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The ''Ligamentization'' Process in Anterior Cruciate Ligament Reconstruction: What Happens to the
Human Graft? A Systematic Review of the Literature
Steven Claes, Peter Verdonk, Ramses Forsyth and Johan Bellemans
Am J Sports Med 2011 39: 2476 originally published online April 22, 2011
DOI: 10.1177/0363546511402662

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 Basic Science Review

The ‘‘Ligamentization’’ Process in M

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 vas-
cularization, 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 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 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

*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:
steven.claes@uzleuven.be).
y ACL reconstruction one of most common surgical proce-
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. 39, No. 11
DOI: 10.1177/0363546511402662
Ó 2011 The Author(s)
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
dures 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 Nonaug-
mented primary ACL repair (ie, just suturing the torn
ends of the ligament) has also been proven to be unsuc-
cessful, thus making ACL reconstruction using tendon

2476
Vol. 39, No. 11, 2011
grafts the current standard of care for the ACL-deficient
knee.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 the
arrangement of matrix macromolecules, however, clearly
differ to provide the specific mechanical properties required
by each structure to function effectively. For example, com-
pared 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 colla-
gen cross-links, and a different distribution of collagen fibril
diameters.14,36 Interestingly, some authors even demon-
strated significant differences between different ligaments
(eg, ACL versus medial collateral ligament),19 while others
were able to discern different tendons on their ultrastruc-
tural 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 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 into
the rabbit knee to replace an excised ACL underwent a pro-
cess of ‘‘ligamentization.’’ They described this phenomenon
as the continuous development of a tissue that was origi-
nally 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 mon-
keys10 have been undertaken to examine the fate of both
autografts and allografts used to replace the ACL. How-
ever, 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 reconstruc-
tion and maintaining long-term joint health, as these fac-
tors 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 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 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
Ligamentization Process in ACL Reconstruction 2477
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 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 syn-
thetic 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 rele-
vant 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 uti-
lization 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 ligamentiza-
tion 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 biol-
ogy 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 with-
out control biopsies of either native tendon or ligament
were 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, 4
studies were determined to be appropriate for systematic
2478 Claes et al The American Journal of Sports Medicine

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

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 biop-
sied 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 tech-
nique in the early 1990s, while Sánchez 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 characteris-
tics 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
could be retrieved, probably because of the invasive nature
Vol. 39, No. 11, 2011 Ligamentization Process in ACL Reconstruction 2479

TABLE 1
Primary Study Characteristicsa

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

Study quality
Level of evidence 4 4 4 3
Primary surgery:
ACL reconstruction
Graft origin Autograft Autograft Autograft Autograft
Graft type BPTB BPTB Hamstrings (n = 8) Hamstrings
BPTB (n = 35)
Surgical technique Technique not Open technique Arthroscopically Arthroscopically
mentioned (n = 11) assisted assisted
Arthroscopically assisted Addition of PRGF
(n = 10) (n = 22)
Graft fixation Fixation over buttons Not mentioned Not mentioned Femur: transcondylar
screw
Tibia: bone plug and
2 metal staples
Surgeon Single surgeon Not mentioned Multiple surgeons Not mentioned
Return to sports Full competition Full competition Full competition from Not mentioned
from 3-6 mo from 10-12 mo when ‘‘the operative
leg was fully
rehabilitated’’
Secondary surgery:
Biopsy procedure
Indication Unrelated knee Screw removal ‘‘New intra-articular Unrelated knee
problems pathology’’ problems (n = 17)
(n = 19)
Volunteers (n = 4) Cyclops (n = 20)
Interval 3 wk–6.5 y 6 wk–15 mo 3 mo–10 y 6-25 mo
No. biopsied ACLs n = 23 n = 21 n = 43 n = 37
Biopsy technique Basket forceps Basket forceps Not mentioned Basket forceps
Biopsy site ‘‘Middle segment Midzone ‘‘superficial Midportion of the graft, ‘‘Standardized with the
either central layer of the core’’ ‘‘1 superficial, 1 deep’’ device under medial
or superficial’’ femoral condyle’’
Biopsy specimen size 1-3 mm 3 10-20 mm 3-5 mm3 Not mentioned 3 mm 3 5-10 mm
Biopsy analysis
No. native controls Native ACL at autopsy Native ACLs at Native ACLs (n = 5) Native hamstring
(n = 1) TKA (n =8) tendons (n = 2)
Native patellar Native patellar
tendons (n = 11) tendons (n = 7)
Independent examiner No No Yes, blinded Yes, blinded
Study method Light microscopy Light microscopy Light microscopy Light microscopy
Electron microscopy
Specimen staining Hematoxylin/eosin Hematoxylin/eosin Hematoxylin/eosin Hematoxylin/eosin
techniques Alcian blue
Quantitative histology Vascularity Vascularity Vascularity Vascularity
Cellularity, nuclear Cellularity, cellular Cellularity Cellularity, nuclear
type shape, cellular shape, and orientation
arrangement
Degeneration Collagen orientation Metaplasia Presence of
glycosaminoglycans
Periodicity of crimp Crimp patterns Fiber pattern Crimp
Polarization
Ligamentization stages 4 3 3 3

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

of the biopsy procedure (level of evidence 2). One report evidence 4).1,13,35 Only half of the included reports used
was set up as a retrospective comparative study (level of a blinded, independent examiner to study the tissue
evidence 3).37 The remaining 3 were case series (level of samples.13,37
2480 Claes et al
Biopsy Procedure
Mostly, the ACL biopsies were performed during second-
look arthroscopy at the time of hardware removal or
when addressing new unrelated knee injury such as menis-
cal 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 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 basket
forceps 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 differs
among these authors: ‘‘. . .middle segment, either central or
superficial,’’35 ‘‘. . .superficial layer of the core,’’1 or by posi-
tioning ‘‘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 con-
cerns regarding the potential deleterious effects of the
biopsy 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 biopsy
site healing.
Biopsy Study Techniques
In all 4 included studies, the procured samples were sub-
jected to light microscopic (LM) evaluation.1,13,35,37 Sample
preparation was similar in these reports with regard to fix-
ation, dehydrating, and embedding of the specimens. All
authors used standard hematoxylin and eosin staining.
Sánchez 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 quantita-
tive approach has the advantage of providing a more or
less objective evaluation of the observed biologic phenom-
ena, 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.
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 compari-
son with native tendon and/or ACL control biopsies.
The American Journal of Sports Medicine
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 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 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 met-
abolically active as shown by their plump nuclei. As remod-
eling continues, the cells become longitudinally aligned
and nuclei become less ovoid as in the control ACL speci-
mens. No report mentions the presence of neural elements
in the studied graft biopsy specimens.
Appearance of ECM. Histologically, disorganized, irreg-
ular 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 trans-
versely 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 diame-
ters 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 cen-
trally 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 pro-
cess, all authors have adapted different ligamentization
stages with characteristic histologic or ultrastructural
changes. Three of 4 reports mention 3 different stages of
ligamentization.1,13,37 Although distinct graft stage ter-
minology 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
Vol. 39, No. 11, 2011
Animals
Early Remodeling
Humans
Early Remodeling
Early Remodeling Maturation
Early Remodeling
Early
0 3 6 9 12
Figure 2. Differing ligamentization time frames in human grafts compared with a recent review of animal reports.38
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 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 hyper-
cellularity 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 Sánchez 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 recon-
structed 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 dif-
ferences 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 necro-
sis 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 gadolinium- enhanced MRI. Howell et al21 could not
detect any revascularization except for the periligamentous
Ligamentization Process in ACL Reconstruction 2481
38
Maturation Scheffler et al. (2008)
Maturation Quiescent Rougraff et al. (1993)35
1
Abe et al. (1993)
13
Maturation Falconiero et al. (1998)
37
Remodeling Maturation Sanchez et al. (2010)
15 18 21 24 30 36 48 Months after ACLR
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 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 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 intra-
articular environment. At any given time point after recon-
struction, 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 ‘‘tendon-
specific’’ 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 differ-
ent 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 differ-
ences regarding the time points between all authors.
In theory, completion of the ligamentization process
implies the generation of a structure that in every respect
2482 Claes et al
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 mat-
ter 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 unimo-
dal collagen fibril diameter distribution in contrast to the
bimodal distribution witnessed in normal ACLs. These find-
ings 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 recon-
struction 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 micros-
copy and that from that point on, no further changes were
evident. However, persistent differences remained at the
ultrastructural level with electron microscopy: mean colla-
gen 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 ultra-
structural 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 rela-
tively 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 rehabil-
itation protocols are recognized between reports. Inter-
study 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
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
The American Journal of Sports Medicine
whether it had undergone degeneration and became acellu-
lar 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 under-
stood in the human knee. Further human studies are
needed to understand normal graft healing after ACL
reconstruction.
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