The ''Ligamentization'' Process in Anterior Cruciate Ligament

AJSM PreView, published on April 22, 2011 as doi:10.1177/0363546511402662

Basic Science Review

The ‘‘Ligamentization’’ Process in Anterior Cruciate Ligament Reconstruction 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, PhD Investigation 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 functionally unstable anterior cruciate ligament–deficient knee. Although tendons clearly differ biologically from ligaments, multiple animal studies have shown that the implanted tendons indeed seem to remodel into a ligamentous ‘‘anterior cruciate ligament–like’’ structure. Purpose: The goal of this study was to systematically review the current literature on the ‘‘ligamentization’’ process in human anterior cruciate ligament reconstruction. Study Design: Systematic review. Methods: A computerized search using relevant search terms was performed in the PubMed, MEDLINE, EMBASE, and Cochrane Library databases, as well as a manual search of reference lists. Searches were limited to studies examining the healing of the intra-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 higher than 3. All reports considered autografts. Biopsy specimens were evaluated by light or electron microscopy and analyzed for vascularization, cellular aspects, and appearance of extracellular matrix. All authors universally agreed that the tendon grafts survive in the intra-articular environment. Based on changes observed in the healing grafts with regard to vascularization, cellular aspects, 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 cruciate ligament undergoes a series of biologic processes termed ‘‘ligamentization.’’ The graft seems to remain viable at any time during this course. Histologically, the mature grafts may resemble the normal human anterior cruciate ligament, but ultrastructural differences regarding collagen fibril distribution do persist. Different stages of the ligamentization process are described, but no agreement exists on their time frame. Problematic direct transmission of animal data to the human situation, the limited number of reports considering the ligamentization process in humans, and the potential biopsy sampling error attributable to superficial graft biopsies 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) are very frequent; in the United States, 250 000 new ACL ruptures are estimated to occur each year, making ACL reconstruction one of most common surgical procedures in sports medicine.16,30 Rupture of the ACL impairs the stability of the knee, resulting in difficulties with sporting activities and increased risk of subsequent meniscal injury and early osteoarthritis.25 It has been shown extensively that a ruptured ACL will not heal spontaneously with nonoperative management.5 Nonaugmented primary ACL repair (ie, just suturing the torn ends of the ligament) has also been proven to be unsuccessful, thus making ACL reconstruction using tendon

*Address correspondence to Dr Steven Claes, Department of Orthopedic Surgery and Traumatology, University Hospitals Leuven Campus, Pellenberg, Weligerveld 1, B-3212 Pellenberg, Belgium (e-mail: [email protected]). y Department of Orthopedic Surgery and Traumatology, University Hospitals Leuven, Belgium. z Department of Orthopedic Surgery and Traumatology, University Hospital Ghent, Belgium. § N. Goormaghtigh Institute of Pathology, University Hospital Ghent, Belgium. The authors declared that they had no conflicts of interest in their authorship and publication of this contribution. The American Journal of Sports Medicine, Vol. XX, No. X DOI: 10.1177/0363546511402662 Ó 2011 The Author(s)

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grafts the current standard of care for the ACL-deficient knee.17 Both tendons and ligaments are composed of dense connective tissue primarily containing types I and III collagen, proteoglycans, and cells. Their precise composition and the arrangement of matrix macromolecules, however, clearly differ to provide the specific mechanical properties required by each structure to function effectively. For example, compared with tendons, ligaments are more metabolically active, contain cells with rounded nuclei, and have higher DNA content, more type III collagen, more proteoglycans, less total collagen, a different amount of nonreducible collagen cross-links, and a different distribution of collagen fibril diameters.14,36 Interestingly, some authors even demonstrated significant differences between different ligaments (eg, ACL versus medial collateral ligament),19 while others were able to discern different tendons on their ultrastructural basis (eg, hamstrings versus patellar tendon).18 Most recently, Pereira et al32 found gender-specific morphologic and (immuno)histochemical differences between hamstring tendons of women and men. Despite these (ultra)structural differences between tendons and ligaments, tendon grafts have become the standard to replace the ruptured ACL. In 1986, Amiel et al3 demonstrated that a tendon autograft transplanted into the rabbit knee to replace an excised ACL underwent a process of ‘‘ligamentization.’’ They described this phenomenon as the continuous development of a tissue that was originally a patellar tendon into a substance very similar to a normal ACL, thus laying the foundation for current ACL reconstruction techniques. To date, numerous animal studies in dogs,4,42 goats,12,31 sheep,6,15 rabbits,3 and monkeys10 have been undertaken to examine the fate of both autografts and allografts used to replace the ACL. However, the complexity of the human ACL anatomy, surgical techniques, postoperative rehabilitation protocols, and testing conditions impede direct transmission of animal data to the human situation. Nevertheless, a thorough understanding of the biologic processes occurring in human grafts is essential for optimizing ACL reconstruction and maintaining long-term joint health, as these factors directly affect the mechanical properties of the reconstructed ligament. Therefore, the purpose of this study is to systematically review the literature with respect to the healing of the intra-articular portion of tendon grafts used in human ACL reconstruction.

METHODS Search Strategy We performed a systematic review of the literature to identify all studies concerning the biology of a successfully reconstructed human ACL. The PubMed, MEDLINE, EMBASE, and Cochrane Library databases were searched from their earliest entry points to April 4, 2010, including articles published online as ‘‘Epub ahead of print.’’ The computerized search was performed using combinations of the following search terms: ‘‘anterior cruciate ligament

The American Journal of Sports Medicine

or ACL reconstruction,’’ ‘‘human,’’ ‘‘histolog*,’’ ‘‘ligamentization,’’ ‘‘graft,’’ ‘‘healing,’’ and ‘‘remodeling.’’

Selection Searches were limited to English-language studies examining the healing of the intra-articular portion of the tendon graft based on biopsies of this graft in a living human. Reports exclusively focusing on the biology of graft-tunnel incorporation were excluded. Reports on the biology of synthetic grafts used in human ACL reconstruction were excluded as well. In addition, each reference list from the identified articles was manually checked to verify that relevant articles were not missed for the current review.

Study Quality Assessment The methodologic quality of the different studies was assessed with respect to study design; the presence of native tendon and/or ligament controls; adequate reporting of the ACL biopsy site, procedure, and sample size; the utilization of independent and/or blinded observers; and the detailed description of the techniques used to examine the tissue samples.

Data Extraction Each study was evaluated for the following variables: study type; method of ACL reconstruction; type and origin of grafted tendon; number of ACLs biopsied; technique, site, and size of the biopsy; study method and techniques used to examine the samples; number of control biopsy samples of native tendon or ligament; interval between ACL reconstruction and biopsy; and proposed ligamentization time frames. Relevant data from each included study were extracted and recorded on multiple worksheets.

RESULTS Study Identification The initial computerized search using the aforementioned search terms identified around 300 potentially relevant articles. Review of all available abstracts revealed 30 articles that were retrieved for further evaluation. These manuscripts were studied in detail and the reference lists were cross-checked by hand in order not to miss a relevant report. After this procedure, 24 articles discussing the biology of the reconstructed ACL based on tissue biopsies of living humans could be withheld. Subsequently, 2 articles reporting on biopsies of a ruptured reconstructed ACL were excluded from the review,7,33 as were 4 articles reporting on a single case.11,23,28,46 Finally, 9 studies without control biopsies of either native tendon or ligament were excluded,2,9,22,24,26,34,39-41 as well as 5 studies not providing a ligamentization timescale.8,20,27,44,45 Therefore, 4

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Studies identified using search terms in Pubmed, Cochrane, and Embase (n = 304) Studies excluded (n = 263) Reason: nonhuman subjects, synthetic grafts, no biopsies taken, nonreconstructed ACLs

Studies retrieved for more detailed evaluation (n = 30)

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

Studies concerning successfully reconstructed human ACLs (n = 22) Studies excluded (n = 4) Reason: case reports Potentially appropriate studies for inclusion in systematic 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 studies were determined to be appropriate for systematic review.1,13,35,37 The Quality of Reporting of Meta-analyses (QUOROM)29 flow diagram depicts the number of studies identified, included, and excluded as well as the reasons for exclusion (Figure 1).

Study Characteristics Two studies were conducted in the United States,13,35 1 study was conducted in Europe,37 and another in Japan.1 The ACL reconstruction procedures were performed between 1984 and 2008. The mean number of ACLs biopsied per study is 31 (range, 21-43), with a total of 124 ACL biopsies. With regard to graft origin, none of the included studies reports on allografts. Two of the included studies consider bone–patellar tendon–bone (BPTB) grafts

exclusively,1,35 1 study considered hamstring grafts,37 and the remaining study performed biopsies on both BPTB and hamstring grafts.13 Surgical approaches and graft fixation techniques greatly differed between reports; Abe et al,1 for example, performed half of their surgeries by an open technique in the early 1990s, while Sańchez et al37 published about 2 decades later on an arthroscopic technique with the addition of a preparation rich in growth factors (PRGF) in half of their cases. The main study characteristics are summarized in Table 1.

Study Quality Assessment of the methodologic quality of these studies revealed that there were no randomized controlled trials (level of evidence 1). Moreover, no prospective studies

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TABLE 1 Primary Study Characteristicsa Rougraff et al (1993)35 Study quality Level of evidence Primary surgery: ACL reconstruction Graft origin Graft type

Abe et al (1993)1

Falconiero et al (1998)13

Sańchez et al (2010)37

4

4

4

3

Autograft BPTB

Autograft BPTB

Autograft Hamstrings

Surgical technique

Technique not mentioned

Graft fixation

Fixation over buttons

Open technique (n = 11) Arthroscopically assisted (n = 10) Not mentioned

Autograft Hamstrings (n = 8) BPTB (n = 35) Arthroscopically assisted

Surgeon Return to sports

Single surgeon Full competition from 3-6 mo

Not mentioned Full competition from 10-12 mo

Multiple surgeons Full competition from when ‘‘the operative leg was fully rehabilitated’’

Unrelated knee problems (n = 19) Volunteers (n = 4) 3 wk–6.5 y n = 23 Basket forceps ‘‘Middle segment either central or superficial’’ 1-3 mm 3 10-20 mm

Screw removal

‘‘New intra-articular pathology’’

6 wk–15 mo n = 21 Basket forceps Midzone ‘‘superficial layer of the core’’

3 mo–10 y n = 43 Not mentioned Midportion of the graft, ‘‘1 superficial, 1 deep’’

3-5 mm3

Not mentioned

Native ACLs at TKA (n =8) Native patellar tendons (n = 7) No Light microscopy Electron microscopy Hematoxylin/eosin

Native ACLs (n = 5)

Native hamstring tendons (n = 2)

Yes, blinded Light microscopy

Yes, blinded Light microscopy

Hematoxylin/eosin

Hematoxylin/eosin Alcian blue Vascularity Cellularity, nuclear shape, and orientation

Secondary surgery: Biopsy procedure Indication

Interval No. biopsied ACLs Biopsy technique Biopsy site

Biopsy specimen size Biopsy analysis No. native controls

Independent examiner Study method Specimen staining techniques Quantitative histology

Native ACL at autopsy (n = 1) Native patellar tendons (n = 11) No Light microscopy Hematoxylin/eosin Vascularity Cellularity, nuclear type Degeneration

Ligamentization stages

Periodicity of crimp Polarization 4

Not mentioned

Arthroscopically assisted Addition of PRGF (n = 22) Femur: transcondylar screw Tibia: bone plug and 2 metal staples Not mentioned Not mentioned

Unrelated knee problems (n = 17) Cyclops (n = 20) 6-25 mo n = 37 Basket forceps ‘‘Standardized with the device under medial femoral condyle’’ 3 mm 3 5-10 mm

Vascularity Cellularity, cellular shape, cellular arrangement Collagen orientation

Vascularity Cellularity

Crimp patterns

Fiber pattern

Presence of glycosaminoglycans Crimp

3

3

3

Metaplasia

a ACL, anterior cruciate ligament; BPTB, bone–patellar tendon–bone; PRGF, preparation rich in growth factors; TKA, total knee arthroplasty.


could be retrieved, probably because of the invasive nature of the biopsy procedure (level of evidence 2). One report was set up as a retrospective comparative study (level of evidence 3).37 The remaining 3 were case series (level of evidence 4).1,13,35 Only half of the included reports used a blinded, independent examiner to study the tissue samples.13,37

Biopsy Procedure Mostly, the ACL biopsies were performed during secondlook arthroscopy at the time of hardware removal or when addressing new unrelated knee injury such as meniscal tears or chondral lesions. Some subjects volunteered to undergo a second-look arthroscopy for the sole purpose of biopsy. In 1 paper,37 more than half of biopsy specimens were obtained during an arthroscopy performed for ACLrelated symptoms (cyclops lesion), thus possibly compromising the representability of the specimens in this series. Most authors used a standard 2.5-mm or 3-mm basket forceps to perform the biopsy. With this technique, a superficial specimen of the reconstructed ACL could be obtained. However, the spatial terminology of the biopsy zone differs among these authors: ‘‘. . .middle segment, either central or superficial,’’35 ‘‘. . .superficial layer of the core,’’1 or by positioning ‘‘the forceps directly under the femoral condyle’’ thus enabling the forceps ‘‘to reach the same area of the graft at any time in all patients.’’37 Although 3 of 4 included reports mention the use of a comparable biopsy device, the mean biopsy sample size between reports ranged from 3 to 5 cubic millimeters to 1 to 3 3 10 to 20 mm. Although some of the included papers mention concerns regarding the potential deleterious effects of the biopsy on graft integrity, such effects have not been witnessed during follow-up by any of these reports. However, none of the studies have provided direct evidence of biopsy site healing.

Biopsy Study Techniques In all 4 included studies, the procured samples were subjected to light microscopic (LM) evaluation.1,13,35,37 Sample preparation was similar in these reports with regard to fixation, dehydrating, and embedding of the specimens. All authors used standard hematoxylin and eosin staining. Sańchez et al37performed additional Alcian blue staining. Three studies describe the histologic changes during the ligamentization process by the use of a self-developed13,35 or adapted37 quantitative scoring system. This quantitative approach has the advantage of providing a more or less objective evaluation of the observed biologic phenomena, thus enabling statistical analysis and comparisons between grafts versus native ligament or tendon controls and between different graft ages, graft types, or surgical techniques. Abe et al,1 on the other hand, reported their histologic findings in a purely descriptive manner, but also reported on an electron microscopic (EM) evaluation of the ACL specimens, focused on collagen fibril diameter distribution.


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Biologic Graft Features Using the aforementioned study techniques, all authors described the changes occurring in the healing graft with regard to (1) vascularization, (2) cellular aspects, and (3) appearance of the extracellular matrix (ECM) in comparison with native tendon and/or ACL control biopsies. Vascularization. All studies found vascularity at least in the periphery of all grafts at any given time point, thus leading to the conclusion that the free tendon graft does survive in the intra-articular environment. The degree of neovascularization seems variable between subjects, although all authors witnessed a hypervascularity early after ACL reconstruction, which only slowly decreases to control ACL vascularization patterns during remodeling. It is suggested that the new blood vessels develop from the synovium, the infrapatellar fat pad, and the pseudoligamentum mucosum; however, no hard evidence to support this hypothesis could be found in the included studies. Cellular Aspects. There is a consensus among all 4 reports that cellular repopulation of the tendon graft does occur after ACL reconstruction, although the exact source of these fibroblast remains unclear. Parallel to the neovascularization, grafts showed increased cell counts during the early postoperative period. In this early phase, fibroblasts are disorganized, randomly arranged, and metabolically active as shown by their plump nuclei. As remodeling continues, the cells become longitudinally aligned and nuclei become less ovoid as in the control ACL specimens. No report mentions the presence of neural elements in the studied graft biopsy specimens. Appearance of ECM. Histologically, disorganized, irregular collagen bundles are witnessed in the early graft. As the graft matures, collagen bundles attain a densely packed, parallel alignment. No report has studied the specimens for collagen type. Abe et al1 showed by electron microscopic analysis that collagen fibrils in the transversely sectioned areas were uniformly small compared with those of normal tendon and ACL. In other words, the bimodality of the collagen fibril diameter distribution of normal tendon changes in unimodal, small fibril diameters over time. One article describes 2 types of dissimilar tissue in the biopsy specimens of more than half of the operated patients: the original, remodeling tendon is observed centrally in the biopsy specimen, enveloped by ‘‘newly formed connective tissue.’’ At first, these 2 areas are discerned by the cell shapes and extent of matrix remodeling, but become hardly distinguishable during maturation.

Ligamentization Stages and End Point The period from ACL surgery to second-look arthroscopy and histologic analysis ranged from 3 weeks to 120 months, with an average interval of 21.1 months. Although graft remodeling is a continuous biologic process, all authors have adapted different ligamentization stages with characteristic histologic or ultrastructural changes. Three of 4 reports mention 3 different stages of

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Animals 38

Scheffler et al. (2008)

Maturation

Early Remodeling

Humans Early

Remodeling Early

Maturation

0

3

1

Remodeling

6

9

13

Falconiero et al. (1998)

Maturation

Early

Remodeling

12

Rougraff et al. (1993)35 Abe et al. (1993)

Maturation

Remodeling Early

Quiescent

15

Sanchez et al. (2010)

Maturation

18

21

24

30

36

48

37

Months after ACLR

Figure 2. Differing ligamentization time frames in human grafts compared with a recent review of animal reports.38 ligamentization.1,13,37 Although distinct graft stage terminology is not always clearly or uniformly formulated, these stages can be termed ‘‘early,’’ ‘‘remodeling,’’ and ‘‘maturation’’ in chronologic order. One study35 added an extra ‘‘quiescent’’ stage. Figure 2 depicts the important differences 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 Scheffler et al38 are shown as well. The ligamentization end point is defined as the time point from which no further changes are witnessed in the remodeled grafts. Falconiero et al13 found no significant differences in the histologic aspect of their 12-month grafts compared with controls, concluding that ‘‘ligamentization occurs over a 12 month period with peak maturity evident at 1 year.’’ Rougraff et al,35 on the other hand, still observed areas of degeneration, neovascularity, and hypercellularity until 3 years after reconstruction. From that time on, these authors describe the grafts as ‘‘quiescent’’ and similar to ACL controls. Abe et al1 state that the ‘‘graft is still undergoing the process of remodeling at 1 year after surgery’’; however, these authors did not study grafts older than 15 months. According to Sańchez et al,37 the grafts reach maturity at around 2 years after surgery.

DISCUSSION Much of the current knowledge on the biologic phenomena occurring in the healing ACL graft have been derived from numerous animal studies. To date, the literature has been fueled by fairly large amounts of data from ACL reconstructed dogs, goats, sheep, rabbits, and monkeys. However, no ideal animal ACL model has been developed so far, as is reflected in this wide variety of studied animal species. Moreover, when comparing the large amounts of animal data with the scarce human biopsy studies, important differences have been revealed.38 For example, the timeline of biologic graft changes between animals and humans appears to be substantially different, with a much slower remodeling activity in human grafts

compared with animals (Figure 2). On the other hand, some in vivo animal reports found that the graft undergoes an early phase of vast necrosis occurring in the center of the graft,3,6 while this necrosis was barely seen in human biopsy specimens. The same important differences have been shown in nonbiopsy studies using gadoliniumenhanced MRI. Howell et al21 could not detect any revascularization except for the periligamentous tissue in human reconstructed ACLs, while Weiler et al,43 using the same technique, did find significantly upregulated neovascularization in ACL-reconstructed sheep. Finally, the complexity of the human ACL anatomy; the rapidly evolving, precise surgical techniques to replicate this anatomy; and the extreme importance of adequate postoperative rehabilitation are all impossible to control in animals. In summary, findings derived from ACL-reconstructed animals should not be transmitted directly to the human knee. When looking at the available human data, the literature is surprisingly limited. Only 4 articles could be withheld for systematic review, with only 1 article reaching a level of evidence higher than 4. An important finding of this systematic review is that a free tendon graft, when implanted in the human knee to replace a ruptured ACL, does survive in the intraarticular environment. At any given time point after reconstruction, the ACL is histologically viable with evidence of nourishing vascularization at least in the graft’s periphery and with no signs of important necrosis. The origin of the neovascularization is thought to be the Hoffa fat pad and the synovium, although no report provides hard evidence to prove this hypothesis. Furthermore, this systematic review shows that the general concept of ‘‘ligamentization’’ of a tendon graft as proposed in animal models is applicable to humans as well. The literature consistently describes a process in which the implanted grafts progressively lose ‘‘tendonspecific’’ biologic features, meanwhile exhibiting more and more ‘‘ligamentous’’ histologic properties. It is clear that this ligamentization process is a continuum of biologic changes rather than a series of distinct, time-dependent biologic events. However, dividing this process into


different stages is considered to be useful, especially with regard to postoperative rehabilitation protocols and timing of return to preinjury sporting activities. As shown in Figure 2, no consensus can be found in the current literature on these ligamentization time frames, with surprising differences regarding the time points between all authors. In theory, completion of the ligamentization process implies the generation of a structure that in every respect is undistinguishable from a native ACL. As no significant statistical differences in light microscopic features could be found between mature grafts and native ACLs, 3 of the included reports indeed concluded that the grafts had become similar to a native ACL,13,35,37 although the time needed by the remodeling graft to reach this status is a matter of debate. On the other hand, Abe et al1 showed that no graft reached ligamentous maturity on an ultrastructural level, as every graft specimen showed a typical depletion of large diameter collagen fibrils, thus leading to an unimodal collagen fibril diameter distribution in contrast to the bimodal distribution witnessed in normal ACLs. These findings were recently confirmed by Zaffagnini et al44,45 in 2 nonincluded reports based on biopsies of a limited number of reconstructed human ACLs up to 10 years after surgery, which is the longest biologic follow-up of human ACL reconstruction currently found in the literature. Briefly, they found that from 24 months after surgery, the graft tissue looked ‘‘very similar’’ to a normal ACL under light microscopy and that from that point on, no further changes were evident. However, persistent differences remained at the ultrastructural level with electron microscopy: mean collagen fibril diameter and bimodality of fibril distribution as in the normal ACL were not reached at any time. In summary, this systematic review has demonstrated that free tendon grafts implanted in the human knee to replace a ruptured ACL remain viable at any time point and undergo a process of ligamentization characterized by progressive biologic changes. Histologically, the mature grafts may resemble the normal human ACL, but ultrastructural differences regarding collagen fibril distribution do persist. Different stages of the ligamentization process are described by many authors, although no agreement exists on their time frame. The limits of this systematic review include the relatively poor level of evidence of the reports. Because ACL surgeries in these studies were performed from 1984 until 2008, major differences in surgical techniques and rehabilitation protocols are recognized between reports. Interstudy heterogeneity could be caused by the different origins of the tendon grafts used to replace the ruptured ACL. Until now, there is no evidence available to assume that BPTB and hamstring autografts will behave similarly during their ligamentization process in humans. Because of the invasive nature of the biopsy procedure and its potential deleterious effect on an otherwise healthy graft, almost all reported biopsies have been taken from the superficial region of the midzone of the reconstructed ACL. The question remains whether this biopsy sample can be representative for the entire 3-dimensional graft structure. Only a few human cases of in toto retrieved grafts


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can be found in the literature. Delay et al11 reported on the retrieval of an entire knee joint at autopsy, 18 months after BPTB ACL reconstruction. Most interestingly, they found ‘‘in contrast to the ‘superficial biopsy’ studies included in this systematic review’’ that the ACL specimen did show vast areas of deep necrosis, most evident where the graft exited the tibial tunnel. The authors state that ‘‘it was unclear whether this area of necrosis represented a portion of the autograft that had never been revascularized or whether it had undergone degeneration and became acellular over time.’’ Clearly, a better understanding of the graft biology in human ACL reconstruction will depend on the possibility to obtain core biopsy samples of these grafts. Accordingly, probably the key finding of this systematic review is that although ACL reconstruction is performed as a routine surgical procedure all around the world, the underlying ligamentization process is still poorly understood in the human knee. Further human studies are needed to understand normal graft healing after ACL reconstruction.

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