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Articular Cartilage Repair With Magnetic Mesenchymal Stem Cells

Goki Kamei, Takaaki Kobayashi, Shingo Ohkawa, Wirat Kongcharoensombat, Nobuo Adachi, Kobun Takazawa,
Hayatoshi Shibuya, Masataka Deie, Koji Hattori, Jeffrey L. Goldberg and Mitsuo Ochi
Am J Sports Med 2013 41: 1255 originally published online April 19, 2013
DOI: 10.1177/0363546513483270

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Articular Cartilage Repair
With Magnetic Mesenchymal Stem Cells
Goki Kamei,* MD, PhD, Takaaki Kobayashi,* MD, PhD, Shingo Ohkawa,* MD,
Wirat Kongcharoensombat,* MD, PhD, Nobuo Adachi,* MD, PhD, Kobun Takazawa,* MD,
Hayatoshi Shibuya,* MD, PhD, Masataka Deie,* MD, PhD, Koji Hattori,y MD, PhD,
Jeffrey L. Goldberg,z MD, PhD, and Mitsuo Ochi,*§ MD, PhD
Investigation performed at the Department of Orthopaedic Surgery,
Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan

Background: Cell therapies are hampered by the difficulty of delivering cells to and retaining them in target tissues long enough
to repair or regenerate local tissues.
Hypothesis: Magnetic-assisted delivery of magnetically labeled mesenchymal stem cells (m-MSCs) would be rapid, allowing for
chondrogenic differentiation and functional joint repair without replacement.
Study Design: Controlled laboratory study.
Methods: Sixteen mini-pigs aged 6 to 7 months were used. A full-thickness cartilage defect was created in the center of the
patella with a cylindrical punch (diameter, 6 mm). At 4 weeks after creation of the cartilage defects, the animals were divided
into 3 treatment groups: In the M group, m-MSCs (5 3 106 cells) were injected and accumulated to the cartilage defect using
an external magnetic force (1.5 T) for 10 minutes; in the G group, the patella was faced upward, filled with MSCs (5 3 106 cells),
and held for 10 minutes; and in the C group, only phosphate-buffered saline was injected. The regenerated cartilage was eval-
uated in 5 knees in each of the 3 groups by arthroscopic surgery at 6 and 12 weeks and histological and ultrasound evaluation at
12 and 24 weeks.
Results: The mean arthroscopic scores at 6 weeks were 10.4 6 1.10 in the M group, 8.8 6 0.84 in the G group, and 7.4 6 0.89 in
the C group. There was a statistically significant difference between the M group and the other 2 groups. The mean arthroscopic
scores at 12 weeks were 12.8 6 1.30 (M group), 10.5 6 1.30 (G group), and 9.5 6 0.58 (C group), with a statistically significant
difference between the M and C groups. The mean histological scores using the Wakitani scoring system at 12 weeks were 2.8 6
0.96 (M group), 5.4 6 0.55 (G group), and 6.0 6 2.20 (C group), and the mean histological scores at 24 weeks were 2.4 6 1.50 (M
group), 3.5 6 0.56 (G group), and 5.3 6 1.50 (C group). The mean histological scores at 12 weeks were significantly better in the M
group than in the other groups, and the M group maintained a significantly better histological score than did the C group at 24
weeks.
Conclusion: The m-MSCs had no adverse effect on chondrogenic differentiation, and m-MSCs delivered by magnetic field appli-
cation repaired cartilage defects.
Clinical Relevance: The clinical application of this novel stem cell delivery system is a potential therapeutic option for treating
cartilage defects and may be more applicable throughout the body than traditional methods.
Keywords: articular cartilage repair; magnetically labeled mesenchymal stem cells; external magnetic force

In spite of many procedures that exist for the treatment of
cartilage injury, such as bone marrow stimulating techni-
ques (drilling, microfracture) and autologous osteochon-
dral grafting,17,27 clinical success has been limited.
Current studies on cartilage regeneration are more focused
on tissue engineering, which usually requires technically
demanding procedures with proper scaffolds or growth fac-
tors.19,21,23,24 Intra-articular cell transplantation without
scaffolds and growth factors is a more attractive option,
The American Journal of Sports Medicine, Vol. 41, No. 6
DOI: 10.1177/0363546513483270
Ó 2013 The Author(s)
as it could reduce surgical intervention to a simple arthro-
scopic cell injection. For example, mesenchymal stem cells
(MSCs) have the ability to differentiate into cells of chon-
drogenic lineage and represent an attractive cell source
for treating such cartilage defects.3,5,25 Recently, numer-
ous studies that evaluated the cartilage repair using
MSCs have been reported.16,20,30 However, from these
studies, it has been observed that confining MSCs to a car-
tilage defect without a scaffold is difficult, and a large
number of MSCs are needed to treat a cartilage defect.
Intra-articular injection of large numbers of MSCs gener-
ates loose bodies of scar tissue in the joint in the rat model,
indicating a significant disadvantage of intra-articular
injection of excessive number of MSCs.1

1255
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1256 Kamei et al The American Journal of Sports Medicine

Therefore, to avoid complications in providing effective
treatment for chondral defects, it may be essential to inject
a small number of MSCs into the joint defect space. Here,
we demonstrate a novel cell delivery system for regenerative
medicine using MSCs with superparamagnetic iron oxide
(SPIO) magnetically labeled MSCs (m-MSCs) and an opti-
mized external magnetic device to accumulate a relatively
small number of MSCs to a specific desired area. Kobayashi
et al11 have reported that m-MSCs can be delivered to a spe-
cific site under an external magnetic force in a rabbit model
and a fresh-frozen porcine model. In addition, Kobayashi
et al10 also found, in an in vitro model, the formation of
a new cell layer attributable to the chondrogenic differentia-
tion of MSCs defined by staining with toluidine blue, safra-
nin O, and type II collagen immunoreactivity. However, the
larger joint spaces and possibility of different articular biol-
ogy found in humans or other large mammals, and the
need for instrumentation to deliver strong, focal magnetic
fields, require investigation in large animals. The purpose
of this study was to evaluate the repair of chronic full-thick-
ness cartilage defects using m-MSCs and an external mag-
netic force in a porcine model and to validate this new
treatment option for osteoarthritis and cartilage injury.
MATERIALS AND METHODS
This study was approved by the Guide for Animal Experi-
mentation and the Committee of Research Facilities for
Laboratory Animal Science (Graduate School of Biomedical
Science, Hiroshima University).
3 days thereafter. The cells proliferated and reached con-
fluence approximately 2 weeks after seeding. Cells were
then harvested by treatment with 0.25% trypsin. To
expand the MSCs, 2 3 105 to 3 3 105 of the harvested cells
were seeded onto 100-mm culture dishes. On reaching con-
fluence again, the cells were reseeded under the same con-
ditions. These adherent cells have been referred to as
MSCs. Cells at passage 3 were used in the current study.
Magnetic Labeling of MSCs
Ferucarbotran (27.9 mg Fe/mL) (Bayer Healthcare Co Ltd,
Osaka, Japan) at a concentration of 97.5 mg Fe/mL was
placed in a tube containing serum-free Roswell Park Memo-
rial Institute (RPMI) 1640 medium (BioSource, Camarillo,
California), consisting of 25 mmol/L of 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES), L-glutamine, mini-
mum essential medium (MEM) nonessential amino acids,
and sodium pyruvate. Protamine sulfate (Mochida Seiyaku
Ltd, Tokyo, Japan) was then added to the solution at a con-
centration of 5 mg/mL. The solution containing ferucarbotran
and protamine was mixed for 3 minutes, with intermittent
manual shaking. Then, an equal volume of solution contain-
ing ferucarbotran-protamine complexes was added to DMEM
in MSC culture. The MSCs were labeled overnight with fer-
ucarbotran and protamine as a transfection agent. After
labeling, all cells were washed twice with sterile phosphate-
buffered saline (PBS), with a final wash containing heparin
(10 U/mL), to dissolve extracellular ferucarbotran-protamine
complexes when present. These magnetically labeled MSCs
were denoted as m-MSCs.

Cell Isolation Adhesion Rate of SPIO-Labeled MSCs (m-MSCs)

The process for isolation and in vitro expansion of bone
marrow–derived MSCs has been described previously.15
In brief, 5 mL of bone marrow from the iliac crest from 6-
to 8-month-old mini-pigs was aspirated with 1 mL of hepa-
rin using an 18-gauge needle. The sample was centrifuged
for 5 minutes at 1500 rpm, and the resulting supernatant,
including heparin sodium, was discarded. The extract was
resuspended in 2 mL of culture medium composed of Dul-
becco’s modified Eagle medium (DMEM) (Gibco BRL,
Carlsbad, California) with 10% fetal bovine serum
(Sigma-Aldrich Corp, St Louis, Missouri) and 1% antibiot-
ics (penicillin, streptomycin, and fungizone) (BioWhit-
taker, Walkersville, Maryland). Then, 2 mL of the
suspension was seeded onto 100-mm culture dishes, and
8 mL of culture medium was added to each dish. The
dishes were incubated for 3 weeks under a humidified
atmosphere and 5% CO2 at 37°C. The medium was not
changed for the first 7 days and then was changed every
To deliver a strong magnetic field in a specific focal space, we
designed a custom-built bulk superconducting magnet sys-
tem (in collaboration with Hitachi Ltd, Ibaraki, Japan). We
opted for a permanent magnet system because the magnetic
force was directed to the center of the disk surface and its
magnitude decreased away from the surface (Figure 1, A
and B). Saho et al26 had previously characterized the force
distribution and gradient measurement with distance. We
performed an ex vivo evaluation using porcine bone
marrow–derived MSCs and porcine patellae to assess the
adhesion rate of m-MSCs with an external magnetic force.
To determine the time required for cell attachment to
the defect, patellar tissue was placed in a vertical fashion
parallel to the surface of the external magnet (Figure
1C). We tested magnetic forces of 0 T, 0.6 T, and 1.5 T
and exposure times of 10 and 60 minutes and then turned
the patellar defect side down for 10 minutes to allow non-
adherent cells to fall into the culture medium. The

§
Address correspondence to Mitsuo Ochi, MD, PhD, Department of Orthopaedic Surgery, Graduate School of Biomedical Science, Hiroshima Univer-
sity, 1-2-3, Kasumi Minami-ku, Hiroshima, Japan (e-mail: ochim@hiroshima-u.ac.jp).
*Department of Orthopaedic Surgery, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.
y
National Institute of Advanced Industrial Science and Technology, Tokyo, Japan.
z
Bascom Palmer Eye Institute and Interdisciplinary Stem Cell Institute, Miller School of Medicine, University of Miami, Miami, Florida.
One or more of the authors has declared the following potential conflict of interest or source of funding: This work was supported by a grant-in-aid to Dr
Ochi for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 21249079), and by ‘‘The Project for Real-
ization of Regenerative Medicine’’ to Dr Ochi from the Ministry of Education, Culture, Sports, Science and Technology in 2012, Japan.

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Vol. 41, No. 6, 2013
A B
D dine blue and safranin O staining) using a pellet culture
100
90
Adhesion rate (%)
80
70
60
C 50
40
30
20
10
0 sox9, and type 3 collagen mRNA expression (n = 8) (Figure
Magnetic force, time of application
Figure 1. A novel, clinically useful bulk superconducting
magnet system. A mobile operator-friendly system (A) with
an operator surface diameter of 10 cm (B) was designed.
The magnetic force at the center of the magnet surface
was approximately 5 T and at 30 mm away from the surface
was approximately 0.5 T. (C) Demonstration of the use of the
magnet in an ex vivo patellar assay. (D) Adhesion rate of
magnetically labeled mesenchymal stem cells (m-MSCs)
with a magnetic force (0.6 T or 1.5 T for 10 or 60 minutes,
as marked) and non–m-MSCs by the gravity adhesion tech-
nique (gravity). ̪P \ .05 compared with gravity.
detached cell number was counted, allowing us to derive an
attached cell number, from which we determined that
approximately 95% of m-MSCs were retained in the target
area with any magnetic field application (Figure 1D). It is
worth noting that no cells are able to move against gravity
when labeled with magnetic particles but no magnetic
force is applied.11 In addition, we evaluated the adhesion
rate of MSCs by a local gravity-assisted technique for 10
minutes.13 The adhesion rate of 4 groups that were
exposed to a magnetic force was statistically significantly
higher than that of the non–m-MSCs by the local adhesion
technique (Figure 1D), but there was no statistically signif-
icant difference among the 4 m-MSC/magnetic field groups
(n = 5 for each group) (P \ .05 compared with the control).
Thus, a very high rate of cell delivery and retention was
possible ex vivo using magnetic cell delivery.
Cell Proliferation and Chondrogenic Differentiation
of SPIO-Labeled MSCs (m-MSCs)
We evaluated cell proliferation using a cell-counting kit
(Dojindo Laboratories, Kumamoto, Japan) and compared
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Articular Cartilage Repair With Magnetic MSCs 1257
the absorbance at day 0 with that at day 7 for non–
m-MSCs, m-MSCs, and m-MSCs that were exposed to
a magnetic force of 1.5 T for 10 minutes. The m-MSC pro-
liferation after exposure to the magnetic force was signifi-
cantly higher than that of m-MSCs alone and non–
m-MSCs (Figure 2A). Thus, magnetic labeling and expo-
sure to a magnetic force did not have any adverse effects
on cell proliferation.
During our 21-day analysis of chondrogenic differentia-
tion, we evaluated chondrogenic gene expression by real-
time polymerase chain reaction (type II collagen, aggrecan,
sox9, and type X collagen) and histological findings (tolui-
system (n = 8) when m-MSCs were grown in chondrogenic
differentiation media. There was no major difference in the
chondrogenic potential of MSCs after magnetic particle
loading and/or magnetic field exposure (1.5 T, 10 minutes),
as indicated by toluidine blue and safranin O staining (Fig-
ure 2B), or by the expression of type II collagen, aggrecan,
2C); indeed mature markers aggrecan and type II collagen
trended towards higher expression after magnetic force
application to m-MSCs. Therefore, magnetic labeling and
magnetic field application at a level high enough to maxi-
mize cell recovery in an ex vivo model did not adversely
affect, and indeed may promote, MSC proliferation and
chondrogenic differentiation.
Animal Model and Creation of a Cartilage Defect
In this study, 16 mini-pigs aged 6 to 7 months were used.
Anesthesia was induced by intramuscular injection of
40 mg/kg medetomidine, 0.2 mg/kg midazolam, and
5 mg/kg ketamine. The medial parapatellar approach was
used to expose the knee joint. A full-thickness cartilage
defect with a diameter of 6 mm was created in the center
of the patella in both knees with a cylindrical punch. At
4 weeks after creation of the cartilage defects, the treat-
ments were performed. They were divided into 3 groups:
In the magnetic force group (M group), m-MSCs (5 3 106
cells) were injected and accumulated to the cartilage defect
using an external magnetic force (1.5 T) for 10 minutes
(Figure 1B); in the gravity group (G group), the patellar
defect was faced upward, filled with MSCs (5 3 106 cells),
and held for 10 minutes; and in the control group (C
group), only PBS was injected. The mini-pigs were then
returned to their cages and were free to exercise. In each
group, mini-pigs were sacrificed at 12 and 24 weeks after
the injection. Eight mini-pigs were sacrificed at 12 weeks,
and the remaining 8 mini-pigs were sacrificed at 24 weeks
after injection. They were divided into 3 groups at 12 and
24 weeks, and 5 knees were included in each group. We
excluded 1 knee at 12 weeks because of infection. In the
G group, 1 knee from 1 of 8 mini-pigs was sacrificed at
24 weeks and used for evaluation, and the other knee
was injected with DiI-labeled m-MSCs, and an external
magnetic force at arthroscopic surgery was applied 1
week before the sacrifice. It was then used for histological
evaluation of the accumulation of m-MSCs after 1 week
under the influence of an external magnetic force. A
1258 Kamei et al The American Journal of Sports Medicine

A B

Type X collagen
Type II collagen
Aggrecan
Sox 9

Figure 2. Magnetic labeling and field application do not adversely affect mesenchymal stem cell (MSC) proliferation or chondro-
genic differentiation. (A) Cell proliferation evaluated by a cell-counting kit (Dojindo Laboratories, Kumamoto, Japan) was compared
between day 0 and day 7 for non–magnetically labeled MSCs (nonlabel), magnetically labeled MSCs (m-MSCs), and m-MSCs that
were exposed to a magnetic force (1.5 T, 10 minutes). Proliferation was statistically significantly greater in m-MSCs exposed to
a magnetic field (̪P \ .05). (B) Chondrogenic differentiation was evaluated by histological findings (safranin O and toluidine blue
as marked) in pellet culture. (C) Chondrogenic differentiation was evaluated by real-time polymerase chain reaction for 1 initial chon-
drogenic promoting marker (sox9) and 2 mature chondrogenic markers (aggrecan, type II collagen) as labeled. Field application to
m-MSCs increased the expression of mature chondrogenic differentiation markers, matching the histology as in B.

flowchart shows the number of pigs and knees used in each
part of this study (Figure 3).
Evaluation of Accumulation of m-MSCs
Under the Influence of an External Magnetic Force
The m-MSCs were injected into the cartilage defect, and
an external magnetic force using arthroscopic surgery
was used to accumulate them in the cartilage defect. We
held the position for 10 minutes, and then the external
magnetic device was released under saline perfusion for
arthroscopic surgery to demonstrate the complete attach-
ment of m-MSCs. At 1 week after injection of the DiI-
labeled m-MSCs, accumulation of the m-MSCs was histo-
logically examined in 1 knee, with the help of an external
magnetic force. The presence of ferucarbotran was evalu-
ated by Berlin blue staining, and nuclei were counter-
stained by nuclear fast red solution (Sigma-Aldrich
Japan, Tokyo, Japan) using 5-mm axial sections of the
patella.
Arthroscopic and Quantitative Ultrasonic Assessment
The regenerated cartilage was evaluated at arthroscopic
surgery in 5 knees in each group at 6 and 12 weeks. The
smooth surface and hardness of the regenerated cartilage
were examined using the ultrasound evaluation system
in 5 knees in each group at 12 and 24 weeks. In fact, the
12-week arthroscopic examination occurred before nec-
ropsy. For arthroscopic assessment, we used the Interna-
tional Cartilage Repair Society (ICRS) macroscopic
evaluation of cartilage repair score and the Oswestry
Arthroscopy Score (OAS) and compared the results from
the 3 groups at 6 and 12 weeks after the treatment.28 We
graded the results as defect repair (0-4), integration to bor-
der zone (0-4), and macroscopic appearance (0-4) on the
ICRS and stiffness on probing (0-2) on the OAS.
In the ultrasonic assessment, we used the ultrasound
evaluation system that was developed by Hattori et al,6,7
and the ultrasonic examination was made with saline using
a transducer and pulsar receiver (Panametrics Japan Co
Ltd, Tokyo, Japan). We evaluated the maximum wavelet

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Vol. 41, No. 6, 2013
Figure 3. A flow diagram shows the number of pigs and knees used in each part of the study.
magnitude and echo duration when the mini-pigs were sacri-
ficed at 12 and 24 weeks. The maximum wavelet magnitude
is closely related to the aggregate module and reflects the
proteoglycan content, and the echo duration is closely related
to the macroscopic fibrillation of the regenerated cartilage.6
We evaluated the adjacent cartilage and regenerated tissue
in the 3 groups and compared the maximum wavelet magni-
tude and echo duration at 12 and 24 weeks after the treat-
ment. The arthroscopic and ultrasonic assessments were
conducted by W.K., who was blinded as to group allocation.
Histological Assessment
The patella was excised and fixed in 4% paraformaldehyde
phosphate-buffered solution (Wako Pure Chemical Industries
Ltd, Osaka, Japan) for 48 hours. The samples were then
decalcified with ethylenediaminetetraacetic acid (EDTA)
solution and embedded in paraffin block. The samples were
cut into 5-mm sections along the axial plane. For histological
assessment, the sections were stained with safranin O and
toluidine blue. For immunohistological assessment, the sec-
tions were stained with type II collagen. Immunohistochem-
istry was started by means of incubation with 10% HistoVT
One (Nacalai USA, San Diego, California) in PBS. The slide
was incubated in hydrogen peroxide block solution (3%H2O2)
for 10 minutes, followed by washing with PBS. Primary anti-
bodies were applied and incubated overnight at 4°C. The
antibody reaction procedures were followed by treatment
with an avidin-conjugated peroxidase (Vectastain ABC-Elite
Lit, Vector Laboratories, Burlingame, California). The reac-
tion for visualization was performed using a 3,3#-diamino-
benzidine (DAB) substrate kit (Vector Laboratories). The
specimens were graded using a histological grading scale
for cartilage regeneration as described by Wakitani et al.29
The presence of ferucarbotran in regenerated tissue was
evaluated by Berlin blue staining, and nuclei were counter-
stained by nuclear fast red solution. K.T., who was not
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Articular Cartilage Repair With Magnetic MSCs 1259
blinded to the group allocation, conducted the histological
evaluation in 5 knees both at 12 and 24 weeks after the
implantation in each group.
Statistical Analysis
Results were expressed as mean 6 standard deviation.
Statistical comparisons among multiple groups were eval-
uated by the Kruskal-Wallis test, and a pairwise compari-
son was performed using the Mann-Whitney test. A P
value of \.05 was considered to indicate a statistically sig-
nificant difference.
RESULTS
Rapid Arthroscopic Accumulation
of m-MSCs With an External Magnetic Force
Arthroscopic observation in this large mammalian preclin-
ical model demonstrated that a magnetic force pulled the
m-MSCs toward the cartilage defect immediately and spe-
cifically after intra-articular injection (Figure 4, A and B;
alao see Video Supplement 1). After 10 minutes, the exter-
nal magnetic device was released, but the m-MSCs were
retained in the cartilage defect, despite the ongoing saline
perfusion used for arthroscopic surgery (see Video Supple-
ment 2).
The retention of m-MSCs at the articular surface was
evaluated by the presence of ferucarbotran by Berlin blue
staining (Figure 4C) and by prelabeling m-MSCs with DiI
(Figure 4D), both of which confirmed cellular integration
into the tissue at 1 week after injection. There were no Ber-
lin blue–positive cells at 12 and 24 weeks, demonstrating
the metabolism and disappearance of the magnetic particles
from the joint space. Similarly, DiI-positive cells disap-
peared at 12 and 24 weeks because of apoptosis. Thus,
1260 Kamei et al The American Journal of Sports Medicine

Figure 4. Short-term outcome of magnetic application of magnetically labeled mesenchymal stem cells (m-MSCs) in the mini-pig
model in vivo. (A) Diagram shows the method of cell and field application. (B) The m-MSCs (arrows) of the magnetic force group (M
group) were injected and accumulated to the cartilage defect (dotted circle) using an external magnetic force (1.5 T) for 10 minutes.
Time-lapse arthroscopic pictures demonstrating the rapid accumulation of m-MSCs in the knee joint upon magnetic field application
(time shown in minutes). (C, D) Histological findings at 1 week after injection of m-MSCs show chondrogenic differentiation of
m-MSCs by Berlin blue staining (C), with confirmation of transplanted cell origin by DiI staining (D).

Figure 5. Arthroscopic findings are maximized by magnetic cell delivery. (A, B) At 6 and 12 weeks, the combined arthroscopic
score based on the International Cartilage Repair Society (ICRS) score and Oswestry Arthroscopy Score (OAS) (stiffness on prob-
ing) was higher in the M group (̪P \ .05). (C, D) Arthroscopic view at 6 (C) and 12 weeks (D) demonstrates that integration at the
border zone and the surface of the regenerated tissue in the M group were smoother than in the other groups; stiffness on probing
was nearly normal in grafts of the M group compared to adjacent cartilage.

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Vol. 41, No. 6, 2013
m-MSCs were rapidly and specifically localized to a cartilage
defect under a magnetic force in vivo.
Arthroscopic Results
The mean arthroscopic scores at 6 weeks were 10.4 6 1.10
in the M group, 8.8 6 0.84 in the G group, and 7.4 6 0.89 in
the C group. There was a statistically significant difference
between the M group and the other 2 groups in the arthro-
scopic assessment (M and G: P = .018, M and C: P = .009)
and between the G and C groups (P = .017) (Figure 5A).
The mean arthroscopic scores at 12 weeks were 12.8 6
1.30 in the M group, 10.5 6 1.30 in the G group, and 9.5
6 0.58 in the C group, with a statistically significant differ-
ence between the M and C groups (P = .019) (Figure 5B). In
the joints of the M group, integration to the border zone
was better, and the surface of the regenerated tissue was
smoother than in the other groups (Figure 5, C and D),
and stiffness on probing was nearly normal compared
with adjacent cartilage. The graft level with surrounding
cartilage was almost equal among the 3 groups.
Ultrasound Assessment
In the ultrasonic assessment of regenerated tissue, at 12
weeks after injection, the maximum wavelet magnitude
was 7.2 6 0.59 in the normal adjacent cartilage (N), 5.1 6
0.50 in the regenerated tissue of the M group, 4.3 6 0.80
in the regenerated tissue of the G group, and 3.0 6 0.90
in that of the C group. At 24 weeks after injection, the
regenerated tissue was 6.9 6 0.40 in the normal area, 5.5
6 0.73 in the M group, 4.3 6 0.48 in the G group, and 4.1
6 0.90 in the C group. There were statistically significant
differences of the maximum wavelet magnitude between
the normal area and the lesions of the other 3 groups at
12 and 24 weeks (N and M: P = .009, N and G: P = .002,
and N and C: P = .002 at 12 weeks; N and M: P = .002, N
and G: P = .001, and N and C: P = .006 at 24 weeks),
between the M and C groups at 12 weeks (P = .009), and
between the M group and the other 2 groups at 24 weeks
(M and G: P = .047, M and C: P = .028) (Figure 6, A and C).
At 12 weeks after injection, the echo duration was 0.41
6 0.02 ms in the normal area, 0.46 6 0.04 ms in the regen-
erated tissue of the M group, 0.58 6 0.06 ms in the regen-
erated tissue of the G group, and 0.50 6 0.06 ms in that of
the C group. At 24 weeks after injection, the echo duration
was 0.40 6 0.06 ms in the normal area, 0.46 6 0.02 ms in
the regenerated tissue of the M group, 0.53 6 0.03 ms in
that of the G group, and 0.52 6 0.06 ms in that of the C
group. There were statistically significant differences
between the normal area and the regenerated tissue of
the other groups at 12 weeks (N and M: P = .04, N and
G: P = .006, N and C: P = .009) and between the normal
and G and C groups at 24 weeks (N and G: P = .01, N
and C: P = .02) (Figure 6, B and D).
Macroscopic Appearance and Histology
Macroscopically, at 12 weeks, the reparative tissue in the M
group had a smooth surface and good integration at the
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Articular Cartilage Repair With Magnetic MSCs 1261
Figure 6. Evaluation of maximum wavelet magnitude (A, C)
and echo duration (B, D) demonstrated regenerated cartilage
indices at 6 (A, B) and 12 weeks (C, D) in normal control (N),
magnetic cells and field application (M), gravity alone (G), and
phosphate-buffered saline–injected control (C) groups. The
maximum wavelet magnitude was closely related to the
aggregate module and reflected the proteoglycan content,
and echo duration was closely related to the macroscopic
fibrillation of regenerated cartilage.
margins, whereas the reparative tissue in the G and C
groups had rough surfaces and discernible edges (Figure
7A). Similarly, excellent histological integration was seen
only in the transplant of the M group, with an almost indis-
cernible interface and no excess or uneven regenerated tis-
sue on the surface (Figure 7, B and C). In addition, the
mean histological score was significantly better in the M
group than in the other groups (M and G: P = .011, M and
C: P = .028) (Figure 7D). Note that almost complete healing
and a yellow tissue color were observed in all groups, with no
signs of synovitis or osteoarthritis. By 24 weeks, the repara-
tive tissue in the M group was white, with a smooth surface
and good integration at the margins, whereas the reparative
tissue in the G and C groups was still slightly yellow, with
rough surfaces and hypertrophic change (Figure 7E). Micro-
scopic findings suggested a similarly improved cellular
response after magnetic cell delivery carried out to 24 weeks
that was not seen in the G or C groups (Figure 7, F and G),
and once again, the M group maintained a significantly bet-
ter histological score than the C group (P = .04) (Figure 7H).
Immunohistochemical staining of collagen type II showed
that the superficial layer regenerated with hyaline-like car-
tilage in the M group, whereas fibrous tissue was seen in the
G and C groups (Figure 7, F and G). Thus, by a variety of
measures from histological to gross and arthroscopic assess-
ments, the magnetic delivery of m-MSCs allowed the best
long-term recovery after joint injury in this preclinical
model.
1262 Kamei et al The American Journal of Sports Medicine

Figure 7. Long-term evaluation of magnetically labeled mesenchymal stem cells (m-MSCs) demonstrates improved integration
and repair at 12 (A-D) and 24 weeks (E-H). Macroscopic findings (A, E) show normalization of the border and whitening of tissue
only in the M group. Histology using safranin O (B, F) reveals improved borders between graft and host tissue (arrows denote
border). Immunohistochemical staining of collagen type II showed that the superficial layer regenerated with hyaline-like cartilage
in the M group, whereas fibrous tissue was seen in the G and C groups (C, G). Wakitani scores at 12 (D) and 24 (H) weeks show
maximal improvement in the M group. ̪P \ .05.

DISCUSSION has reported on cartilage regeneration using a combination

This study is the first to evaluate the repair process of
a chronic full-thickness cartilage defect using m-MSCs
and an external magnetic force in a large porcine model.
Specifically, we found that the arthroscopic score at 6 and
12 weeks, and histological and ultrasonic scores at 12 and
24 weeks, were better in the M group than in the G and C
groups. Arthroscopic findings showed that integration to
the border zone was better and the surface of regenerated
tissue was smoother than the other groups, and stiffness
on probing was nearly normal compared with adjacent car-
tilage. Furthermore, with the highly efficient magnetic cell
delivery tested here, the superficial layer regenerated with
hyaline-like cartilage in the M group, whereas fibrous tissue
was seen in the G and C groups. Taken together, these data
suggest that this novel cell delivery system promotes early
regeneration of the cartilage layer and regenerates the car-
tilage defect with hyaline-like cartilage.
Stem cell therapy has been established as a potential
method for cartilage regeneration.4,12,19 Recent literature
of MSCs, scaffolds, and growth factors.19,21-24 For example,
intra-articular injection of MSCs plus bone marrow stimu-
lation repaired chronic osteochondral defects of the knee in
rats.20 An intra-articular injection of MSCs suspended in
hyaluronic acid was also tested for the treatment of large
cartilage defects.16
However, intra-articular injection of a large number of
MSCs also generates loose bodies of scar tissue in the joint
in the rat model.1 Furthermore, scaffolds and growth factors
are expensive, complex to administer in the intra-articular
space, and add an extra layer to translation for human dis-
ease. A previous cell delivery system used an intra-articular
magnet to localize magnetically labeled, synovium-derived
cells using a magnetic force to a small area and demon-
strated that this magnetic delivery system improved carti-
lage regeneration, but such an intra-articular magnet
must be removed after repairing the articular cartilage,
a procedure that is too invasive for translation to human
application.8 A local adhesion and gravity technique was
also tested in which the cartilage defect was faced upward

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Vol. 41, No. 6, 2013
and filled with synovial MSCs and held for 10 minutes,
which was less invasive; however, only 60% of the cells
attached.13
Here, we hypothesized that magnetic delivery of MSCs
using an external magnet might bring together the best
aspects of these prior studies. Generally, MSCs have been
labeled using SPIO nanoparticles alone or SPIO complexed
to transfection agents, such as ferumoxide, ferucarbotran,
ferumoxide-protamine, poly-L-lysine(PLL)–ferumoxide, and
others.2,9,14 Ferucarbotran, used in our study, is approved
for clinical use as a contrast medium in Japan, and it is
also metabolized completely in about 48 hours.
Before experimenting in the large animal model, we
evaluated the adhesion rate of m-MSCs to a chondral
defect using fresh-frozen porcine patellae. There was no
statistically significant difference in the magnetic fields
or the exposure times. From this, we decided to use
a 1.5-T, 10-minute exposure because a stronger magnetic
field is expected to be more effective to accumulate the
m-MSCs to the cartilage defect, and a shorter exposure
time is better for clinical use in regard to surgical time,
for example, to minimize the risk of infection.
Cell proliferation and differentiation of m-MSCs
exposed to an external magnetic force have not previously
been studied. We found that cell proliferation and chondro-
genic differentiation of m-MSCs exposed to magnetic fields
(1.5 T, 10 minutes) were not statistically significantly dif-
ferent from controls and may have trended toward
improved proliferative and differentiation properties.
This may be caused by physical, mechanical stimulation
of the cells by the field application. Previous work using
a microarray analysis showed that gene expression of
m-MSCs exposed to a magnetic force upregulates cell adhe-
sion molecules including integrin a2 (ITG a2), integrin a6
(ITG a6), integrin b3 binding protein (ITG b3BP), intercel-
lular adhesion molecule–2 (ICAM-2), and platelet/
endothelial cell adhesion molecule–1 (PECAM-1).18 From
these results, we suppose that m-MSCs adhere to the
defect site physically at first and then they gradually
engraft to the defect site biologically. The lack of adverse
effects on the proliferation and chondrogenic differentia-
tion of MSCs supports their safe translation.
The adhesion rate using our novel technique was about
95% higher than that of the local adhesion technique pre-
viously reported.8 In addition, the current study demon-
strated that the magnetically delivered m-MSCs
remained at the site of the cartilage defect under arthro-
scopic saline perfusion even after removing the external
magnetic device. There is a risk of attached cells undergo-
ing peeling from the defect site by the friction of knee
motion after adhesion to the defect site, but we have shown
that the cells remained at the defect site at 1 week after
injection using cell tracking by DiI.
The bulk superconducting magnet system created for this
application had enough force to accumulate the m-MSCs to
the cartilage defect. As the size of this magnet system is
small, it is easy to handle, making the procedure clinically
feasible. That this procedure can be completed under arthro-
scopic examination without the need for surgical opening of
the knee joint is another advantage. Thus, a cartilage injury
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Articular Cartilage Repair With Magnetic MSCs 1263
can be treated more safely and easily using a less invasive
technique and relatively fewer materials.
Further examination is necessary because of some of the
study’s limitations. The first limitation is the small num-
ber of mini-pigs for evaluation in each group. This study
did not allow a powerful comparison between groups; how-
ever, this study showed the statistically significant differ-
ences between 3 groups. We believed that the number of
mini-pigs was sufficient to evaluate cartilage regeneration
when this method was compared with those in previous
reports in large animals. The second limitation is the
follow-up period. A longer follow-up such as 12 months is
ideal to evaluate matrix organization and cell-type matura-
tion. However, we could obtain better results at 12 and 24
weeks in the M group compared with the G and C groups
and thought that the validity of this procedure was demon-
strated, even though the follow-up period was relatively
short. Third, in the current study, although there were
not any observable effects of the magnetic field on the sur-
gical tools, care will have to be taken to minimize potential
field effects on surgical probes, arthroscopic equipment,
television monitors, and anesthesia apparatus. It is likely
that this approach cannot be applied to patients with
a pacemaker or other magnetically susceptible implants.
Finally, magnetic cell delivery for joint repair may be diffi-
cult in certain sites of cartilaginous defects such as the tib-
ial plateau, as the magnetic force becomes weak far from
the center of the surface and as the magnetic force is gen-
erated in only one direction. Future experiments could
explore using 2 external magnetic devices to change the
vector and size of magnetic force application to overcome
these theoretical limitations.
CONCLUSION
This study showed that m-MSCs had no adverse effect on
cell proliferation and differentiation and that m-MSCs
delivered by magnetic field application repaired cartilage
defects. Thus, we suggest that the clinical application of
this novel stem cell delivery system using m-MSCs and
an external magnetic force is a potential therapeutic option
for treating cartilage defects and may be applicable
throughout the body.
A Video Supplement for this article is available in the online
version or at http://ajsm.sagepub.com/supplemental.
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