Asia-Pacific Journal of Sports Medicine, Arthroscopy, Rehabilitation and Technology 25 (2021) 8e15

Contents lists available at ScienceDirect

Asia-Pacific Journal of Sports Medicine, Arthroscopy,

Rehabilitation and Technology
journal homepage: www.ap-smart.com

Review Article

Graft healing after anterior cruciate ligament reconstruction (ACLR)

Shiyi Yao, Bruma Sai-Chuen Fu, Patrick Shu-Hang Yung*
Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Hong Kong

a r t i c l e i n f o a b s t r a c t

Article history: Anterior cruciate ligament reconstruction (ACLR) is a commonly performed procedure in Orthopaedic
Received 9 December 2020 sports medicine. With advances in surgical techniques providing better positioning and fixation of the
Received in revised form graft, subsequent graft failure to certain extent should be accounted by poor graft healing. Although
5 February 2021
different biological modulations for enhancement of graft healing have been tried in different clinical and
Accepted 21 March 2021
animal studies, complete graft incorporation into bone tunnels and the “ligamentization” of the intra-
articular part have not been fully achieved yet. Based on the understanding of graft healing process
and its failure mechanism, the purpose of this review is to combine both the known basic science &
Keywords:
Anterior cruciate ligament reconstruction
clinical evidence, to provide a much clearer picture of the obstacle encountered in graft healing, so as to
(ACLR) facilitate researchers on subsequent work on the enhancement of ACL graft healing.
Graft healing process. biological modulation © 2021 Asia Pacific Knee, Arthroscopy and Sports Medicine Society. Published by Elsevier (Singapore) Pte
Graft failure Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/
by-nc-nd/4.0/).

Introduction phase with the most intensive cell infiltration, and finally a matu-
ration phase with slow matrix remodeling.5,6 For ACLR using free
There is an estimated incidence of 200,000 anterior cruciate tendon grafts, complete tunnel closure and ossification of graft
ligament (ACL)injuries per year in the United States,1 with inside bone tunnels have never been truly observed, as only certain
approximately 60,000 to 150,000 annually requiring ACL recon- graft incorporation into tunnel wall is found as Sharpey's fibers or
struction (ACLR). However, the rate of graft failure for this via fibrocartilage zone only.7,8 Also, the biological changes in the
commonly performed surgical procedure was still considerably intra-articular region of the graft, which is described as “ligamen-
high with reports up to 13.3%,2 with evidence suggesting that tization”,4 could not be fully achieved as well. Because of that, over
despite advances in surgical techniques and optimizing rehabili- the years, different biological modulations have been advocated by
tation protocols, unfavorable healing of graft may probably one of different researchers, in order to improve the graft healing and thus
the major contributing factors. the final clinical outcome.9 A successful ACLR with a tendon graft
Previous studies have demonstrated different ways of clinical requires solid healing of the tendon graft in the bone tunnel and
and biological assessment of the healing process of ACL graft at fully “ligamentization” in the intra-articular region of the graft as
different stages. Clinically, different imaging techniques, arthros- soon as possible after surgery. Enhancing the healing of the tendon
copy and biopsy have been used to detect the changes of the tendon graft is crucial to facilitate an early and aggressive rehabilitation
graft after ACLR. Meanwhile, a myriad of histological and and a rapid return to full activity.10
biochemical pre-clinical studies have demonstrated the different Based on systematic review on all the previous per-clinical &
molecular and cellular response of the tendon graft after ACL clinical evidence on the study of ACL graft healing, we are pro-
reconstruction. posing a clearer picture of the whole process of ACL graft healing
Graft healing in ACL reconstruction has been conventionally after reconstruction, which is indeed a multitude of molecular and
categorized as a 3-stage process, namely early healing, prolifera- cellular events, taking place at different region of the graft, at
tion, and maturation phase.3,4 The early healing phase is charac- different time points, leading to sequential changes in the original
terized by graft necrosis and hypocellularity without any significant tendon, to become the new ACL. It is thus important to understand
detectable revascularization occurs, followed by the proliferation the regulation of all these events, and their clinical relevance during
the rehabilitation, to facilitate future research directions and
* Corresponding author.
established the right targets, on improvement of the graft healing,
E-mail addresses: [email protected] (S. Yao), [email protected]. and thus the final clinical outcome.
hk (P.S.-H. Yung).

https://doi.org/10.1016/j.asmart.2021.03.003
2214-6873/© 2021 Asia Pacific Knee, Arthroscopy and Sports Medicine Society. Published by Elsevier (Singapore) Pte Ltd. This is an open access article under the CC BY-NC-
ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Yao, B.S.-C. Fu and P.S.-H. Yung Asia-Pacific Journal of Sports Medicine, Arthroscopy, Rehabilitation and Technology 25 (2021) 8e15

Graft failure as a result of problematic graft healing healing process can still be concluded based on this MRI evidence
(Fig. 1), and objective quantitative MRI biomarkers of graft healing
The true incidence of ACL graft failure after implantation is would be desirable for further studies.
unknown at present although as high as 24.4% has been reported.11
Most studies have reported graft failure rates in the range of 0.7%e Computerized tomography (CT)
14%.12e14 Several recent systematic reviews by Spindler et al.,15
Lewis et al.16 and Wright et al.,17 have reported failure rates of CT has been recommended to evaluate bone-tunnel changes
3.6%, 4%, and 5.8%, respectively. A study by the University of Pitts- during the ACL graft healing since plain radiograph is often difficult
burgh18 showed that after single-bundle ACL reconstruction, the to reliably identify the tunnel and measure the width of the tun-
most common rupture pattern seen at the time of revision surgery nel,32,33 Suzuki et al.34 evaluated the bone plug was almost
is proximal rupture, followed by mid-substance rupture. They completely integrated into the rectangular femoral tunnel by 8
also19 classified the mechanisms of ACL graft failure as related to (a) weeks after anatomical ACL reconstruction using a bone-patellar
surgical technique; (b) graft incorporation; and (c) trauma. Also, a tendon-bone (BTB) graft by CT scans. Christian Fink et al.35 used
Multicenter ACL Revision Study (MARS) Group developed a multi- CT sequentially to monitor the time course of changes over 2 years.
surgeon, multicenter prospective longitudinal study, and the The percentage of change in tunnel size (Fig. 3) was significantly
MARS cohort (460 patients) showed the etiology of failure, as higher within the first 6 weeks following surgery compared with all
deemed by the revising surgeon, including traumatic, technical, other time intervals and the tunnel size was almost stable after 1
biologic, etc.20 year. For autologous hamstring tendons, at a mean follow-up of 10
Since advances have been made in surgical techniques21 and months, the CT scan showed a 3% femoral tunnel diameter increase,
rehabilitation methods,22 when graft failure happens following ACL and sclerotic tunnel boundary can be revealed.36 CT imaging has
reconstruction without traumatic events, problematic graft healing also been used to compare the extent of widening using different
should be considered. So, a better understanding of the biological tunnel placement methods as well as different fixation
healing process is needed. methods.37,38 Besides, low bone mineral density (BMD) may in-
crease the risk of incident knee osteoarthritis after ACLR, which
Clinical evidence of graft healing cannot be detected by the conventional CT scan. Peripheral quan-
titative computed tomography (pQCT) captures not only the bone
Magnetic resonance imaging (MRI) mineral content but also volumetric trabecular and cortical bone
microstructure which is directly related to bone strength.39 We can
Clinically, MRI is the most commonly used imaging technique see that conventional CT and pQCT detect the properties of bony
for monitoring the healing process after ACL reconstruction.23 changes after ACLR, while researchers found that dual-energy
Howell et al.24 conducted the first prospective study to serially computed tomography (DECT)40 has the potential to evaluate soft
observe the MR appearance of ACL autografts during the first year tissue changes by generating gemstone spectral imaging (GSI) im-
of implantation. A four-level grading system (Fig. 2) based on the ages and creating material-specific color mapping and dual-energy
MR signal of the graft was developed and it was reported that in- bone removal. So, with the development of the CT technique, we
creases in magnetic resonance graft signal were time-dependent, may be able to evaluate the bony and soft tissue changes simulta-
becoming well established by 3 months and remaining un- neously with high accuracy.
changed at 1 year. The increased MR signal has been thought to be
related to an increase in water concentration representing graft Second-look knee arthroscopy & biopsy
edema.25 Later, the Howell team also designed a study to assess the
degree of revascularization after administration of Gd-DPTA Since the healing status provided by the non-invasive methods
contrast agent with T1-weighed MRI.26 The unimpinged ACL graft such as MRI and CT scans is still limited, second-look knee
acquired no discernible blood supply during the 2 years of im- arthroscopy after ACL reconstruction is one of the most reliable
plantation and the periligamentous soft tissues were richly vascu- types of examination to provide valuable information on ACL grafts
larized and covered the graft by 1 month. However, Biercevicz such as synovialization and vascularization.41 Nakamae et al.42
et al.27 found that the use of signal intensity (SI) as an outcome demonstrated significantly better synovial coverage of the graft
measure was limited by its dependence on image acquisition pa- 18 months after ACL reconstruction using second-look arthroscopy
rameters and scanner manufacturer. Later, Li et al.28 evaluated the (Fig. 4). Synovialization plays an important role in graft healing and
MRI signal/noise quotient (SNQ) of ACL grafts at 3, 6, and 12 months is considered to positively affect the survival of the graft. Studies
after ACLR (a high graft SI represents high SNQ value, which in- reported that hamstring autografts showed considerably better
dicates inferior graft maturity) and demonstrated that the graft synovial coverage than soft tissue allograft based on second-look
SNQ value has a significant negative correlation with postoperative arthroscopic evaluation.43,44 Furthermore, arthroscopy has been a
time from 12 to 114 months postoperatively. Recently, with tool to get biopsy samples for examinations to study the healing
increasing interest in biological treatments to enhance ACL graft process.45e47 Histology through biopsy specimens procedure dur-
healing, there is a clinical need for improved quantitative MRI ing second-look arthroscopy48 has been examined to investigate
measures to follow up the healing process. MRI ultra-short echo the fate of ACL allografts on a long-term basis. In the 6-month, the
time T2* (UTE-T2*) is sensitive to collagen matrix integrity and surface blood flow was significantly higher than that for the control
organization.29,30 Chu et al.31 showed that quantitative MRI ultra- ACLs and declined with time from 6 months post-surgery onward,
short echo time T2* (UTE-T2*) and T2* mapping suggested sub- reach a plateau by 12 months, and maintained a level equivalent to
stantial changes within the graft during the first 6 months post- that of the normal ACLs. In 24e30, 36e45, 48-89-month grafts, the
surgery and relatively stable graft composition from 6 months to blood flow values were also statistically insignificant compared
1 year, consistent with remodeling, followed by decreases from 1 to with those for the normal control ACLs. Histologically, the speci-
2 years, suggestive of continuing maturation. As above-mentioned, mens at 24e89 months closely resembled those at 18 months,
MRI results differed greatly across the studies due to the wide suggesting that the allografts had reached stability by 18 months
heterogeneity of the acquisition and interpretation methods, which post-surgery and remained viable thereafter. Besides from syno-
will impede the comparison of SI. However, the time frames of the vialization and vascularization, innervation after ACLR also raises
9
S. Yao, B.S.-C. Fu and P.S.-H. Yung Asia-Pacific Journal of Sports Medicine, Arthroscopy, Rehabilitation and Technology 25 (2021) 8e15

Fig. 1. Graft healing time frames in human grafts demonstrated by MRI.

Fig. 2. A, at 1 week post implantation the entire graft had a normal MR signal (Grade I). B, by 3 months, the graft exiting from the femoral tunnel in the proximal intraarticular zone
(1) has remained unchanged (Grade I); the middle intraarticular zone (2) had acquired an increased signal involving approximately 50% of the width of the graft (Grade II); the distal
intraarticular zone (3) had only a few strands of normal-appearing ligament with more than 50% of the ligament having an increased signal (Grade III). The portion of the graft
within the tibial tunnel (4) was normal in appearance (Grade I). C, the increase in the MR signal of the graft persisted at 1 year with no evidence of returning to normal.24

Fig. 3. Tunnel enlargement in the sagittal plane. L1, tibia plateau; L4, proximal end of the bone block; L2, 33% of L1 to L4; L3, 66% of L1 to L4; L5, P33% ofL1 to L4.35

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S. Yao, B.S.-C. Fu and P.S.-H. Yung Asia-Pacific Journal of Sports Medicine, Arthroscopy, Rehabilitation and Technology 25 (2021) 8e15

Fig. 4. Second-look arthroscopic view of the reconstructed anterior cruciate ligament. The synovial coverage of the grafts was classified as follows: (a) good (synovial coverage
of>80% of the graft), (b) fair (50e80% coverage), and (c) poor (<50% coverage).41

interests from researchers. However, biopsy after ACLR using part in the early healing phase, which may lead to a poor healing
Achilles tendon showed that neural tissue analogs could only be outcome if the subsequent healing process is not optimized.
found on the H-E stains in the control group and in the Achilles It is important to actually know about the biochemical & cellular
tendon allograft group, mechanoreceptors were not observed.49 response in this early healing phase after ACL reconstruction, as a
Although the biopsy can demonstrate the vascular, nerval, and lot of surgeons are in favor of providing NSAID or COX-2 Inhibitors
other cellular level changes during the healing process, it is an immediately after ACL reconstruction, to minimize the symptoms
invasive technique and cannot do a follow-up of the healing process (pain & swelling) after the operation. A recent systematic review
frequently. indeed demonstrated that the selective COX-2 inhibitors could
Despite different imaging techniques such as MRI & CT scan, as negatively affect the healing process, though some other studies
well as arthroscopic assessment have been widely employed for demonstrated no negative effect.56 That's why cautions is needed
assessment of ACL graft healing, the most ultimate solid evidence of for surgeons when administering NSAIDs/COX-2 inhibitors after
proper graft healing still relies on histological & biochemical ACL reconstruction, probably good to keep the duration and dosage
assessment. Over the years, there have been a lot of scientific work of NSAIDs as short and low as possible to while avoiding unpleasant
on studying the different cellular & biochemical response of the effect on the graft.57
graft healing, which took place at different parts of the graft, at
different time point after the ACL reconstruction.
The proliferation phase

Histological and biochemical characterization of graft healing Since the ACL graft undergoes necrosis following implantation,
adequate revascularization is critical for successful graft healing by
Some papers define the graft healing process as the combination allowing cellular repopulation and subsequent matrix remodeling.
of several biological events, including inflammatory response, graft A deficient revascularization process will result in a lack of available
necrosis, revascularization, cell repopulation, osseous integration, oxygen for cells, thus impeding the cell repopulation.50 Researchers
collagen remodeling, and ligamentization.50 These biological have emphasized the importance of the blood supply and revas-
events can be categorized into three different healing phases, cularization of the autograft in the maintenance of graft
namely early healing, proliferation, and maturation phase. viability.58,59 Vascular ingrowth forms as early as 3 weeks and in-
filtrates even the central portion of the autograft. It is suggested
The early healing phase that new blood vessels develop from the synovium, the infrapa-
tellar fat pad, and the pseudo-ligamentum mucosum.60 And
The early cellular response following surgical implantation of a vascular endothelial growth factor (VEGF) expression is accompa-
tendon graft involves the accumulation of host inflammatory cells. nied by the level of vascular density. The observed VEGF production
Shortly after graft implantation, neutrophils and ED1þ macro- in vivo might be induced by the previous Inflammatory reactions in
phages51 are recruited to the periphery of the implanted graft and tendon grafts.61
various cytokines like interleukin-6(IL-6), tumor necrosis factor- The replacement cells are from a source other than the auto-
alpha (TNF-a), transforming growth factor-beta (TGF-b) are graft. From drilling maneuver, bone marrow stem cells are released
released.52 It has also been shown that the level of matrix- in the bone tunnels for osteoblasts and there is no question that
metalloproteinase-1, 13 (MMP-1, 13) released by cells increased some of these cells end up in the intraarticular space and could
after ACL reconstruction,53 which will digest the collagen and help contribute to graft cellularity. Seeding fibroblasts from the residual
the repopulated cells with infiltration. stumps of the ACL can survive the synovial fluid and produce the
At the same time, researchers agree that the tendon graft un- extra-cellular biochemical products of the ACL. Meanwhile, pleuri-
dergoes avascular necrosis mainly in its central portion.5 As part of potential mesenchymal cells from the articular cartilage could
this necrotic process, several cytokines are released and initiate the potentially express fibroblastic properties which are well suited to
cascade of growth factors that guide the different subsequent survive in synovial fluid. The fibroblast-like cells (Type B cells)
steps.51,54 Different from the inside tunnel part, the intra-articular originating from the synovial membrane are adapted for survival in
substance is exposed to synovial fluid, which contains a lot of cat- synovial fluid and are present within the joint throughout the
alytic enzymes, cytokines, and growth factor inhibitors that inter- postoperative period. This suggests that these fibroblasts are the
fere with the healing mechanisms.55 Such differences may result in most likely candidates for the source of replacement cells that seed
extended necrosis, collagen disturbance (disintegration, fragmen- the autograft.58 These cells are initially seen at the periphery of the
tation, disorganization), myxoid degeneration in the intra-articular graft, then migrated to more loosely woven areas of the matrix
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S. Yao, B.S.-C. Fu and P.S.-H. Yung Asia-Pacific Journal of Sports Medicine, Arthroscopy, Rehabilitation and Technology 25 (2021) 8e15

where they proliferated and finally repopulated the vacant con- different host and graft responses in the two different sites of the
nective tissue matrix.5 The cell repopulation correlates with the graft (intra-articular part and bone tunnels) has been proposed
presence of PDGF-AA, PDGF-BB, and TGF-b1 in the reconstructed (Fig. 5):
graft. At the early healing phase, inflammation of the host and cell
As shown from the evidence, the blood vessels and cells during necrosis of the graft happens immediately as a response to grafting
this phase are coming from various tissues in the knee joint, such as after ACLR. In the next phase of proliferation, depending on the
the synovium, the infrapatellar fat pad, and the pseudo- peri-graft environment, different cell types are recruited inside the
ligamentum mucosum, indicating that it is indeed important to bone (osteoprogenitors) and in the intra-articular space (fibro-
preserve these tissues during operation and over-debridement blasts). These cells repopulate the necrotic graft tissues with neo-
should be prevented. There is also evidence showing that supe- vascularization to gain nutrients supplies. The subsequent
rior postoperative knee stability and clinical outcomes were maturation phase represents matrix remodeling processes medi-
observed for remnant-preserving ACLR, with the native ACL stump ated by these repopulated cells, under mechanical and biochemical
preserved during the operation, when compared with standard influences that exhibit regional variations along with the graft.
ACLR.62 Bone tendon junction healing inside bone tunnels and liga-
mentization in the intra-articular part are reactive matrix remod-
The maturation phase eling processes influenced by regional responses to grafting. In
contrast to the conventional concepts of graft healing in ACLR, we
The changes at the wall of the bone tunnel are similar to the highlight the regional variations in the peri-graft environment that
process of endochondral ossification, with the environment of the influence the matrix remodeling. Biological modulation should
bone tunnel similar to that of a fracture. Bone morphogenetic target specifically either advantageous cell repopulation or the
proteins (BMP-2, BMP-7) are specifically involved in bone remod- favorable regional peri-graft environment to achieve matrix
eling leading to osseous integration.63 Bone ingrowth plays an remodeling to regain the original function of ACL.
important role in graft-to-bone healing because this stage of
healing coincides with improved load-to-failures. Several studies Biological enhancement of graft healing: are we there yet?
have investigated strategies to improve bone ingrowth into a
tendon graft. Osteoinductive factors (BMP-2, BMP-7),64e66 osteo- Although tremendous biological modulations have been used to
conductive agents such as calcium-phosphate cement,67 and oste- improve the above-mentioned healing process, good ACL graft
oclast inhibition68 have been studied as potential strategies to healing is still far from ideal:
improve bone formation around a tendon graft.
Basic fibroblast growth factor (bFGF) is expressed from the
margins of the tendon that signals the migration of spindle-shaped Site of biological enhancement: graft tunnel interface vs. intra-
fibroblasts from the bone tunnel into the graft that then produce articular mid substance
type III collagen. Then, total collagen content and the non-
reducible/reducible crosslink ratio increase during this process. Biological modulations like mesenchymal stem cells, growth
The collagen fibrils in the reconstructed ligament are differently factors, biomaterials, or biophysical intervention have been applied
organized than those of the native ACL, having a unimodal, small to improve the healing outcome and these biological strategies
diameter collagen-fibril diameter profile and the remodeling pro- have long been reviewed.71,72 From different reviews, we can
cess never results in exact reproduction of the original ligament conclude that majority targeted graft incorporation inside bone
organization.47 What should be mentioned here is that, Andreas tunnels or tendon-bone interface healing. However, it was reported
Weiler et al. showed that the histologic data indicated that that the graft ruptured most frequently at the femoral insertion73
anatomic interference bio-screw would lead to the development of and was followed by intra-articular mid-substance.18 Animal
a direct type of ligament insertion.69 Thus, the tendon-to-bone studies have shown the same result.74,75 So, more attention may
incorporation process may be improved by the use of bio-screw need to be paid to try to modulate the biological events which may
fixation method. improve the healing results of the intra-articular mid-substance of
During the above-mentioned whole healing process, Smith the graft.
et al.70 conducted anterior laxity test to see the increase in knee
laxity between the day of surgery and each monthly follow-up Targeted outcomes of biological enhancement
interval in the first year using tibialis allograft in ACLR. The result
showed the maximum increase in anterior laxity was at 6 months, As one of the most important outcomes of the ACL reconstruc-
which is correlated with the timeline of the healing process when tion, the mechanical property is always measured to compare the
the graft is between the early healing phase and the beginning of results after operation. However, unfortunately, the strength of the
the proliferation phase.60 And there is no increase in the knee laxity graft simulating native ACL has never been achieved. McFarland
in the late proliferation phase and the following maturation phase. et al.76 developed a dog ACL reconstruction model and by 16 weeks,
the grafts remained only 40% as strong as controls. Another study77
ACL graft healing is characterized by matrix remodeling examined the biomechanics of goats for as long as 3 years after
influenced by regional responses at different phases after surgery and the strength and stiffness of the grafts were 44 and 49%
graft implantation those of the control ligaments, respectively. Rhesus monkeys78
were also studied, the tendon had approximately 80% of the ten-
As above-mentioned, although a pyramid of biological modu- sile strength that they had before transfer. And for small animals
lations has been tried, the healing outcome has never been perfect. like rats and rabbits, they could achieve around 20% strength when
So, a better understanding & differentiation of the graft healing compared with the native ACL.74,79 We have to be cautious that
process, in terms of the time and location of the responses, may be indeed the initial ultimate “Strength to failure” of various graft is
helpful for all the researchers in this area. Here, a clearer picture of much higher than that of the native ACL before implantation, which
graft healing, categorized into three different phases (early healing again suggests that the weakest link is at the bone tendon junction,
phase, proliferation phase, and maturation phase), followed by which justify the efforts on researching better ACL graft healing.80
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S. Yao, B.S.-C. Fu and P.S.-H. Yung Asia-Pacific Journal of Sports Medicine, Arthroscopy, Rehabilitation and Technology 25 (2021) 8e15

Fig. 5. ACL Graft healing is characterized by matrix remodeling influenced by regional responses at different phases after graft implantation.

Delivery of biological enhancement Declaration of competing interest

Since most of the existing modulations were delivered at the None.

early healing phase but were proposed to act on the proliferation
phase or maturation phase, sustained delivery of modulations was Acknowledgements
required but few studies have demonstrated whether they act on
the desired phases. Taking the cell supplementation as examples, SYY drafted the manuscript, BSF provided writing assistance and
the proliferation phase is identified by cell infiltration and repo- PSY revised and proof read the article.
pulation in the graft. To enhance the cell repopulation, mesen-
chymal stem cells (MSC),81 adipose-derived stem cells (ADSC),82
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S. Yao, B.S.-C. Fu and P.S.-H. Yung Asia-Pacific Journal of Sports Medicine, Arthroscopy, Rehabilitation and Technology 25 (2021) 8e15

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