Morphological Assessment of MACI Grafts in Patients with Revision Surgery and Total Joint Arthroplasty

Abstract

Objective
To compare the histological and immunohistochemical characteristics of matrix-assisted chondrocyte implantation (MACI) grafts between patients with revision surgery and patients with total joint arthroplasty.

Methods
Biopsies of MACI grafts from patients with revision and total joint arthroplasty. The graft tissue characteristics and subchondral bone were examined by qualitative histology, ICRS (International Cartilage Repair Society) II scoring and semiquantitative immunohistochemistry using antibodies specific to type I and type II collagen.

Results
A total of 31 biopsies were available, 10 undergoing total knee arthroplasty (TKA) and 21 patients undergoing revision surgery. Patients in the clinically failed group were significantly older (46.3 years) than patients in the revision group (36.6 years) ( P = 0.007). Histologically, the predominant tissue in both groups was of fibrocartilaginous nature, although a higher percentage of specimens in the revision group contained a hyaline-like repair tissue. The percentages of type I collagen (52.9% and 61.0%) and type II collagen (66.3% and 42.2%) were not significantly different between clinically failed and revised MACI, respectively. The talar dome contained the best and patella the worst repair tissue. Subchondral bone pathology was present in all clinically failed patients and consisted of bone marrow lesions, including edema, necrosis and fibrosis, intralesional osteophyte formation, subchondral bone plate elevation, intralesional osteophyte formation, subchondral bone cyst formation, or combinations thereof.

Conclusions
MACI grafts in patients with revision and total joint arthroplasty were predominantly fibrocartilage in repair type, did not differ in composition and were histologically dissimilar to healthy cartilage. Clinically failed cases showed evidence of osteochondral unit failure, rather than merely cartilage repair tissue failure. The role of the subchondral bone in relation to pain and failure and the pathogenesis warrants further investigation.

Full text

https://doi.org/10.1177/1947603519890754
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Article
Introduction
Articular cartilage of diarthrodial joints is supported by the
subchondral bone. Between the hyaline articular cartilage
and the subchondral bone, are several structures connecting
these two different tissues to form a tight functional associa-
tion. Together these structures have been commonly referred
to as the osteochondral unit.1 Isolated articular cartilage
defects without subchondral bone changes are common2,3
and their lack of intrinsic healing warrants treatment when
functionally symptomatic. Isolated cartilage defects not only
cause morbidity comparable to advanced osteoarthritis4
but can also initiate secondary osteoarthritic changes.5,6
Moreover, cartilage defects have a propensity to progress
when clinical osteoarthritis has been established.7
The contribution of the subchondral bone in the develop-
ment and progression of osteoarthritis has long been recog-
nized,1,8-10 but its role in cartilage repair techniques has only
recently received more attention.11,12
Autologous chondrocyte implantation (ACI) was first
reported in 1994,13 with the initial technique consisting of a
chondrocyte suspension injected under a periosteal flap
890754CARXXX10.1177/1947603519890754CARTILAGEBeck et al.
research-article2019
1Centre for Orthopaedic Research, Faculty of Health and Medical
Sciences, The University of Western Australia, Nedlands, Western
Australia, Australia
2Knee Research Australia, Benowa, Queensland, Australia
3School of Human Sciences, Faculty of Science, The University of
Western Australia, Nedlands, Western Australia, Australia
4Perth Orthopaedic and Sports Medicine Research Institute, West Perth,
Western Australia, Australia
5Foot & Ankle Department, St. Vincent’s Clinic, Sydney, New South
Wales, Australia
Corresponding Author:
Ming-Hao Zheng, Centre for Orthopaedic Research, Faculty of Health
and Medical Sciences, The University of Western Australia, 2nd Floor
M-Block QEII Medical Centre, Nedlands, Western Australia 6009,
Australia.
Email: minghao.zheng@uwa.edu.au
Morphological Assessment of MACI
Grafts in Patients with Revision
Surgery and Total Joint Arthroplasty
Aswin Beck1, David Wood1, Christopher J. Vertullo2,
Jay Ebert3, Greg Janes4, Martin Sullivan5, and Ming-Hao Zheng1
Abstract
Objective. To compare the histological and immunohistochemical characteristics of matrix-assisted chondrocyte implantation
(MACI) grafts between patients with revision surgery and patients with total joint arthroplasty. Methods. Biopsies of
MACI grafts from patients with revision and total joint arthroplasty. The graft tissue characteristics and subchondral
bone were examined by qualitative histology, ICRS (International Cartilage Repair Society) II scoring and semiquantitative
immunohistochemistry using antibodies specific to type I and type II collagen. Results. A total of 31 biopsies were available,
10 undergoing total knee arthroplasty (TKA) and 21 patients undergoing revision surgery. Patients in the clinically failed
group were significantly older (46.3 years) than patients in the revision group (36.6 years) (P = 0.007). Histologically, the
predominant tissue in both groups was of fibrocartilaginous nature, although a higher percentage of specimens in the
revision group contained a hyaline-like repair tissue. The percentages of type I collagen (52.9% and 61.0%) and type II
collagen (66.3% and 42.2%) were not significantly different between clinically failed and revised MACI, respectively. The
talar dome contained the best and patella the worst repair tissue. Subchondral bone pathology was present in all clinically
failed patients and consisted of bone marrow lesions, including edema, necrosis and fibrosis, intralesional osteophyte
formation, subchondral bone plate elevation, intralesional osteophyte formation, subchondral bone cyst formation, or
combinations thereof. Conclusions. MACI grafts in patients with revision and total joint arthroplasty were predominantly
fibrocartilage in repair type, did not differ in composition and were histologically dissimilar to healthy cartilage. Clinically
failed cases showed evidence of osteochondral unit failure, rather than merely cartilage repair tissue failure. The role of the
subchondral bone in relation to pain and failure and the pathogenesis warrants further investigation.
Keywords
MACI, osteochondral unit, revision surgery, subchondral bone, total knee arthroplasty

2 CARTILAGE 00(0)
sutured to the surrounding healthy cartilage, known as peri-
osteum-covered ACI (PACI).13 Subsequent advancements
in biomaterials allowed the replacement of the periosteum
with fibrin glue–stabilized collagen and hyaluronan-based
scaffolds, known as matrix-assisted chondrocyte implanta-
tion (MACI) technique.14
While the scientific literature increasingly supports the clin-
ical outcomes and usage of PACI and MACI techniques,15-19
far less attention has been paid to the failures of this tech-
nique. A recent systematic review reported an overall fail-
ure rate of 14.9% in 4294 patients undergoing ACI, most
commonly within the first 5 years following implantation.20
LaPrade et al.21 illustrated that failed ACI is predominantly
composed of fibrous tissue and fibrocartilage with variable
positivity for both type I and type II collagen. This study
evaluated osteochondritis dissecans lesions repaired with
PACI, not MACI. Histological differences in repair tissue
could be expected between these 2 techniques, as the for-
mer utilizes a cell solution covered with living tissue (peri-
osteum) to initiate repair, whereas the latter initiates repair
with a cell-scaffold construct adhered to the subchondral
bone.13,22
Subchondral bone alterations following ACI techniques
have been observed,11,12,23-26 although their clinical signifi-
cance remain subject of debate. Recognizing articular carti-
lage and the subchondral bone as one functional unit, we
investigated the histological aspects of the subchondral
bone.
The aim of our study was therefore to examine the histo-
logical and immunohistological features of MACI grafts in
patients with revision and total joint arthroplasty and to
investigate the subchondral bone status in patients with
total joint arthroplasty.
Material and Methods
Patients
Between 2000 and 2006, our institution served as the
Australian national reference center for examination of
biopsy tissues obtained from MACI grafts. Included biop-
sies were obtained either from (1) MACI grafts requiring
revision arthroscopy due to graft failure or another reason
requiring arthroscopy or from (2) knees that had further
degenerated and had progressed toward total knee arthro-
plasty (TKA). The decision as to whether the graft had clin-
ically failed and/or revision arthroscopy was indicated, or
whether joint arthroplasty was indeed now the best surgical
option for the patient, was undertaken by the treating
surgeons.
The tissues obtained from patients undergoing repeat
arthroscopy were either cartilage repair tissue biopsies or
osteochondral biopsies (3 mm diameter) taken as part of a
chondroplasty of symptomatic hypertrophic tissue at the
graft site of patients with the primary complaint of “pain,
clicking, and catching” of the graft. For clinically failed
patients, osteochondral slabs including the graft area were
collected as part of a TKA procedure. Patients undergoing
TKA presented with recurring and worsening pain follow-
ing MACI, which failed to respond to conservative treat-
ment, with evidence of degenerative joint disease (and
progression thereof) such that TKA was considered the
most appropriate treatment. Patient consent for treatment
and use of the biopsy for research purposes was obtained
and ethical approval was given by the Human Research
Ethics Committee of our institution.
Histology
Routine histology was performed to evaluate and character-
ize the repair tissue. Samples were placed in 4% paraformal-
dehyde for 48 hours prior to processing. Samples containing
mineralized tissue were decalcified in 10% EDTA (ethylene-
diaminetetraacetic acid), pH 7. For osteochondral slabs from
arthroplasty patients, multiple sections 3 mm apart through
the entire graft were performed prior to paraffin embedding.
Samples were sectioned to 4-µm-thick slices and routinely
stained with hematoxylin and eosin, toluidine blue/fast
green, and safranin O/fast green stain.
All stained samples were evaluated by bright-field
microscopy to characterize tissue as hyaline-like, fibrocarti-
lage, or fibrous tissue based on cellularity (density, shape,
and presence of lacunae) and matrix appearance as previ-
ously reported by Zheng et al.22 For osteochondral samples,
qualitative analysis of the subchondral bone was performed
as described by Zanetti et al.27 Bone marrow edema was
defined as presence of swollen fat cells surrounded by
eosinophilic staining, marrow fibrosis as replacement of fat
cells by fibrous tissue, and marrow necrosis as formation of
foam cells and presence of swollen fat cells with loss of
nuclei. Trabecular bone structure was evaluated for pathol-
ogy, consisting of necrosis (characterized as loss of nuclei),
sclerosis, and repeated remodeling (characterized by bone
formation with reversal lines and bone resorption with
increased osteoclastic activity). Alterations of the subchon-
dral bone, recognized as clinical conditions by Orth et al.,28
were reported accordingly. The grading system as described
by Aho et al.29 was applied on the subchondral bone below
and distant to the repair tissue in normal tissue as control. In
brief, grade 0 is characterized by a thin subchondral bone
plate (SBP) with direct connections between the bone mar-
row and the articular cartilage via open fenestrae, grade 1
by some subchondral bone sclerosis with remaining open
fenestrae, grade 2 by a distinct increase in SBP thickness,
absence of fenestrae, and presence of SBP fibrillation and
grade 3 by severe sclerosis and flattening of the SBP.
Additionally, ICRS (International Cartilage Repair Society)
II scoring was performed as previously described.30 As

Beck et al. 3
blinding was impossible due to obvious differences in the
nature of the sample, they were analyzed twice by the same
investigator, 6 months apart. The average score was taken if
the difference between the 2 time points was less than 10
units. If the difference between the 2 scores was greater
than 10 units, the sample was evaluated by a board-certified
pathologist and consensus between the 2 evaluators was
reached.
Immunohistochemistry
Sections deparaffinized in xylene, rehydrated with decreas-
ing ethanol solutions, and rinsed in distilled water.
Enzymatic antigen retrieval (Pepsin, Abcam ab128991) was
performed at 37°C for 15 minutes and endogenous peroxi-
dase activity was blocked using 3% H2O2 in 96% ethanol
for 10 minutes at room temperature. Nonspecific binding
sites were blocked with 10% fetal bovine serum (FBS) in
0.1% Triton X100 in TBS (Tris-buffered saline) for 1 hour
at room temperature. Samples were then incubated at 4°C
overnight with primary antibodies against rabbit anti-human
collagen type I (Abcam, ab34710, dilution 1:500) and
mouse anti-chicken collagen type II (Developmental
Studies Hybridoma Bank, Iowa, strain II-II6B3, dilution to
2 µg/ml). Antibody was detected using anti-rabbit (Sigma
A0545) and anti-mouse antibodies (Sigma A9917) diluted
at 1:1000 for Col I and II, respectively. Peroxidase staining
was done following the instructions of the manufacturer
(Liquid DAB+ substrate chromogen system, DAKO).
Sections were counterstained using Gill’s hematoxylin and
saturated in lithium carbonate until they turned blue, dehy-
drated in ethanol, cleared in xylene, and mounted. Negative
control samples were incubated in 5% FBS instead of the
primary antibodies. Positive control samples consisted of
an osteochondral section from a nonarthritic joint.
For evaluation of the immunostained sections, the total
area of cartilage was measured using Bioquant Osteo soft-
ware (v13.2.6, Bioquant, Nashville, TN). Areas of positive
staining were measured using semiautomatic thresholding
and the percentage of positively stained tissue, regardless of
the staining intensity, was calculated.
Micro-Computed Tomography
If performed, osteochondral blocks underwent micro-com-
puted tomography (micro-CT) scanning prior to decalcifi-
cation. Samples were scanned in 70% ethanol in a micro-CT
scanner (SkyScan 1176; Bruker-microCT, Kontich, Belgium)
at 50 kV, 500 µA, 240 ms exposure time, rotation step of
0.3, 360° degree of rotation, and an isotropic resolution of
17.78 μm, using a 0.5 mm-thick Al filter.
Images of each specimen were reconstructed using the
manufacturer’s software (NRecon v 1.7.1.0; Bruker-microCT,
Kontich, Belgium). For evaluation of the trabecular bone
microarchitecture, cylinder-shaped volumes of interest (VOI)
with a diameter of 7 mm were selected semiautomatically to
mimic core-extraction samples in the grafted area and in an
area distant to the graft, excluding cysts manually, if pres-
ent. The height of the cylinders did not exceed 4 mm. Bone
morphometric parameters (bone volume fraction [BV/
TV], BS/BV, trabecular thickness, and trabecular separa-
tion) were calculated using CTAnalyzer software (Bruker-
microCT, Kontich, Belgium).
Statistics
Results are expressed as mean ± SD. Data were not nor-
mally distributed, and therefore nonparametric analysis was
performed (Kruskal Wallis and Mann-Whitney U test). The
Fisher exact test was used to investigate relations between
demographic data. Differences were considered significant
at P ≤ 0.05. All calculations were performed with SPSS
(version 22.0; IBM Corp.). Post hoc power analysis was
performed using the G*Power computer program.31
Results
Demographics
Biopsies were available from 31 patients, including 10
undergoing total knee arthroplasty (TKA) and 21 patients
undergoing revision surgery. Seventeen of the revision sur-
geries were performed on knee joints and 4 on ankle joints.
Demographic characteristics are described in Table 1.
Micro-CT was performed on 2 clinically failed patients who
underwent TKA.
The average age at MACI implantation was older in the
clinically failed group who underwent subsequent TKA
(46.3 years) than the group who underwent revision (36.6
years, P = 0.007).
The mean graft survival time was 26.8 months for those
who underwent knee replacement, which was not signifi-
cantly longer (P = 0.94) than survival time for the revision
patients (17.8 months). Defect sizes were on average 4.3
and 4.8 cm2 for TKA and revision patients, respectively, and
were not significantly different (P = 0.68). There was no
significant association between gender and classification
group (P = 0.697) and lesion location and classification
group (P = 0.464).
Histological and Histopathological Observations
Representative histological features of the TKA patients are
illustrated in Figure 1. The predominant cartilage type in
TKA was fibrocartilage (70%), only 10% had a predomi-
nantly hyaline-like cartilage and 20% had a predominantly
fibrous nature. The repair tissue morphology often con-
sisted of a mixture of areas with randomly organized fibers

4 CARTILAGE 00(0)
and areas with less birefringence. Increased birefringence
coincided with more elongated cell types, and thus more
fibrous nature of the tissue, although vascularization was
only occasionally seen.
The tissue surface often showed several degrees of fibril-
lation (Fig. 1), cleft formation, poor organization of colla-
gen fibers, and elongated cells (Fig. 1). Deeper layers
similarly showed poor organization, but cells were often
rounder (Fig. 1) and tidemark formation was rarely
observed. Integration of the repair tissue with the subchon-
dral bone was highly variable and often a thin layer of
fibrous tissue could be observed if integration was incom-
plete (Fig. 1), separating the repair tissue from the subchon-
dral bone. Abnormal mineralization within the basal layers
of the defect was frequently observed and could more spe-
cifically be described as intralesional osteophyte formation
(30%) and elevation of the subchondral bone plate (40%)
(Fig. 1) according to Orth et al.28 Subchondral bone cyst
formation was observed in 2 patients (20%) (Fig. 1). Using
the grading system recently developed by Aho et al.,29 the
subchondral bone plate below the graft was grade 2 or 3 in
all specimens and was always higher than the grade of the
perigraft area (grade 0 or 1 in all cases).
In comparison with TKA patients, the different cartilage
types in revision patients were more evenly distributed with
hyaline-like cartilage counting for 28.5%, fibrocartilage
43%, and fibrous tissue 28.5%. Tissue characteristics were
similar to TKA patients, although generally the tissue sur-
face, basal integration of the tissue scored better and less
vascularization and abnormal mineralization was seen (rep-
resentative image in Fig. 2). From the observed differ-
ences, ICRS II scores for abnormal calcification within the
cartilage defect area and vascularization were significantly
different between the 2 groups (Table 2), thus indicating
more abnormal mineralization within the basal layers of
the repair tissue in TKA patients. Excluding ankle cases
retained the same significant differences, but additionally
showed a significantly better basal integration in knee revi-
sion surgery compared with TKA (P = 0.014).
Interestingly, the average percentage of collagen type 2
was higher in the TKR group (61.0%) compared with the
revision group (44.2%), although the difference was not
significant (Table 2). The reverse was similarly true for col-
lagen type I. For TKA patients, 70% had a ratio Col II %/
Col I % higher >1, compared with 38% in revision patients.
Using the qualitative evaluation criteria of the subchon-
dral bone as outlined by Zanetti et al.,27 all TKA patients
had various extents of subchondral bone marrow edema,
necrosis, and fibrosis, albeit minimal to mild in most cases.
Trabecular abnormalities were also frequently present with
predominantly trabecular necrosis, increased bone remodel-
ing as evidenced by new bone formation, reversal lines, and
osteoclastic activity. All revision surgery patients had bone
marrow fibrosis, which varied from minimal to marked and
only a small number had some bone marrow edema and
necrosis. Trabecular bone changes consisted primarily of
trabecular necrosis, and there was only minimal bone
remodeling in a few patients.
Comparison of MACI Histology between
Anatomical Sites
The prevalence of graft tissue type, regardless of undergo-
ing arthroplasty or revision surgery, in the different ana-
tomic locations is illustrated in Figure 3 and the ICRS II
tissue morphology and overall scores, and percentage col-
lagen types I and II in Figure 4.
Knee Joints. Within the knee joints, patients with lesions on
the patella scored significantly worse than patients with
lesions in the medial femoral condyle (MFC) for tissue mor-
phology, fill type, deep zone and overall assessment. Fibrous
tissue was present in all patients (5/5) undergoing MACI
treatment for patella lesions (Figs. 3-5), but only 2 out of 5
patients underwent joint replacement surgery. Collagen type
I accounted for more than 95% of the repair tissue in all
patella samples and less than 5% was collagen type II. Sub-
chondral bone pathology was rarely present (Fig. 5).
Ankle Joints. Ankle revisions contained overall better quality
of tissue with none of the 4 patients receiving MACI treat-
ment developing fibrous repair tissue. Ankle revisions
scored significantly better in cartilage parameters (tissue
morphology, P = 0.018; deep zone assessment, P = 0.047;
and overall assessment, P = 0.018) compared with knee
revision surgeries, but worse in basal integration (P = 0.005)
(Fig. 6) and subchondral bone health (P = 0.039) (Fig. 6).
Table 1. Patient Demographics for Patients Undergoing Total
Knee Arthroplasty (TKA) and Revision Surgery.
Characteristic TKA (n = 10) Revision (n = 21)
Age, years, mean (SD) 46.3 (5.00) 36.6 (9.43)
Graft survival time,
months, mean (SD)
26.8 (25.54) 17.8 (10.3)
Sex, n (%)
Male 3 (30) 9 (43)
Female 7 (70) 12 (57)
Defect size, cm2,
mean (SD)
4.3 (2.00) 4.8 (4.00)
Lesion location, n (%)
Knee 10 (100) 17 (81)
Medial femoral
condyle
7 (70) 10 (59)
Patella 2 (20) 3 (17)
Trochlea 1 (10) 2 (12)
Unknown 2 (12)
Ankle 4 (19)

Beck et al. 5
Figure 1. (A) Clinically failed matrix-assisted chondrocyte implantation (MACI) graft of the medial femoral condyle (hematoxylin
and eosin [H&E] stain; scale bar = 4 mm). Arrows indicate the margins of the MACI graft. Asterisk indicates the original cement
line and calcified cartilage layer with the area of subchondral bone plate elevation above this line, including grafted and nongrafted
area. Trabecular bone sclerosis and subchondral bone cyst formation are present. Inset 1: Cells within the deeper layers of the
MACI graft were generally rounder, representing a more chondrocyte phenotype. Inset 2: Cells within the superficial layers of
the MACI graft were generally more elongated, representing a more fibroblast phenotype. Inset 3: Incomplete lateral integration
between the graft and the remaining cartilage. Inset 4: Incomplete integration of the graft with the underlying subchondral bone
plate with fibrous tissue within the separation (arrowheads). Inset 5: Marked bone remodeling and osteoclast-mediated (arrows)
subchondral bone cyst formation. Inset 6: Edge of the subchondral bone plate elevation characterized by multiple small blood
vessels extending into the calcified and noncalcified cartilage. Insets 7 and 8: More extensive subchondral bone plate fibrillation
and thickening within the grafted area (Inset 7) compared with the nongrafted and nonelevated area (Inset 8). All insets: scale
bar = 200 μm.
(B) Collagen type II immunohistochemistry (scale bar = 4 mm). Collagen type II is distributed throughout the basal layers of the graft, except for the
surface. (C) Collagen type I immunohistochemistry (scale bar = 4 mm). Collagen type I is present throughout most part of the graft, but less toward
the base of the graft.

6 CARTILAGE 00(0)
The subchondral bone pathology in ankle patients was charac-
terized primarily by marked bone marrow fibrosis (Fig. 6).
Age at implantation was negatively associated with the
score for chondrocyte clustering (ρ = −0.622, P = 0.001)
and abnormal calcification (ρ = −0.511, P = 0.03) when all
samples were included. A negative association between age
at implantation and chondrocyte clustering (ρ = −0.577,
P = 0.010) persisted when TKA patients were excluded.
MACI survival time was positively associated with the
formation of a tidemark (ρ = 0.542, P = 0.037) when all
samples were included. For revision patients subchondral
bone health was positively associated with chondrocyte
clustering (ρ = 0.790, P = 0.020) and basal integration
(ρ = 0.713, P = 0.031).
Subchondral Bone Structure of MACI with TKA
Patients who underwent TKA had specimens of subchon-
dral bone tissue, which enabled the assessment of the sub-
chondral bone structure. One specimen was characterized
Figure 2. Matrix-assisted chondrocyte implantation (MACI) knee revision surgery specimen (left to right: hematoxylin and eosin
[H&E] stain, toluidine blue–safranin O stain, and Col type II stain, scale bar = 4 mm). The original calcified cartilage is clearly visible on
the toluidine blue stain (asterisk) with newly formed bone above. The repair tissue contains collagen type II throughout. Insets 1 and
2: Cells are primarily round-shaped in different layers of the graft, but the matrix is poorly organized. Inset 3: the subchondral bone
marrow shows bone marrow edema and mild fibrosis. All insets: scale bar = 200 μm.
Table 2. ICRS II Scores and Percentage Col Type II and Col Type I for TKA and Revision Surgery.
TKA (n = 10), Mean (± SD) Revision Surgery (n = 21), Mean (± SD) P
Tissue morphology 48.3 (± 24.2) 48.8 (± 25.6) 0.69
Matrix staining 50.0 (± 27.8) 46.5 (± 31.7) 0.82
Cell morphology 49.0 (± 28.1) 43.2 (± 31.3) 0.62
Chondrocyte clustering 60.3 (± 31.2) 73.3 (± 28.5) 0.28
Surface architecture 30.3 (± 37.4) 58.6 (± 37.6) 0.14
Basal integration 70.9 (± 32.0) 90.0 (± 21.2) 0.09
Formation tidemark 24.7 (± 27.5) 16.9 (± 24.8) 0.32
Subchondral bone abnormalities 49.7 (± 35.8) 65.3 (± 30.7) 0.37
Abnormal calcification/ossification 35.3 (± 44.5) 91.5 (± 22.3) <0.01*
Vascularization 56.8 (± 37.3) 81.3 (± 33.4) 0.04*
Surface/superficial assessment 30.0 (± 30.5) 53.9 (± 34.5) 0.12
Mid/deep zone assessment 53.3 (± 27.2) 63.8 (± 23.9) 0.31
Overall assessment 47.0 (± 25.4) 55.8 (± 24.5) 0.35
Col type II 61.0 (± 33.4) 44.2 (± 37.9) 0.37
Col type I 52.8 (± 37.5) 60.238 (± 35.6) 0.69
ICRS II = International Cartilage Repair Society II; TKA = total knee arthroplasty.
*Significant differences.

Beck et al. 7
by marked subchondral bone plate elevation and subchon-
dral bone cyst formation in the grafted area (Fig. 7) and the
other one by subchondral bone plate erosion (Fig. 7).
Bone morphometric parameters showed a 2.2-fold increase
in bone volume percentage in the grafted area, characterized
by increased trabecular thickness and decreased trabecular
Figure 3. Repair tissue type distribution within different anatomical locations (MFC = medial femoral condyle). All patellar grafts
consisted of primarily fibrous tissue whereas none of the ankles contained fibrous tissue. The majority of MFC specimens consisted of
fibrocartilaginous tissue.
Figure 4. Average (±SD) ICRS II scores for tissue morphology and overall assessment and percentage collagen type II and collagen
type I in 4 different anatomic locations (MFC = medial femoral condyle). Tissue morphology and overall assessment scores were
highest for ankle grafts and lowest for patellar grafts and were significantly different from each other. There was significantly more
collagen type II in MFC graft tissue compared to patellar graft tissue.

8 CARTILAGE 00(0)
separation (Table 3). The Structure Model Index was closer
to 0 in the grafted area indicative of trabeculae to be more
plate-like than rod-like (Table 3).
Power Calculation
A 1-sided post hoc power analysis using the G*Power com-
puter program31 indicated that a total of 102 specimens
would be needed to detect medium effects (d = 0.51) with
80% power using a Mann-Whitney U test with α at 0.05.
Discussion
The data of the present study show that there was no signifi-
cant difference in histological features of MACI grafts
between patients with revised surgery and arthroplasty. The
majority of cases in both groups contained fibrocartilagi-
nous repair tissue, although hyaline-like tissue was more
frequently observed in the revision group. Clinically failed
TKA patients were significantly older, had more abnormal
calcification and vascularization within the graft tissue
compared with patients undergoing revision surgery. The
observed subchondral bone pathology of differing degrees
was present in all TKA patients, and hence we propose that
the mechanism of failure is of the entire osteochondral unit
and joint, rather than just failure of the cartilage repair tis-
sue. The MACI grafting procedure succeeded in generating
and maintaining repair tissue within symptomatic cartilage
defects as evidenced by histology but failed to preserve
joint function in patients therefore proceeding to TKA.
Other studies have observed similar graft tissue to this
study following failed cartilage restoration procedures. For
ACI procedures, LaPrade et al. evaluated repair tissue of 6
periosteal-ACI failures for the treatment of osteochondral
dissecans.21 All specimens were characterized as primarily
fibrocartilaginous tissue with variable amounts of type II
and type I collagen. The failure mode however differed,
with patients experiencing pain following dislodged or par-
tially dislodged graft tissue.
In contrast to our study, LaPrade et al.21 reported a ratio
of Coll II %/Col I % of <1 in all ACI patients, whereas we
found this ratio <1 in only 30% of the cases. A minimal
degree of graft integration with the subchondral bone may
be needed to form or maintain type II collagen as nondis-
lodged tissue in the microfracture-treated specimens in the
study by LaPrade et al.21 formed more collagen type II com-
pared with collagen type I. This is further supported by find-
ings of Nehrer et al.32 who similarly demonstrated fibrous
tissue in detached repair tissue compared to more hyaline-
like tissue where the PACI graft had bonded with the sub-
chondral bone. Nehrer et al.32 reported fibrous tissue as the
most common type in PACI, accounting for 60% of the
cross-sectional area.32 None of the patients in our study were
evaluated because of graft detachment, a failure mechanism
Figure 5. Clinically failed patella matrix-assisted chondrocyte implantation (MACI). (A) Hematoxylin and eosin (H&E) stain,
overview (scale bar = 4 mm). Arrows indicate the margins of the MACI graft. Marked surface fibrillation and clefting and poor
lateral integration of the graft with the surrounding cartilage are present. Besides a small subchondral bone plate erosion (asterisk)
and intralesional osteophyte (arrow head) at the junction of the graft tissue with the surrounding cartilage tissue, there are minimal
changes within the trabecular bone. Inset 1 (scale bar = 200 μm): Graft tissue consisted primarily of high-cellular fibrous tissue with
an abundance of blood vessels throughout. (B) Scale bar = 4 mm. Collagen type II immunohistochemistry. Minimal collagen type II is
present and is primarily localized within the base of the repair tissue. (C) Scale bar = 4 mm. Collagen type I is present within all layers
of the graft tissue.

Beck et al. 9
now largely avoided by improved fixation technique with
fibrin glue and collagen matrix. Differences in ACI tech-
nique (MACI vs. PACI) may account for different failure
mechanisms, but tissue characteristics do not seem to differ.
Tissue characteristics in “failed cases” may also not depend
on treatment method as Kaul et al. similarly found fibrocar-
tilaginous tissue following bone marrow stimulation proce-
dures in patients with early osteoarthritis.33
We found fibrous tissue in all patients undergoing MACI
treatment for patella lesions and although considered infe-
rior quality tissue, it was interesting to note that only 2 out
of 5 patients underwent joint replacement surgery. While a
certain degree of poorly regenerated tissue thus seems to be
acceptable in the patellofemoral joint, it is also probable
that surgeons are less likely to offer TKA for patellofemoral
lesions, a selection bias evident in this study.
Of interest, despite the fact that more revision patients
contained hyaline like repair tissue, 62% of revision patients
had a Col I%/Col II % >1 compared with 30% of TKA
patients. In many instances, specimens from revision
patients consisted of biopsies from the surface of the repair
site. It is unclear whether this hypertrophic surface repre-
sents a tissue composition indicative of the repair as a
whole, or only the upper portion of the repair. We frequently
observed increased levels of type 1 collagen toward the sur-
face of the repair tissue. In our study, patients undergoing
TKA were significantly older than patients undergoing revi-
sion surgery. The question whether age is a limiting factor
for MACI remains controversial. Some studies have found
a negative association of age with outcome,34,35 while others
have found no such association.36-38 Recent guidelines
advise on careful selection above the age of 50 years.39 Our
Figure 6. Revision ankle matrix-assisted chondrocyte implantation (MACI) graft. Hematoxylin and eosin (H&E) stain, scale bar
= 3 mm. The graft surface is smooth and cells are primarily round shaped throughout the entire graft (Insets 1 and 2). Cell
clustering is present at the surface. Inset 3 (toluidine blue–fast green staining) Clefting at the base of graft tissue (asterisk) near
the tidemark. The subchondral bone plate shows marked proliferative islands within the calcified cartilage layer (arrows). Scale bar
insets = 200 μm.

10 CARTILAGE 00(0)
study included 5 patients ≥50 years (3 TKA patients 51, 51,
and 52 years and 2 revision patients 50 and 50 years).
The effect of preexisting osteoarthritis on MACI treat-
ment could not be examined in our study due to its design;
however, ACI in the presence of osteoarthritis is controver-
sial in the literature.
Minas et al.40 reported an overall failure rate of 12 out of
155 knees in 153 patients (8%) with early osteoarthritic
changes undergoing ACI, selected with the specific aim of
delaying arthroplasty surgery. Arthroplasty in failed cases
was performed at an average of 38 months after ACI, which
was longer compared with our study. Interestingly, almost
half of the patients receiving ACI in the authors’ series
included patients with early osteoarthritic changes (153 out
of 328 patients with follow-up over 2 years) indicating a
greater tendency to perform ACI in early arthritic knees than
in other series. Morphological and qualitative information
on the graft tissue was not given. Moreover, Hollander
et al.41 did not find a negative effect of osteoarthritis on graft
repair, but on the contrary osteoarthritis was shown to
enhance tissue regeneration. Despite these interesting obser-
vations, Filardo et al.42 observed a failure rate of 27.5% in
Figure 7. Micro-computed tomography (micro-CT) images (scale bar = 4 mm). (A) Marked subchondral bone overgrowth and 3
subchondral bone cysts are present. (B) The grafted area contains subchondral bone plate erosion with a steep sharp osseous rim
(osteophyte) protruding toward the cartilage.
Table 3. Bone Morphometric Parameters for the Trabecular Bone in the Graft and Perigraft Area.
Specimen BV/TV (%) BS/BV (1/mm) Tb.Th (mm) Tb.Sp (mm) Tb.N (1/mm) SMI
MFC, 55 year old female, 72
months post-MACI graft area
34.4 15.7 0.22 0.44 1.60 0.6
MFC, 55 year old female, 72
months post-MACI peri-graft area
15.5 25.0 0.14 0.65 1.11 1.2
MFC, 46 year old male, 43 months
post-MACI peri-graft area
22.3 20.9 0.17 0.50 1.30 1.2
BV/TV = percentage bone volume; BS/BV = bone surface/bone volume ratio; Tb.Th = trabecular thickness; Tb.Sp = trabecular separation;
Tb.N = trabecular number; SMI = Structure Model Index.

Beck et al. 11
44 patients with osteoarthritis (Kellgren grade 2 and 3)
treated with ACI and suggested that once the osteoarthritic
process had started, the potential of ACI is compromised
regardless of the severity of the joint condition.
The observation of different degrees of subchondral
bone pathology in all clinically failed TKA patients, signifi-
cantly worse abnormal calcification and vascularization in
TKA patients are interesting and could suggest subchondral
bone and endochondral ossification may play a pivotal role
in clinically failed patients.
In recent years, the importance of the subchondral bone
in cartilage repair strategies has gained interest.11,12,24,43
Despite the frequent observation of subchondral bone alter-
ations and pathology,24 its clinical significance and correla-
tion with pain remain controversial, while in osteoarthritis
the subchondral bone is a well-recognized source of pain.1,8-10
We observed subchondral bone changes in all patients that
underwent TKA due to clinical failure. The changes were
often localized and more extensive below the graft tissue,
although subchondral bone plate elevation also extended
beyond the grafted area. Vasiliadis et al.24 reported intrale-
sional osteophyte formation in 64% of patients and sub-
chondral bone cyst formation in 39% of patients, 9 to 18
years following ACI treatment, but failed to find signifi-
cant correlation between clinical outcome and subchondral
bone abnormalities. However, they considered its occur-
rence a negative prognostic factor.24 Impaired bone and
cartilage regeneration processes, altered biomechanical
loading, disturbed mechanisms of cartilage–subchondral
bone crosstalk, and pathological vascularization or angio-
genesis have been proposed as potential causes.28 Although
it has been proposed that overgrowth of the subchondral
bone leads to narrowing of the repair tissue,28 this was not
our observation, as the repair tissue was always at least as
thick as the nongrafted cartilage, but repair tissue protruded
above the normal position of the cartilage surface. More
research needs to be undertaken to investigate the patho-
physiology and clinical significance of the subchondral
bone pathology.
Patella repair tissue had the worst tissue composition. In
the original report of the MACI technique, Brittberg et al.13
similarly reported significantly worse clinical and morpho-
logical outcomes for patients with patella lesions. Adressing
patella malalignment has led to improved outcomes, similar
to other anatomic locations.44 A certain degree of patella
malalignment could not be ruled out in our cohorts.
Ankle MACI grafts in our study performed overall better
and none of the specimens had fibrous repair tissue. Other
studies have similarly reported good to excellent outcomes
and a negative association with age >40 years.45,46 Three
out of 4 patients in our study were younger than 40 years.
Interestingly, Dixon et al.45 observed a high proportion of
patients with magnetic resonance imaging evidence of
complete defect filling, complete integration of borders, an
intact graft surface and homogeneous signal within the graft
in patients with persistent pain, implying that failure to
relieve pain may not necessarily indicate technical failure
of the procedure.
Our data suggest that repair tissues in revised MACI
repair and clinically failed MACI are similar. However, this
study has several limitations and conclusions need to be
approached with caution.
Because of the retrospective nature of the study, the
number of specimens in each group could not be controlled
for and limited statistical power because of small sample
size may have contributed to limiting the significance of
some of the statistical comparisons. Specimens from revised
MACI grafts contained several biopsies from the surface of
the graft site. This tissue may not represent the composition
within the entire graft. Similarly, 3-mm diameter osteo-
chondral samples from the center of the defect may also not
represent the entire graft. Indeed, similar to Laprade et al.,21
we found large variation in tissue composition throughout
the entire sample in the knee arthroplasty slabs.
Patients classified as clinical failures may be completely
or partially a consequence of other pathologic changes
within the joint and thus sources of pain not directly related
to the MACI treatment. In other words, clinically failed
cases may contain biologically and functionally adequate
repair tissue, but the repair tissue failed to prevent degen-
eration of the entire joint. After all, cartilage does not con-
tain nerve endings and pain can persist despite excellent
cartilage defect infill.45,47
Although the histological and immunohistological eval-
uation of biopsies yield important data regarding the qual-
ity of repair tissue, their correlation with clinical symptoms
and function remains unclear. Intuitively, tissue with char-
acteristics closer to native hyaline articular should be
superior in function and more durable, but frequently
fibrocartilaginous repair tissue seems sufficient. Hollander
et al.,41 for example, collected 2mm diameter biopsies of
23 patients undergoing follow-up arthroscopy for nonclini-
cal reasons following ACI procedures and found that 43%
of patients had fibrocartilaginous tissue 6 to 25 months
after the ACI procedure. Peterson et al.48 found primarily
fibrous tissue in biopsy samples of 4 out of 12 patients with
good to excellent clinical grading at a mean follow-up of
54.3 months.
Similarly, Saris et al.17 evaluated 116 biopsies in a pro-
spective randomized trial comparing MACI with microfrac-
ture. Two years following treatment both treatments showed
very good structural repair and similar mean ICRS II over-
all assessment scores (±63.8). Despite similar histological
appearance, MACI-treated patients performed clinically
significantly better than microfracture-treated patients.17
The mean overall assessment scores in revised MACI in our
study (55.8) was lower, although similar to MACI treated
patients in the study by Saris et al.17 It is clear that, besides

12 CARTILAGE 00(0)
cartilage repair tissue composition, other factors must con-
tribute to clinical success or failure.
As a noninvasive technique and with the ability to follow
patients over time, magnetic resonance imaging has pro-
vided valuable information in regards to cartilage repair and
avoids some of the limitations of biopsies, but studies have
similarly shown a poor correlation between repair tissue
structural parameters and clinical outcome.24,49,50
Another limitation of the current study is the inability to
blind the investigators to the groups, an inherent limitation
of the sampling method. Additionally, the different sizes in
specimens (blocks for TKA vs. cylinders for clinically
intact grafts) and the nature of tissue (osteochondral and
cartilage shavings for clinically intact grafts) leads to a
comparison of heterogeneous sample groups. Similarly,
pathology of the subchondral bone was heterogeneous and
certain aspects such as the position of the subchondral bone
plate in the osteochondral biopsy samples relative to the
surrounding subchondral bone could not be determined.
The decision to offer TKA or revision arthroscopy was
undertaken by the treating surgeons. Although specific
clinical scores and/or patient-reported outcome measures
(PROMs) collected at the time of revision surgery would
have been useful for the analysis and interpretation of our
data, this information is not routinely collected from
patients in a clinical setting and is rarely used as part of the
decision-making process for arthroplasty surgery. There
are no agreed criteria for patients to have a knee replace-
ment and the decision to proceed with TKA is typically a
patient’s preference-based decision.51
We used immunohistochemistry to measure the per-
centage of type I and II collagen within the repair tissue by
measuring the total area of positive immunostaining,
regardless of staining intensity, within the total grafted
area. Although immunohistochemistry provides informa-
tion about the distribution of the collagen subtypes and an
indication of the amount of positively stained tissue, it can
be argued that this is not a true quantitative method,52 as
results may be affected by potential inconsistencies in
methodology (such as thickness of tissue in sections and
antigen retrieval protocols).53 Additionally, the limited asso-
ciation between antigen presence and staining intensity
above and below a certain threshold of bound antibody
combined with the subjectivity in assessment, make stain-
ing intensity an unreliable method.53
Micro-CT imaging of clinically failed specimens was
performed in only 2 out of 10 cases and illustrations may
therefore not be representative of all clinically failed MACI
grafts. The low number of available micro-CT data was due
to delayed recognition of the subchondral bone involved in
the pathology and therefore only the most recent samples
underwent micro-CT scanning.
In summary, the most prevalent tissue in revised and
clinically failed MACI biopsies was of fibrocartilaginous
nature and there was no significant difference in composi-
tion between the 2 groups. Subchondral bone pathology of
differing degrees was present in all clinically failed MACI
biopsies and failure of the osteochondral unit, rather than
the graft repair tissue is probably more appropriate termi-
nology. Further investigation into the cause of joint pain
and tighter definition of failure are needed.
Acknowledgments and Funding
The author(s) received no financial support for the research,
authorship, and/or publication of this article.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Ethical Approval
Ethical approval was given by the Human Research Ethics
Committee of our institution (approval number: 2003-31).
Informed Consent
Patient consent for treatment and use of the biopsy for research
purposes was obtained.
Trial Registration
Not applicable.
ORCID iD
Aswin Beck https://orcid.org/0000-0002-0095-6454
References
1. Goldring SR, Goldring MB. Changes in the osteochondral
unit during osteoarthritis: structure, function and cartilage-
bone crosstalk. Nat Rev Rheumatol. 2016;12(11):632-44.
doi:10.1038/nrrheum.2016.148
2. Curl WW, Krome J, Gordon ES, Rushing J, Smith BP,
Poehling GG. Cartilage injuries: a review of 31 516 knee
arthroscopies. Arthroscopy. 1997;13(4):456-60. doi:10.1016/
s0749-8063(97)90124-9
3. Widuchowski W, Widuchowski J, Trzaska T. Articular car-
tilage defects: study of 25 124 knee arthroscopies. Knee.
2007;14(3):177-82. doi:10.1016/j.knee.2007.02.001
4. Heir S, Nerhus TK, Røtterud JH, Loken S, Ekeland A,
Engebretsen L, et al. Focal cartilage defects in the knee impair
quality of life as much as severe osteoarthritis: a comparison
of knee injury and osteoarthritis outcome score in 4 patient
categories scheduled for knee surgery. Am J Sports Med.
2010;38(2):231-7. doi:10.1177/0363546509352157
5. Schinhan M, Gruber M, Vavken P, Dorotka R, Samouh L,
Chiari C, et al. Critical-size defect induces unicompart-
mental osteoarthritis in a stable ovine knee. J Orthop Res.
2012;30(2):214-20. doi:10.1002/jor.21521
6. Lee CR, Grodzinsky AJ, Hsu HP, Martin SD, Spector M.
Effects of harvest and selected cartilage repair procedures

Beck et al. 13
on the physical and biochemical properties of articular car-
tilage in the canine knee. J Orthop Res. 2000;18(5):790-9.
doi:10.1002/jor.1100180517
7. Davies-Tuck ML, Wluka AE, Wang Y, Teichtahl AJ, Jones
G, Ding C, et al. The natural history of cartilage defects in
people with knee osteoarthritis. Osteoarthritis Cartilage.
2008;16(3):337-42. doi:10.1016/j.joca.2007.07.005
8. Arnoldi CC, Lemperg K, Linderholm H. Intraosseous
hypertension and pain in the knee. J Bone Joint Surg Br.
1975;57(3):360-3.
9. Dieppe P. Subchondral bone should be the main target for
the treatment of pain and disease progression in osteoarthri-
tis. Osteoarthritis Cartilage. 1999;7(3):325-6. doi:10.1053/
joca.1998.0182
10. Torres L, Dunlop DD, Peterfy C, Guermazi A, Prasad P,
Hayes KW, et al. The relationship between specific tissue
lesions and pain severity in persons with knee osteoarthritis.
Osteoarthritis Cartilage. 2006;14(10):1033-40. doi:10.1016/j.
joca.2006.03.015
11. Gomoll AH, Madry H, Knutsen G, van Dijk N, Seil R,
Brittberg M, et al. The subchondral bone in articular carti-
lage repair: current problems in the surgical management.
Knee Surg Sports Traumatol Arthrosc. 2010;18(4):434-47.
doi:10.1007/s00167-010-1072-x
12. Madry H. The subchondral bone: a new frontier in articu-
lar cartilage repair. Knee Surg Sports Traumatol Arthrosc.
2010;18(4):417-8. doi:10.1007/s00167-010-1071-y
13. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O,
Peterson L. Treatment of deep cartilage defects in the knee
with autologous chondrocyte transplantation. N Engl J Med.
1994;331(14):889-95. doi:10.1056/NEJM199410063311401
14. Gomoll AH, Farr J. Autologous chondrocyte implantation
(ACI). In: Farr J, Gomoll AH, editors. Cartilage restoration.
Cham, Switzerland: Springer; 2018. p. 265-274.
15. National Institute for Health and Care Excellence. Autologous
chondrocyte implantation for treating symptomatic articular
cartilage defects of the knee. Available from: https://www.
nice.org.uk/guidance/ta477
16. Brittberg M, Recker D, Ilgenfritz J, Saris DBF; SUMMIT
Extension Study Group. Matrix-applied characterized autolo-
gous cultured chondrocytes versus microfracture: five-year
follow-up of a prospective randomized trial. Am J Sports
Med. 2018;46(6):1343-51. doi:10.1177/0363546518756976
17. Saris D, Price A, Widuchowski W, Bertrand-Marchand M,
Caron J, Drogset JO, et al; SUMMIT Study Group. Matrix-
applied characterized autologous cultured chondrocytes
versus microfracture: two-year follow-up of a prospective
randomized trial. Am J Sports Med. 2014;42(6):1384-94.
doi:10.1177/0363546514528093
18. Basad E, Ishaque B, Bachmann G, Sturz H, Steinmeyer J.
Matrix-induced autologous chondrocyte implantation ver-
sus microfracture in the treatment of cartilage defects of the
knee: a 2-year randomised study. Knee Surg Sports Traumatol
Arthrosc. 2010;18(4):519-27. doi:10.1007/s00167-009-1028-1
19. Vanlauwe J, Saris DB, Victor J, Almqvist KF, Bellemans J,
Luyten FP, et al. Five-year outcome of characterized chondro-
cyte implantation versus microfracture for symptomatic carti-
lage defects of the knee: early treatment matters. Am J Sports
Med. 2011;39(12):2566-74. doi:10.1177/0363546511422220
20. Andriolo L, Merli G, Filardo G, Marcacci M, Kon E. Failure
of autologous chondrocyte implantation. Sports Med Arthrosc
Rev. 2017;25(1):10-18. doi:10.1097/JSA.0000000000000137
21. LaPrade RF, Bursch LS, Olson EJ, Havlas V, Carlson CS.
Histologic and immunohistochemical characteristics of failed
articular cartilage resurfacing procedures for osteochondritis
of the knee: a case series. Am J Sports Med. 2008;36(2):360-
8. doi:10.1177/0363546507308359
22. Zheng MH, Willers C, Kirilak L, Yates P, Xu J, Wood D,
et al. Matrix-induced autologous chondrocyte implantation
(MACI): biological and histological assessment. Tissue Eng.
2007;13(4):737-46. doi:10.1089/ten.2006.0246
23. Brown WE, Potter HG, Marx RG, Wickiewicz TL, Warren RF.
Magnetic resonance imaging appearance of cartilage repair
in the knee. Clinical Orthop Relat Res. 2004;(422):214-23.
doi:10.1097/01.blo.0000129162.36302.4f
24. Vasiliadis HS, Danielson B, Ljungberg M, McKeon B,
Lindahl A, Peterson L. Autologous chondrocyte implanta-
tion in cartilage lesions of the knee: long-term evaluation
with magnetic resonance imaging and delayed gadolinium-
enhanced magnetic resonance imaging technique. Am J Sports
Med. 2010;38(5):943-9. doi:10.1177/0363546509358266
25. Saris DB, Vanlauwe J, Victor J, Haspl M, Bohnsack M,
Fortems Y, et al. Characterized chondrocyte implantation
results in better structural repair when treating symptomatic
cartilage defects of the knee in a randomized controlled trial
versus microfracture. Am J Sports Med. 2008;36(2):235-46.
doi:10.1177/0363546507311095
26. Minas T, Gomoll AH, Rosenberger R, Royce RO, Bryant T.
Increased failure rate of autologous chondrocyte implantation after
previous treatment with marrow stimulation techniques. Am J
Sports Med. 2009;37(5):902-8. doi:10.1177/0363546508330137
27. Zanetti M, Bruder E, Romero J, Hodler J. Bone marrow edema
pattern in osteoarthritic knees: correlation between MR imag-
ing and histologic findings. Radiology. 2000;215(3):835-40.
doi:10.1148/radiology.215.3.r00jn05835
28. Orth P, Cucchiarini M, Kohn D, Madry H. Alterations of the
subchondral bone in osteochondral repair—translational data
and clinical evidence. Eur Cells Mater. 2013;25:299-316.
29. Aho OM, Finnilä M, Thevenot J, Saarakkala S, Lehenkari P.
Subchondral bone histology and grading in osteoarthritis. PLoS
One. 2017;12(3):e0173726. doi:10.1371/journal.pone.0173726
30. Mainil-Varlet P, Van Damme B, Nesic D, Knutsen G, Kandel R,
Roberts S. A new histology scoring system for the assessment
of the quality of human cartilage repair: ICRS II. Am J Sports
Med. 2010;38(5):880-90. doi:10.1177/0363546509359068
31. Faul F, Erdfelder E, Buchner A, Lang AG. Statistical power
analyses using G*Power 3.1: tests for correlation and regression
analyses. Behav Res Methods. 2009;41(4):1149-60. doi:10.3758/
BRM.41.4.1149
32. Nehrer S, Spector M, Minas T. Histologic analysis of tissue
after failed cartilage repair procedures. Clin Orthop Relat Res.
1999;(365):149-62.
33. Kaul G, Cucchiarini M, Remberger K, Kohn D, Madry H. Failed
cartilage repair for early osteoarthritis defects: a biochemical, his-
tological and immunohistochemical analysis of the repair tissue
after treatment with marrow-stimulation techniques. Knee Surg
Sports Traumatol Arthrosc. 2012;20(11):2315-24. doi:10.1007/
s00167-011-1853-x

14 CARTILAGE 00(0)
34. Nawaz SZ, Bentley G, Briggs TW, Carrington RW, Skinner
JA, Gallagher KR, et al. Autologous chondrocyte implanta-
tion in the knee: mid-term to long-term results. J Bone Joint
Surg Am. 2014;96(10):824-30. doi:10.2106/JBJS.L.01695
35. McNickle AG, L’Heureux DR, Yanke AB, Cole BJ.
Outcomes of autologous chondrocyte implantation in a diverse
patient population. Am J Sports Med. 2009;37(7):1344-50.
doi:10.1177/0363546509332258
36. Niemeyer P, Köstler W, Salzmann GM, Lenz P, Kreuz PC,
Sudkamp NP. Autologous chondrocyte implantation for treat-
ment of focal cartilage defects in patients age 40 years and older:
a matched-pair analysis with 2-year follow-up. Am J Sports
Med. 2010;38(12):2410-6. doi:10.1177/0363546510376742
37. Bhosale AM, Kuiper JH, Johnson WE, Harrison PE,
Richardson JB. Midterm to long-term longitudinal outcome of
autologous chondrocyte implantation in the knee joint: a multi-
level analysis. Am J Sports Med. 2009;37(Suppl 1)131S-138S.
doi:10.1177/0363546509350555
38. Rosenberger RE, Gomoll AH, Bryant T, Minas T. Repair of
large chondral defects of the knee with autologous chondro-
cyte implantation in patients 45 years or older. Am J Sports
Med. 2008;36(12):2336-44. doi:10.1177/0363546508322888
39. Niemeyer P, Albrecht D, Andereya S, Angele P, Ateschrang
A, Aurich M, et al. Autologous chondrocyte implanta-
tion (ACI) for cartilage defects of the knee: a guideline by
the working group “Clinical Tissue Regeneration” of the
German Society of Orthopaedics and Trauma (DGOU). Knee.
2016;23(3):426-35. doi:10.1016/j.knee.2016.02.001
40. Minas T, Gomoll AH, Solhpour S, Rosenberger R, Probst C,
Bryant T. Autologous chondrocyte implantation for joint pres-
ervation in patients with early osteoarthritis. Clin Orthop Relat
Res. 2010;468(1):147-57. doi:10.1007/s11999-009-0998-0
41. Hollander AP, Dickinson SC, Sims TJ, Brun P, Cortivo
R, Kon E, et al. Maturation of tissue engineered cartilage
implanted in injured and osteoarthritic human knees. Tissue
Eng. 2006;12(7):1787-98. doi:10.1089/ten.2006.12.1787
42. Filardo G, Vannini F, Marcacci M, Andriolo L, Ferruzzi A,
Giannini S, et al. Matrix-assisted autologous chondrocyte
transplantation for cartilage regeneration in osteoarthritic
knees: results and failures at midterm follow-up. Am J Sports
Med. 2013;41(1):95-100. doi:10.1177/0363546512463675
43. Demange MK, Minas T, von Keudell A, Sodha S, Bryant
T, Gomoll AH. Intralesional osteophyte regrowth following
autologous chondrocyte implantation after previous treatment
with marrow stimulation technique. Cartilage. 2017;8(2):
131-8. doi:10.1177/1947603516653208
44. Ebert JR, Schneider A, Fallon M, Wood DJ, Janes GC. A
comparison of 2-year outcomes in patients undergoing tibio-
femoral or patellofemoral matrix-induced autologous chon-
drocyte implantation. Am J Sports Med. 2017;45(14):3243-53.
doi:10.1177/0363546517724761
45. Dixon S, Harvey L, Baddour E, Janes G, Hardisty G. Functional
outcome of matrix-associated autologous chondrocyte implan-
tation in the ankle. Foot Ankle Int. 2011;32(4):368-74. doi:
10.3113/FAI.2011.0368
46. Giannini S, Buda R, Vannini F, Di Caprio F, Grigolo B.
Arthroscopic autologous chondrocyte implantation in osteo-
chondral lesions of the talus: surgical technique and results.
Am J Sports Med. 2008;36(5):873-80. doi:10.1177/036354
6507312644
47. Andjelkov N, Riyadh H, Wretenberg P. Neuralgic and noci-
ceptive pain in the knee as a cause of the treatment failure
after cartilage repair surgery: two case reports. J Pain Relief.
2018;7(4):325. doi:10.4172/2167-0846.1000325
48. Peterson L, Brittberg M, Kiviranta I, Akerlund EL, Lindahl
A. Autologous chondrocyte transplantation. Biomechanics
and long-term durability. Am J Sports Med. 2002;30(1):2-12.
doi:10.1177/03635465020300011601
49. Ebert JR, Smith A, Fallon M, Wood DJ, Ackland TR.
Correlation between clinical and radiological outcomes after
matrix-induced autologous chondrocyte implantation in the
femoral condyles. Am J Sports Med. 2014;42(8):1857-64.
doi:10.1177/0363546514534942
50. Ebert JR, Robertson WB, Woodhouse J, Fallon M, Zheng
MH, Ackland T, et al. Clinical and magnetic resonance imag-
ing-based outcomes to 5 years after matrix-induced autolo-
gous chondrocyte implantation to address articular cartilage
defects in the knee. Am J Sports Med. 2011;39(4):753-63.
doi:10.1177/0363546510390476
51. Barlow T, Scott P, Thomson L, Griffin D, Realpe A. The
decision-making threshold and the factors that affect it:
a qualitative study of patients’ decision-making in knee
replacement surgery. Musculoskeletal Care. 2018;16(1):3-12.
doi:10.1002/msc.1190
52. Hollander AP, Dickinson SC, Sims TJ, Soranzo C, Pavesio
A. Quantitative analysis of repair tissue biopsies following
chondrocyte implantation. Novartis Found Symp. 2003;249
:218-41.
53. Walker RA. Quantification of immunohistochemistry—issues
concerning methods, utility and semiquantitative assessment I.
Histopathology. 2006;49(4):406-10. doi:10.1111/j.1365-2559
.2006.02514.x