Hamstrings

BPTB (n = 35)Surgical technique Technique not

mentionedOpen technique(n = 11)

Arthroscopicallyassisted

Arthroscopicallyassisted

Arthroscopically assisted(n = 10)

Addition of PRGF(n = 22)

Graft fixation Fixation over buttons Not mentioned Not mentioned Femur: transcondylarscrew

Tibia: bone plug and2 metal staples

Surgeon Single surgeon Not mentioned Multiple surgeons Not mentionedReturn to sports Full competition

from 3-6 moFull competitionfrom 10-12 mo

Full competition fromwhen the operativeleg was fullyrehabilitated

Not mentioned

Secondary surgery:Biopsy procedureIndication Unrelated knee

problems(n = 19)

Screw removal New intra-articularpathology

Unrelated kneeproblems (n = 17)

Volunteers (n = 4) Cyclops (n = 20)Interval 3 wk6.5 y 6 wk15 mo 3 mo10 y 6-25 moNo. biopsied ACLs n = 23 n = 21 n = 43 n = 37Biopsy technique Basket forceps Basket forceps Not mentioned Basket forcepsBiopsy site Middle segment

either centralor superficial

Midzone superficiallayer of the core

Midportion of the graft,1 superficial, 1 deep

Standardized with thedevice under medialfemoral condyle

Biopsy specimen size 1-3 mm 3 10-20 mm 3-5 mm3 Not mentioned 3 mm 3 5-10 mmBiopsy analysisNo. native controls Native ACL at autopsy

(n = 1)Native ACLs atTKA (n =8)

Native ACLs (n = 5) Native hamstringtendons (n = 2)

Native patellartendons (n = 11)

Native patellartendons (n = 7)

Independent examiner No No Yes, blinded Yes, blindedStudy method Light microscopy Light microscopy Light microscopy Light microscopy

Electron microscopySpecimen stainingtechniques

Hematoxylin/eosin Hematoxylin/eosin Hematoxylin/eosin Hematoxylin/eosinAlcian blue

Quantitative histology Vascularity Vascularity Vascularity VascularityCellularity, nucleartype

Cellularity, cellularshape, cellulararrangement

Cellularity Cellularity, nuclearshape, and orientation

Degeneration Collagen orientation Metaplasia Presence ofglycosaminoglycans

Periodicity of crimp Crimp patterns Fiber pattern CrimpPolarization

Ligamentization stages 4 3 3 3

aACL, anterior cruciate ligament; BPTB, bonepatellar tendonbone; PRGF, preparation rich in growth factors; TKA, total kneearthroplasty.



Biopsy Procedure

Mostly, the ACL biopsies were performed during second-look arthroscopy at the time of hardware removal orwhen addressing new unrelated knee injury such as menis-cal tears or chondral lesions. Some subjects volunteered toundergo a second-look arthroscopy for the sole purpose ofbiopsy. In 1 paper,37 more than half of biopsy specimenswere obtained during an arthroscopy performed for ACL-related symptoms (cyclops lesion), thus possibly compro-mising the representability of the specimens in this series.

Most authors used a standard 2.5-mm or 3-mm basketforceps to perform the biopsy. With this technique, a super-ficial specimen of the reconstructed ACL could be obtained.However, the spatial terminology of the biopsy zone differsamong these authors: . . .middle segment, either central orsuperficial,35 . . .superficial layer of the core,1 or by posi-tioning the forceps directly under the femoral condylethus enabling the forceps to reach the same area of thegraft at any time in all patients.37 Although 3 of 4included reports mention the use of a comparable biopsydevice, the mean biopsy sample size between reportsranged from 3 to 5 cubic millimeters to 1 to 3 3 10 to20 mm. Although some of the included papers mention con-cerns regarding the potential deleterious effects of thebiopsy on graft integrity, such effects have not been wit-nessed during follow-up by any of these reports. However,none of the studies have provided direct evidence of biopsysite healing.

Biopsy Study Techniques

In all 4 included studies, the procured samples were sub-jected to light microscopic (LM) evaluation.1,13,35,37 Samplepreparation was similar in these reports with regard to fix-ation, dehydrating, and embedding of the specimens. Allauthors used standard hematoxylin and eosin staining.Sanchez et al37performed additional Alcian blue staining.Three studies describe the histologic changes during theligamentization process by the use of a self-developed13,35

or adapted37 quantitative scoring system. This quantita-tive approach has the advantage of providing a more orless objective evaluation of the observed biologic phenom-ena, thus enabling statistical analysis and comparisonsbetween grafts versus native ligament or tendon controlsand between different graft ages, graft types, or surgicaltechniques. Abe et al,1 on the other hand, reported theirhistologic findings in a purely descriptive manner, butalso reported on an electron microscopic (EM) evaluationof the ACL specimens, focused on collagen fibril diameterdistribution.

Biologic Graft Features

Using the aforementioned study techniques, all authorsdescribed the changes occurring in the healing graft withregard to (1) vascularization, (2) cellular aspects, and (3)appearance of the extracellular matrix (ECM) in compari-son with native tendon and/or ACL control biopsies.

Vascularization. All studies found vascularity at least inthe periphery of all grafts at any given time point, thusleading to the conclusion that the free tendon graft doessurvive in the intra-articular environment. The degree ofneovascularization seems variable between subjects,although all authors witnessed a hypervascularity earlyafter ACL reconstruction, which only slowly decreases tocontrol ACL vascularization patterns during remodeling.It is suggested that the new blood vessels develop fromthe synovium, the infrapatellar fat pad, and the pseudo-ligamentum mucosum; however, no hard evidence to sup-port this hypothesis could be found in the included studies.

Cellular Aspects. There is a consensus among all 4reports that cellular repopulation of the tendon graftdoes occur after ACL reconstruction, although the exactsource of these fibroblast remains unclear. Parallel to theneovascularization, grafts showed increased cell countsduring the early postoperative period. In this early phase,fibroblasts are disorganized, randomly arranged, and met-abolically active as shown by their plump nuclei. As remod-eling continues, the cells become longitudinally alignedand nuclei become less ovoid as in the control ACL speci-mens. No report mentions the presence of neural elementsin the studied graft biopsy specimens.

Appearance of ECM. Histologically, disorganized, irreg-ular collagen bundles are witnessed in the early graft. Asthe graft matures, collagen bundles attain a denselypacked, parallel alignment. No report has studied thespecimens for collagen type. Abe et al1 showed by electronmicroscopic analysis that collagen fibrils in the trans-versely sectioned areas were uniformly small comparedwith those of normal tendon and ACL. In other words,the bimodality of the collagen fibril diameter distributionof normal tendon changes in unimodal, small fibril diame-ters over time.

One article describes 2 types of dissimilar tissue in thebiopsy specimens of more than half of the operatedpatients: the original, remodeling tendon is observed cen-trally in the biopsy specimen, enveloped by newly formedconnective tissue. At first, these 2 areas are discerned bythe cell shapes and extent of matrix remodeling, butbecome hardly distinguishable during maturation.

Ligamentization Stages and End Point

The period from ACL surgery to second-look arthroscopyand histologic analysis ranged from 3 weeks to 120months, with an average interval of 21.1 months.Although graft remodeling is a continuous biologic pro-cess, all authors have adapted different ligamentizationstages with characteristic histologic or ultrastructuralchanges. Three of 4 reports mention 3 different stages ofligamentization.1,13,37 Although distinct graft stage ter-minology is not always clearly or uniformly formulated,these stages can be termed early, remodeling, andmaturation in chronologic order. One study35 added anextra quiescent stage. Figure 2 depicts the importantdifferences in stage time frames between these 4 reports.For comparison, the graft healing phases occurring in



animal models as described in a recent overview by Schef-fler et al38 are shown as well.

The ligamentization end point is defined as the timepoint from which no further changes are witnessed in theremodeled grafts. Falconiero et al13 found no significantdifferences in the histologic aspect of their 12-month graftscompared with controls, concluding that ligamentizationoccurs over a 12 month period with peak maturity evidentat 1 year. Rougraff et al,35 on the other hand, stillobserved areas of degeneration, neovascularity, and hyper-cellularity until 3 years after reconstruction. From thattime on, these authors describe the grafts as quiescentand similar to ACL controls. Abe et al1 state that the graftis still undergoing the process of remodeling at 1 year aftersurgery; however, these authors did not study grafts olderthan 15 months. According to Sanchez et al,37 the graftsreach maturity at around 2 years after surgery.

DISCUSSION

Much of the current knowledge on the biologic phenomenaoccurring in the healing ACL graft have been derivedfrom numerous animal studies. To date, the literature hasbeen fueled by fairly large amounts of data from ACL recon-structed dogs, goats, sheep, rabbits, and monkeys. However,no ideal animal ACL model has been developed so far, as isreflected in this wide variety of studied animal species.Moreover, when comparing the large amounts of animaldata with the scarce human biopsy studies, important dif-ferences have been revealed.38 For example, the timelineof biologic graft changes between animals and humansappears to be substantially different, with a much slowerremodeling activity in human grafts compared with animals(Figure 2). On the other hand, some in vivo animal reportsfound that the graft undergoes an early phase of vast necro-sis occurring in the center of the graft,3,6 while this necrosiswas barely seen in human biopsy specimens. The sameimportant differences have been shown in nonbiopsy studiesusing gadolinium- enhanced MRI. Howell et al21 could notdetect any revascularization except for the periligamentous

tissue in human reconstructed ACLs, while Weiler et al,43

using the same technique, did find significantly upregulatedneovascularization in ACL-reconstructed sheep. Finally, thecomplexity of the human ACL anatomy; the rapidly evolv-ing, precise surgical techniques to replicate this anatomy;and the extreme importance of adequate postoperative reha-bilitation are all impossible to control in animals. In sum-mary, findings derived from ACL-reconstructed animalsshould not be transmitted directly to the human knee.When looking at the available human data, the literatureis surprisingly limited. Only 4 articles could be withheldfor systematic review, with only 1 article reaching a levelof evidence higher than 4.

An important finding of this systematic review is thata free tendon graft, when implanted in the human kneeto replace a ruptured ACL, does survive in the intra-articular environment. At any given time point after recon-struction, the ACL is histologically viable with evidence ofnourishing vascularization at least in the grafts peripheryand with no signs of important necrosis. The origin of theneovascularization is thought to be the Hoffa fat pad andthe synovium, although no report provides hard evidenceto prove this hypothesis.

Furthermore, this systematic review shows that thegeneral concept of ligamentization of a tendon graft asproposed in animal models is applicable to humans aswell. The literature consistently describes a process inwhich the implanted grafts progressively lose tendon-specific biologic features, meanwhile exhibiting moreand more ligamentous histologic properties. It is clearthat this ligamentization process is a continuum of biologicchanges rather than a series of distinct, time-dependentbiologic events. However, dividing this process into differ-ent stages is considered to be useful, especially with regardto postoperative rehabilitation protocols and timing ofreturn to preinjury sporting activities. As shown in Figure2, no consensus can be found in the current literature onthese ligamentization time frames, with surprising differ-ences regarding the time points between all authors.

In theory, completion of the ligamentization processimplies the generation of a structure that in every respect

Animals

Early Scheffler et al. (2008)38

Humans

Rougraff et al. (1993)35

Abe et al. (1993)1

Falconiero et al. (1998)13

Sanchez et al. (2010)37

Months after ACLR

Remodeling Maturation

0 3 6 9 12 18 21 24 30

Early Remodeling

Maturation

36

Maturation

48

Early Remodeling Maturation Quiescent

15

Early Remodeling Maturation

Early Remodeling

Figure 2. Differing ligamentization time frames in human grafts compared with a recent review of animal reports.38



is undistinguishable from a native ACL. As no significantstatistical differences in light microscopic features couldbe found between mature grafts and native ACLs, 3 of theincluded reports indeed concluded that the grafts hadbecome similar to a native ACL,13,35,37 although the timeneeded by the remodeling graft to reach this status is a mat-ter of debate. On the other hand, Abe et al1 showed that nograft reached ligamentous maturity on an ultrastructurallevel, as every graft specimen showed a typical depletionof large diameter collagen fibrils, thus leading to an unimo-dal collagen fibril diameter distribution in contrast to thebimodal distribution witnessed in normal ACLs. These find-ings were recently confirmed by Zaffagnini et al44,45 in 2nonincluded reports based on biopsies of a limited numberof reconstructed human ACLs up to 10 years after surgery,which is the longest biologic follow-up of human ACL recon-struction currently found in the literature. Briefly, theyfound that from 24 months after surgery, the graft tissuelooked very similar to a normal ACL under light micros-copy and that from that point on, no further changes wereevident. However, persistent differences remained at theultrastructural level with electron microscopy: mean colla-gen fibril diameter and bimodality of fibril distribution asin the normal ACL were not reached at any time.

In summary, this systematic review has demonstratedthat free tendon grafts implanted in the human knee toreplace a ruptured ACL remain viable at any time pointand undergo a process of ligamentization characterizedby progressive biologic changes. Histologically, the maturegrafts may resemble the normal human ACL, but ultra-structural differences regarding collagen fibril distributiondo persist. Different stages of the ligamentization processare described by many authors, although no agreementexists on their time frame.

The limits of this systematic review include the rela-tively poor level of evidence of the reports. Because ACLsurgeries in these studies were performed from 1984 until2008, major differences in surgical techniques and rehabil-itation protocols are recognized between reports. Inter-study heterogeneity could be caused by the differentorigins of the tendon grafts used to replace the rupturedACL. Until now, there is no evidence available to assumethat BPTB and hamstring autografts will behave similarlyduring their ligamentization process in humans.

Because of the invasive nature of the biopsy procedureand its potential deleterious effect on an otherwise healthygraft, almost all reported biopsies have been taken fromthe superficial region of the midzone of the reconstructedACL. The question remains whether this biopsy samplecan be representative for the entire 3-dimensional graftstructure. Only a few human cases of in toto retrieved graftscan be found in the literature. Delay et al11 reported on theretrieval of an entire knee joint at autopsy, 18 months afterBPTB ACL reconstruction. Most interestingly, they foundin contrast to the superficial biopsy studies included inthis systematic review that the ACL specimen did showvast areas of deep necrosis, most evident where the graftexited the tibial tunnel. The authors state that it wasunclear whether this area of necrosis represented a portionof the autograft that had never been revascularized or

whether it had undergone degeneration and became acellu-lar over time. Clearly, a better understanding of the graftbiology in human ACL reconstruction will depend on thepossibility to obtain core biopsy samples of these grafts.

Accordingly, probably the key finding of this systematicreview is that although ACL reconstruction is performedas a routine surgical procedure all around the world, theunderlying ligamentization process is still poorly under-stood in the human knee. Further human studies areneeded to understand normal graft healing after ACLreconstruction.

An online CME course associated with this article isavailable for 1 AMA PRA Category 1 CreditTM at http://ajsm-cme.sagepub.com. In accordance with the standardsof the Accreditation Council for Continuing Medical Edu-cation (ACCME), it is the policy of The American Ortho-paedic Society for Sports Medicine that authors, editors,and planners disclose to the learners all financial rela-tionships during the past 12 months with any commercialinterest (A commercial interest is any entity producing,marketing, re-selling, or distributing health care goodsor services consumed by, or used on, patients). Any andall disclosures are provided in the online journal CMEarea which is provided to all participants before theyactually take the CME activity. In accordance withAOSSM policy, authors, editors, and planners participa-tion in this educational activity will be predicated upontimely submission and review of AOSSM disclosure. Non-compliance will result in an author/editor or planner to bestricken from participating in this CME activity.

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The ‘‘Ligamentization’’ Process In

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