Murata et al. Journal of Orthopaedic Surgery and Research (2015) 10:35
DOI 10.1186/s13018-015-0173-0

RESEARCH ARTICLE

Open AccessA preliminary study of osteochondral regeneration
using a scaffold-free three-dimensional constructof porcine adipose tissue-derived mesenchymal
stem cells
Daiki Murata1, Satoshi Tokunaga2, Tadashi Tamura3, Hiroaki Kawaguchi4, Noriaki Miyoshi4, Makoto Fujiki1,
Koichi Nakayama5 and Kazuhiro Misumi1*

Abstract
Background: Osteoarthritis (OA) is a major joint disease in humans and many other animals. Consequently, medical
countermeasures for OA have been investigated diligently. This study was designed to examine the regeneration ofarticular cartilage and subchondral bone using three-dimensional (3D) constructs of adipose tissue-derived mesenchymal
stem cells (AT-MSCs).
Methods: AT-MSCs were isolated and expanded until required for genetical and immunological analysis and construct
creation. A construct consisting of about 760 spheroids that each contained 5.0 × 104 autologous AT-MSCs was implanted
into an osteochondral defect (diameter: 4 mm; depth: 6 mm) created in the femoral trochlear groove of two adultmicrominipigs. After implantation, the defects were monitored by computed tomography every month for 6 months
in animal no. 1 and 12 months in animal no. 2.
Results: AT-MSCs were confirmed to express the premature genes and to be positive for CD90 and CD105 and negative
for CD34 and CD45. Under specific nutrient conditions, the AT-MSCs differentiated into osteogenic, chondrogenic, andadipogenic lineages, as evidenced by the expressions of related marker genes and the production of appropriate matrix
molecules. A radiopaque area emerged from the boundary between the bone and the implant and increased more
steadily upward and inward for the implants in both animal no. 1 and animal no. 2. The histopathology of the implants
after 6 months revealed active endochondral ossification underneath the plump fibrocartilage in animal no. 1. The
histopathology after 12 months in animal no. 2 showed not only that the diminishing fibrocartilage was as thick as thesurrounding normal cartilage but also that massive subchondral bone was present.
Conclusions: The present results suggest that implantation of a scaffold-free 3D construct of AT-MSCs into an
osteochondral defect may induce regeneration of the original structure of the cartilage and subchondral boneover the course of 1 year, although more experimental cases are needed.
Keywords: Regeneration, Cartilage, Bone, Scaffold-free, Three-dimensional construct, Stem cell, Adipose tissue,
Computed tomography, Histopathology, Porcine* Correspondence: kaz_msm@vet.kagoshima-u.ac.jp
1
Veterinary Surgery, Department of Veterinary Clinical Science, Joint Faculty of
Veterinary Medicine, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima
890-0065, Japan
Full list of author information is available at the end of the article© 2015 Murata et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.

Murata et al. Journal of Orthopaedic Surgery and Research (2015) 10:35

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Background
Osteoarthritis (OA) is a major joint disease contributing
to midlife and geriatric locomotor dysfunction, and theassociated disability can decrease quality of life inhumans [1]. OA slowly progresses not only as a result of
traumatic injuries to joint structures [2] but also throughmany exacerbating factors such as age, sex, body massindex, occupation, bone shape, and genetic factors regulating proteolytic enzymes [3-5]. In advanced OA, cartilage degeneration and subchondral bone sclerosis may beworsened by the usual mechanical load of daily activities[6], and therefore surgical strategies to reconstruct boththe bone and cartilage have been investigated to restore
joint structure and function [7]. A particular issue of interest in recent studies has been the complete regenerationof hyaline cartilage covering the subchondral bone.
Although a clinical study of osteochondral autografts
from non-load-bearing sites implanted into deteriorated
sites showed favorable outcomes following surgery (clinical improvement in 79%–94% of OA patients) [8], a lossof clinically sound cartilage at the donor sites is associatedwith autologous osteochondral transfer [9]. Studies onsurgical procedures using a combination of artificial bone
and autologous chondrocytes seeded into a collagen scaffold have also shown favorable restoration of cartilage[10,11]. However, some studies have suggested associatedproblems such as isolation of few chondrocytes from asmall piece of normal cartilage [10] and dedifferentiationof chondrocytes during passages in culture [12]. To solvethese problems, stem cells have recently received attention
in a study [13].
Stem cells are defined as immature cells that have theability for self-renewal and the potential for multilineage
differentiation into specific cells. Mesenchymal stem cells(MSCs) derived from bone marrow (BM) and adipose tissue (AT) have mostly been used to demonstrate differentiation into bone and cartilage in vitro [14,15]. BM-derived
MSCs (BM-MSCs) appear to have some disadvantages including decreased numbers and deterioration of the cells
depending on senescence and natural transformation
caused by genomic instability [16]. Previous experiments
have shown age-related decreases in the yield rate, growthrate, and differentiation potential of BM-MSCs in rats andhumans [17,18]. On the other hand, the advantages of ATderived MSCs (AT-MSCs) are that abundant cells can beisolated from AT and their cellular proliferation rate may
be higher in mature animals [19]. Furthermore, given thatobesity is undesirable in OA patients, the regenerative
strategy for bone and cartilage using unwanted AT could
be reasonable and acceptable by many OA patients. It has
been reported that AT-MSCs hardly differentiate into
chondrocytes [20]. However, a recent study using rabbits
demonstrated the regeneration of bone and cartilage after
implantation of scaffold-free three-dimensional (3D)

constructs of AT-MSCs into osteochondral defects [21].
This report also contains a novel strategy for scaffold-freecell implantation.
Previous studies indicated that scaffolds composed ofmaterials such as collagen and hyaluronic acid could be
useful for promoting cell adhesion, proliferation, andchondrogenic differentiation [22,23], and bone regeneration using AT-MSCs seeded into hydroxyapatite has
also been investigated [24]. However, artificial materials
may induce xenobiotic reactions through immune reactions in the tissue [25].
In many previous studies, bone and cartilage regeneration through various cell therapies has been evaluatedin the knee joint of rabbits [26-30]. However, to obtainmeaningful results that are appropriate for extrapolatingbone and cartilage regeneration to human OA, we expect that pigs will provide a more appropriate animalmodel than rabbits. Microminipigs (MMPigs) have similar behavior patterns to human daily life, as they spend
time standing and walking in the daytime and sleeping
at night [31,32]. In contrast, rabbits usually sit in cages.
This study was designed to evaluate the regeneration ofarticular cartilage and subchondral bone using 3D constructs of autologous AT-MSCs in MMPigs.

Materials and methods
Animals

Two MMPigs (Fuji Micra, Shizuoka, Japan), designatedanimal no. 1 (male) and animal no. 2 (female), were usedin this study. Their body weights and ages were 13.8 kg
and 25 months, and 14.6 kg and 23 months, respectively.
All procedures in this study were approved by the Animal
Care and Use Committee of Kagoshima University(Approval No. A11037). Ten to fifteen grams of cervicalAT per animal was aseptically obtained under generalanesthesia.
Isolation and expansion of AT-MSCs

The AT samples were minced and digested for 90 min in
phosphate-buffered saline (PBS) containing 0.1% collagenase (Collagenase Type I; Worthington Biochemical,
Lakewood, NJ). The digested cell suspensions were filteredthrough a 70-μm-pore-diameter membrane (Cell Strainer;
BD, Franklin Lakes, NJ) and centrifuged at 160 × g for
5 min at room temperature. After decanting the supernatant, the pellet was resuspended with PBS and centrifuged. The supernatant was removed, and the pellet was
resuspended and plated on a 150-cm2 culture dish (Tissue
Culture Dish φ 150; TPP, Trasadingen, Switzerland) incomplete culture medium (CCM): Dulbecco’s modified
Eagle’s medium (DMEM; Life Technologies, Carlsbad,CA) containing 10% fetal bovine serum (FBS; Thermo
Fisher Scientific, Waltham, MA) and 1% antibioticantifungal preparation (100 U/ml penicillin G, 100 μg/ml

Murata et al. Journal of Orthopaedic Surgery and Research (2015) 10:35

streptomycin, 0.25 μg/ml amphotericin B; AntibioticAntimycotic; Life Technologies). Following incubation at
37 °C under 5% CO2 for 7 days, the cells adhering to thebottom of the dish were washed with PBS and cultured in
CCM. The medium was changed on day 7 at passage 0.
At day 10, the cells were harvested with 0.25% trypsin and
1 mM EDTA (Trypsin-EDTA; Life Technologies) diluted
by adding five volumes of PBS and centrifuged. Afterdecanting the supernatant, the pellet was rinsed with
CCM, and the cells were replated at 5 × 105 cells per
150-cm2 dish and cultured for 6 days. The medium waschanged every 3 days for 6 days during passage 1. This
serial process of passaging was repeated until the cells
were required for analysis and construct creation. The
cells were used for creating the constructs at passage 4.
Immunological surface markers and multipotency of thecells were analyzed at passage 5.

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Genetic and molecular specificity of AT-MSCs

Ten thousand cells were used to analyze the specific gene
expressions in MSCs. Total RNA from the cells was prepared with an RNA isolation kit (MirVana miRNA Isolation
Kit; Life Technologies), according to the manufacturer’s instructions. The isolated RNA was converted to cDNA andamplified with a TAKARA RT-PCR system (PCR Thermal
Cycler MP; Takara Bio, Otsu, Japan) and RT-PCR kit(ReverTra Dash; Toyobo, Osaka, Japan). Specific PCRprimers were used to amplify octamer-binding transcription
factor 4 (OCT-4), sex-determining region Y box 2 (SOX-2),
Krüppel-like factor 4 (KLF-4), cellular myelocytomatosis
oncogene (C-MYC), and homeobox protein NANOG(NANOG) as premature marker genes. The conditions andexpected sizes of the products are summarized in Table 1.
Ten thousand cells were resuspended in 500 μl of staining
buffer (SB; PBS containing 1% FBS) and incubated forTable 1 List of PCR primers
Marker

GeneSequence (forward/reverse)

Ann. temp. (°C)

Fragment (bp)Premature

OCT-45′-GTCGCCAGAAGGGCAAAC-3′

57.0

157

59.0

85

59.0

119

59.0

613

55.1

184

56.0

131

68.0

204

57.5

187

57.5

119

65.5

194

59.8

238

59.8

167

60.0

450

5′-CAGGGTGGTGAAGTGAGGG-3′SOX-25′-CCCTGCAGTACAACTCCATGAC-3′5′-GGTGCCCTGCTGCGAGTA-3′

KLF-45′-CGGCAAAACCTACACGAAGAGT-3′5′-AGTTCATCTGAGCGGGCAAAT-3′

NANOG

5′-CTTATTCAGGACAGCCCTGATTCTTC-3′
5′-AAGACGGCCTCCAAATCACTG-3′C-MYC

5′-GGATTCCGCCTCGTT-3′5′-TCTCCAAGCATCACTCG-3′

Osteogenic

ALP

5′-ATGAGCTCAACCGGAACAA-3′5′-GTGCCCATGGTCAATCCT-3′

OC5′-TCAACCCCGACTGCGACGAG-3′5′-TTGGAGCAGCTGGGATGATGG-3′

ON

5′-TCCGGATCTTTCCTTTGCTTTCTA-3′5′-CCTTCACATCGTGGCAAGAGTTTG-3′

Chondrogenic

SOX-95′-CCGGTGCGCGTCAAC-3′5′-TGCAGGTGCGGGTACTGAT-3′

AGG

5′-TTCCCTGAGGCCGAGAAC-3′5′-GGGCGGTAATGGAACACAAC-3′

Adipogenic

PPAR-γ25′-GCGCCCTGGCAAAGCACT-3′5′-TCCACGGAGCGAAACTGACAC-3′AP25′-GGCCAAACCCAACCTGA-3′5′-GGGCGCCTCCATCTAAG-3′

Housekeeping

GAPDH

5′-ACCACAGTCCATGCCATCAC-3′5′-TCCACCACCCTGTTGCTGTA-3′

OCT-4 octamer-binding transcription factor 4, SOX-2 sex-determining region Y box 2; KLF-4 Krüppel-like factor 4, NANOG, homeobox protein NANOG, C-MYC, cellularmyelocytomatosis oncogene, ALP alkaline phosphatase, OC osteocalcin, ON osteonectin, SOX-9 sex-determining region Y-box 9, AGG aggrecan, PPAR-γ2 peroxisome
proliferator-activated receptor γ2, AP2 adipocyte fatty acid-binding protein 2, GAPDH glyceraldehyde-3-phosphate dehydrogenase.

Murata et al. Journal of Orthopaedic Surgery and Research (2015) 10:35

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30 min at 4 °C with 20 μg/ml FITC-conjugated antibodies
against CD34 (BD), CD45 (BD), CD90 (BD), or CD105(Abcam, Cambridge, UK). Non-specific FITC-conjugatedmouse immunoglobulin G1κ (BD) was used as a negative
control. The characteristics of the antibodies are listed inTable 2. The FITC-labeled cells were washed with SB andresuspended in 500 μl of SB for fluorescence-activated cellsorting (FACS) analysis. Cell fluorescence was evaluated asa strong shift in the mean fluorescence intensity (MFI) on
flow cytometry using a FACSAria II instrument (BD). The
data were analyzed using FACSDiva software (BD).

genes were sex-determining region Y-box 9 (SOX-9) andaggrecan (AGG). The adipogenic marker genes were adipocyte fatty acid-binding protein 2 (AP2) and peroxisome
proliferator-activated receptor γ2 (PPAR-γ2). The reaction products were electrophoresed in a 2% agarose gel(Agarose XP; Wako Pure Chemical Industries, Osaka,
Japan), and the expressions of the specific genes were determined based on the expected sizes of the bands labeledwith SYBR Green (Takara Bio).
Production of calcium apatite crystals in the osteogenic
extracellular matrix was evaluated with alizarin red staining in the wells of culture plates. The chondrogenic cellpellets were fixed with 10% neutral buffered formalin(NBF), embedded in paraffin, and cut into 5-μm sections
using a microsection instrument. The sections were
stained with alcian blue to detect cartilage-specific proteoglycans. Adipocyte-specific intracellular lipids were
stained with oil red O.

Tri-lineage analysis

To investigate osteogenic differentiation, the AT-MSCs
were placed in six-well plates (6 Well Plate-N; Nest
Biotech, Wuxi, China) in CCM at an initial density of
5,000 cells/cm2. After 24 h of incubation, the medium was
replaced with osteogenic induction medium (Differentiation
Basal Medium—Osteogenic; Lonza, Walkersville, MD),
supplemented with 100 μM ascorbic acid, 10 mM βglycerophosphate, and 1 μM dexamethasone, for 2 weeks.
To investigate chondrogenic differentiation, AT-MSCs(5 × 105) were resuspended in a 15-ml culture tube
(SuperClear centrifuge tubes; Labcon, Petaluma, CA) in
500 μl of chondrogenic induction medium (Differentiation
Basal Medium—Chondrogenic, Lonza), supplemented
with 4.5 g/l D-glucose, 350 μM L-proline, 100 nM dexamethasone, and 0.02 g/l transforming growth factor beta
3. Chondrogenic differentiation was induced in pellet cultures for 2 weeks. Adipogenic differentiation began when
AT-MSCs reached a density of 5,000 cells/cm2 in six-wellplates in CCM. Following a 24-h preincubation, themedium was replaced with Adipogenic Induction Medium(Lonza), supplemented with 4.5 g/l D-glucose, 100 μM
indomethacin, 10 μg/ml insulin, 0.5 mM 3-isobutyl-1methylxanthine, and 1 μM dexamethasone, for 3 days for
induction of specific genes and molecules.
The PCR primers and conditions, and the expected sizesof the products are summarized in Table 1. The osteogenic
marker genes were osteocalcin (OC), osteonectin (ON),and alkaline phosphatase (ALP). The chondrogenic marker
Table 2 List of antibodies
Antibody Company Clone

Epitope

Dilution

CD34

BD

581

O-glycosylatedtransmembrane
glycoprotein

1:5

CD45

BD

2D1

T200 family

1:2.5

CD90

BD

5E10

N-glycosylated GPI-linked
membrane glycoprotein

1:10

CD105

Abcam

MEM229Disulfide-linked
glycoprotein homodimer

1:20

IsotypeBD

MOPC-21 (Not confirmed)

1:10

Preparation and implantation of 3D constructs of AT-MSCs

At least 4 × 107 AT-MSCs were used to produce eachautologous construct. The cells were inoculated into
eight 96-well plates (Sumitomo Bakelite, Tokyo, Japan)
with 5 × 104 cells/well. After undisrupted incubation for
48 h, the cells formed spheroids with a diameter of about
700 μm in the bottom of the wells. About 760 spheroids
were placed in a cylindrical mold and incubated in CCM
until implantation (7 days). When the mold was carefully
removed, a columnar construct of 4 mm in diameter and
6 mm in height appeared and was used for autologous implantation (Figure 1A). The general outline of this methodof construction has already been reported [21,33].
The implant surgery was performed under generalanesthesia using oxygen and isoflurane inhalation following premedication with sedatives and analgesics. Bothfemoropatellar joints were incised from the outside, andthe femoral trochlear groove was exposed. Using a surgicaltrephine with an outer diameter of 4 mm, the articularcartilage and subchondral bone were drilled to a depth of
6 mm at the center of the groove. After removing a column of cartilage and bone, a cylindrical osteochondral defect was created in each groove (Figure 1B). A columnar
construct (4 mm in diameter and 6 mm in height) composed of spheroids of AT-MSCs was autografted into the
osteochondral defect in the right hind limb (Figure 1C),
while no graft was implanted into the defect in the left
limb (control defect, Figure 1B).
Assessment of osteochondral defects

Postoperatively, the implants and osteochondral defects
were followed up every month for 6 months in animal no.
1 and 12 months in animal no. 2 using computed tomography (CT) scans of both stifles. For assessment, longitudinal section images were obtained at the maximum

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Figure 1 Surgical procedure. A columnar construct (4 mm in diameter and 6 mm in height) for the implantation (A). A cylindrical osteochondral
defect in each groove before implantation (B). The construct composed of about 760 spheroids of AT-MSCs was autografted into the osteochondral
defect in the right hind limb (C). Nothing was implanted into the left limbs (control defects; B).

diameter in lateral views of the cylindrical defect and the
maximum diameters of the radiolucent area in the images
were evaluated at 0.5-mm intervals between 0 and 9 mm.
Animal no. 1 was euthanized at 6 months after surgery,and animal no. 2 was euthanized at 12 months after surgery. The macroscopic findings were scored with theInternational Cartilage Repair Society (ICRS) gross grading scale (Table 3). Both distal femurs were fixed in 10%

NBF for 1 week and then longitudinally sectioned parallelto the trochlear groove. The tissue was decalcified withformic acid for 1 week and embedded in paraffin. Serial
sections (3-μm thickness) were placed on glass slides
and evaluated by Masson’s trichrome staining, alcian
blue staining, and immunohistochemistry using specificantibodies against collagen type II (Col-II; 1:100 dilution; Daiichi Fine Chemicals, Takaoka, Japan) and an

Table 3 ICRS gross grading scale
FeatureCoverage

Neocartilage color

Defect margins

Surface

Average (0–4)

Score>75% fill4

50%–75% fill3

25%–50% fill2

<25% full1

No fill0

Normal4

25% yellow/brown

3

50% yellow/brown

2

75% yellow/brown

1

100% yellow/brown

0

Invisible

425% circumference visible

3

50% circumference visible

2

75% circumference visible

1

Entire circumference visible0

Smooth/level with normal

4

Smooth but raised3

Irregular 25%–50%2

Irregular 50%–75%

1

Irregular >75%

0Animal no. 1

Animal no. 2

Control site

Implanted site

Control site

Implanted site

2

3

4

4

1

2

3

4

1

2

3

3

0

2

3

3

1.0

2.25

3.25

3.5

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Avidin-Biotin Enzyme Complex system (VECTASTAINABC Standard Kit; Vector Laboratories, Southfield, MI).
The histopathologic findings were scored with the ICRS
histological grading scale (Table 4).

AGG (Figure 4B). Histological observation of the cell pellets showed a hyaline cartilage-like structure that was
positively stained with alcian blue (Figure 2C). Adipogenic
induction of the AT-MSCs resulted in adipocyte-like flattened cells with small lipid vesicles that were positively
stained with oil red O (Figure 2D). RT-PCR revealed significant increases in adipogenic marker gene expressions
such as AP2 and PPAR-γ2 (Figure 4B).

Results
Genetic and molecular characteristics and tri-lineage potentialof AT-MSCs

Porcine AT-MSCs adhering to the bottom of the culture
dish were spindle-shaped and proliferated well (Figure 2A),
reaching over 1 × 106 and 1 × 107 cells at passage 3 andpassage 4, respectively. A strong shift in MFI on flow cytometry was detected with antibodies against CD90 andCD105 (Figure 3A, B), while no signals were detected with
antibodies against CD34 and CD45 (Figure 3C, D). The
genetic markers of OCT-4, SOX-2, KLF-4, C-MYC, and
NANOG were all positive (Figure 4A). Following osteogenic induction, AT-MSCs aggregated and contracted toform colonies (Figure 2B), and expressions of specificmarker genes, including ALP, OC, and ON, were detected(Figure 4B). These cells also showed appropriate characteristics of the stroma, including staining with alizarin
red, indicating the presence of calcium apatite crystals(Figure 2B). Reverse transcription PCR (RT-PCR) of ATMSCs placed in chondrogenic induction medium revealed
the expressions of marker genes, including SOX-9 andCT images

The reduction in the subchondral radiolucent area of
the implanted site became more dramatic at 2 or
3 months after surgery compared with the control site in
the both animals (Figure 5). CT images at 6 months
after surgery for animal no. 1 are shown in Figure 5A. A
radiopaque area emerged from the boundary between
the bone and the implant and increased more steadily
upward and inward for the implanted defect (the rightfemur) as time passed after surgery, compared with thecontrol site. The radiolucent area of the implant diminished in a stepwise manner and then degraded to adiameter of 1 mm by 5 months after surgery. CT images
at 12 months after surgery for animal no. 2 are shown in
Figure 5B. A radiopaque area of the implant emerged in
the same manner as in animal no. 1, gradually progressed, and then filled the entire osteochondral defect

Table 4 ICRS histological grading scale
FeatureScore Animal no. 1

Animal no. 2

Control site Implanted site Control site Implanted site
Surface

MatrixCell distribution

Viability of cell population

Subchondral bone

Smooth/continuous

3

Discontinuities/irregularity

0

Hyaline3

Mixture; hyaline/fibrocartilage

2

Fibrocartilage

1

Fibrous tissue0

Columnar3

Mixed/columnar clusters

2

Clusters

1

Individual cells/disorganized0

Predominantly viable3

Partially viable1<10% viable0

Normal3

Increased remodeling

2

Bone necrosis/granulation tissue1

Detached/fracture/callus at base0

Cartilage mineralization (calcified cartilage) Normal

3

0

3

3

3

0

1

1

2

0

0

0

2

3

3

3

3

1

2

0

2

0

3

3

3

0.67

2

1.67

2.5

Abnormal/inappropriate location 0
Average (0–3)

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Figure 2 Representative images of special staining and RT-PCR results of tri-lineage differentiation in AT-MSCs. AT-MSCs adhering to thebottom of the culture dish were spindle-shaped (A). Following 2 weeks of osteogenic induction, MSCs also showed characteristics of the stroma,
including staining with alizarin red, indicating the presence of calcium apatite crystals (B). Observation of the cell pellets that were induced bychondrogenic induction medium for 2 weeks showed a cartilage-like structure that was positively stained with alcian blue (C). Adipogenic induction of
the MSCs resulted in adipocyte-like flattened cells with small lipid vesicles that stained positively with oil red O (D).

at 12 months after surgery. On the other hand, in thecontrol site, a radiopaque area emerged in the shallow
layer, but bone formation was not completed in the deep
layer. The maximum diameter of the radiolucent area in
the implanted site diminished in a stepwise manner andbecame 0 mm at 12 months after surgery. The control
site remained at a diameter of 2.5 mm.
Macroscopic appearance and histopathology of theosteochondral defects

Macroscopic examination of animal no. 1 revealed thatthe surface of the implanted defect was covered with
abundant cartilaginous white tissue (Figure 6A), while cartilaginous tissue was scarce and the surface was depressedin the control site (Figure 6B). Similarly, in animal no. 2,
the surface was quite uniformly covered with abundantcartilaginous white tissues and the boundary to the surrounding normal cartilage was unclear in the implantedsite (Figure 6C), compared with the findings at the control
site (Figure 6D). The average macroscopic scores for the
implanted site were higher than those for the control site
in animal no. 1, while the differences between the scoresfor the implanted site and the control site were decreased
in animal no. 2 (Table 3).
Histopathological sections of animal no. 1 at 6 months
after surgery showed that thickened fibrocartilage had developed over the subchondral bone that was regeneratingin the implanted site (Figure 7A, B). The surface of the
cartilage was smooth, and the boundary with the surrounding normal cartilage was obscure at the implantedsite (Figure 7A). Meanwhile, the surface was collapsed andirregular at the control site (Figure 7C, D). The fibrocartilage showed more intense alcian blue staining and Col-II
immunostaining at the implanted site (Figure 7E, F) compared with the control site (Figure 7G, H). In animal no. 2
at 12 months after surgery, partially thickened fibrocartilage was mounted on developed subchondral bone at the
implanted site (Figure 7I, J). The surface of the cartilage
was smooth, and the boundary with the surrounding normal cartilage was obscure, although small areas of endochondral ossification persisted at the center, and small
amounts of AT had differentiated at the bottom part ofthe site (Figure 7I). Subchondral bone was symmetricallyreconstructed in the defect and was covered by a mixed
matrix of hyaline cartilage and fibrocartilage, in whichclusters and columnar clusters of cells were observed(Figure 7J). In the control site, fibrocartilage had immediately covered the defect, but the subchondral ossification
was poor (Figure 7K, L). The hyaline cartilage showed more
intense and uniform alcian blue staining and Col-II immunostaining at the implanted site (Figure 7M, N) comparedwith the control site (Figure 7O, P). The averages of histologic scores for the implanted site were distinctly higher
than those for the control site in both animals (Table 4).

Figure 3 Flow cytometry results of immunological markers in AT-MSCs. A strong shift in MFI was detected with antibodies against CD90 (A)and CD105 (B), whereas no signal reaction was detected with antibodies against CD34 (C) and CD45 (D).

Murata et al. Journal of Orthopaedic Surgery and Research (2015) 10:35

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Figure 4 RT-PCR results of gene expression in AT-MSCs. Premature gene expression in AT-MSCs (A) and specific marker gene expression in
AT-MSCs induced by tri-lineage differentiation medium (B) were confirmed.

Discussion
Human AT-MSCs have been shown to be positive for
CD90, which suppresses the cancerization of stem cells[34], and CD105, which is associated with cellular responses to blood vessel formation and TGF-β1 [34]. The
porcine AT-derived and spindle-shaped cells adhering to
the bottom of the culture dish in the present study werestrongly positive for CD90 and CD105. CD34, which is

involved in cell adhesion and is expressed in hematopoietic
stem cells [34], and CD45, which activates T and B
lymphocyte receptors in hematopoietic cells [34], wereboth negative in the porcine AT-derived cells. Because human hematopoietic cells, but not human MSCs, were positive for these molecules [35,36], the porcine AT-derived
cells may not be contaminated with hematopoietic cells[37]. Genetic markers specific for human MSCs, such as

Figure 5 CT assessment of osteochondral defects. The upper image shows one cross section of the multiplanar reconstruction images 1, 3, and6 months after the surgery in animal no. 1 (A). The lower image shows one cross section of the multiplanar reconstruction images 1, 6, 12 months after
the surgery in animal no. 2 (B).

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Figure 6 Macroscopic findings of the surface of the implanted and control sites. In animal no. 1, the surface of the implanted defect wascovered with the abundant cartilaginous white tissues (A), whereas the cartilaginous tissue was scarce and the surface was depressed in the control
site (B). In animal no. 2, the surface was more uniformly covered with abundant cartilaginous white tissues and the boundary to the surrounding
normal cartilage was unclear in the implanted site (C), comparing to those of the control site (D).

Figure 7 Histopathology of osteochondral defects using Masson’s trichrome, alcian blue, and immunohistochemical staining of type II
collagen. In animal no. 1, the articular surface was smooth and fibrocartilage developed on the subchondral bone at the implanted site (A, B, E,
F), whereas the surface was irregular and fibrous tissue lay over the subchondral bone at the control site (C, D, G, H). At the implanted site inanimal no. 2, the subchondral bone was symmetrically reconstructed and was covered by matrix including hyaline cartilage, which was suggested by
the clusters (arrowhead) and columnar clusters (arrow) of cells (I, J, M, N). On the other hand, smooth and continuous surface was restored due tofibrocartilage formation, but subchondral bone was absent in the bottom half of the defect, at the control site in animal no. 2 (K, L, O, P). Black dottedlines indicate the areas of osteochondral defects immediately after the surgery. Masson’s trichrome staining sections (B, D, J, L) were enlarged from red
dotted square in the images A, C, I, and K, respectively. The insert images in sections B, D, J, and L were enlarged from white dotted square in images B,
D, J, and L, respectively. The bars in the insert images indicate 50 μm.

Murata et al. Journal of Orthopaedic Surgery and Research (2015) 10:35

Page 10 of 12

SOX-2, OCT-4, NANOG [38], KLF-4, and C-MYC [39],were detected in the porcine cells by RT-PCR [40]. Moreover, the osteogenic, chondrogenic, and adipogenic potential of the cells was confirmed, and we therefore definedthem as porcine AT-MSCs.
In accordance with a previously described procedure[21,33], we constructed scaffold-free 3D implants (diameter:
4 mm; height: 6 mm) composed of 760 spheroids each containing 5 × 104 autologous AT-MSCs. The cross-sectional
CT images obtained at 6 and 12 months after implantationin animal no. 1 and animal no. 2, respectively, may mirror
the histology because the localization, size, and shape of theradiolucent and radiopaque areas entirely correspondedwith those of the fibrocartilage and regenerated bone. To
further discriminate between cartilaginous and fibrous tissues in the radiolucent area, magnetic resonance imaging
should be used.
The higher average macroscopic scores may suggest
better improvement in superficial features at the implanted site, compared with the control site (Table 3).
However, the differences in the average scores between
the control and implanted sites were lower in animal no.
2 (euthanized at 12 months after surgery) than in animal
no. 1 (euthanized at 6 months after surgery). All fourfeatures in the ICRS gross grading scale system were improved at the implanted site compared with the control
defect site in animal no. 1, whereas a difference in neocartilage color only was seen between the two sites inanimal no. 2. The results in animal no. 2 were not consistent with a previous study using rabbits, in which amore degraded macroscopic appearance of the control
defect (diameter: 4.8 mm; depth: 5 mm) was observed at
12 months after implantation [21]. Based on the results,
we speculate that the superficial features may improve
spontaneously from 6 to 12 months after surgery for thissize of osteochondral defect (diameter: 4 mm; depth:6 mm) in MMPigs. To discriminate the superficial features caused by spontaneous repair from those caused
by MSC-based regeneration in this size of defect, further
evaluation of the pathology at 6 months after surgery
will be appropriate in MMPigs. Other studies are needed
to determine methods for repairing osteochondral defects with larger diameters and depths, which could
never repair by themselves (as shown in the Additional
files 1 and 2).

We also obtained higher average histologic scores at
the implanted sites in both animals, which may indicate
desirable osteochondral recovery compared with thecontrol site (Table 4). As summarized in Table 5, regarding the histological features in animal no. 1, a smoothand continuous surface was restored by thickened fibrocartilage at the implanted site, whereas the surface wascollapsed and irregular at the control site. Fibrocartilage
formation and endochondral ossification during theprocess of MSC-based regeneration were present at the
implanted site, compared with fibrous granulation
matrix and inadequate bone formation in the control defect. On the other hand, in animal no. 2, a smooth andcontinuous articular surface was restored through cartilage formation at both sites, but subchondral bone formation was distinctly more satisfactory at the implantedsite than at the control site, in which the trabecular pattern was completely absent (bone was detached) in thebottom half of the defect. Subchondral bone was coveredby a mixed matrix of hyaline cartilage and fibrocartilage at
the implanted site, while fibrocartilage had immediately
covered the defect at the control site. These findings weresimilar to data reported previously reported in rabbits [21]and may suggest transformation of fibrocartilage into hyaline cartilage during the process of MSC-based osteocartilage regeneration. Because neither hyaline cartilage nor cellclusters were seen in the implanted defect site in animal
no. 1, transformation of fibrocartilage into hyaline cartilage
may begin between 6 and 12 months after implantation.
However, more time may be required to regenerate pure,high-quality hyaline cartilage as well as complete subchondral regeneration in the implanted defect.
Consistent with a previous study on rabbits [21], we report here the successful outcome of osteochondral regeneration with scaffold-free AT-MSC constructs in MMPigs.
Although further studies will be required, we conclude that
implantation of a scaffold-free 3D construct of AT-MSCs
into an osteochondral defect can regenerate the original
structure of the bone and cartilage.

Conclusions
This pilot study suggests that implantation of a scaffoldfree 3D construct of AT-MSCs into an osteochondral defect can induce regeneration of the original structure of the
cartilage and subchondral bone over the course of 1 year.

Table 5 Summary of histological features
Animal no. 1
Cartilage

Animal no. 2

Control site

Implanted site

Control site

Implanted site

Surface

Irregularity

SmoothSmoothSmoothMatrixFibrous tissue

Fibrocartilage

Fibrocartilage

Mixture; hyaline/fibrocartilage (transformation)

Granulation tissue

Increased remodeling

Detached (in the bottom
half of the defect)

Increased remodeling (endochondral
ossification)

Subchondral boneMurata et al. Journal of Orthopaedic Surgery and Research (2015) 10:35

Additional files
Additional file 1: Surgical procedure, CT images, macroscopic
findings of the articular surface, and histopathology of osteochondral
defects in animal no. 3. Figure S1. surgical procedure: A columnar
construct (6 mm in diameter and 8 mm in height) composed of about 1,150
spheroids of AT-MSCs (A). An elliptic cylindrical osteochondral defect in eachgroove (B). Two constructs were autografted into the defect of the right hindlimb (C). No implantation was in the left limb (B). Figure S2. CT images after
the surgery: One cross section of the multi-planar reconstruction images 1, 6,
and 12 months after the surgery in animal no. 3. In the implanted site, theradiopaque area gradually progressed and filled throughout the osteochondral
defect after 12 months. However, in the control site, the spread of theradiopaque area was limited in the shallow layer, and no bone formation wasin the deep layer. Figure S3. macroscopic findings of the articular surface: The
surface was completely covered with abundant cartilaginous white tissues.
The boundary to the surrounding normal cartilage was not different between
the implanted site (A) and the control site (B). Figure S4. histopathology ofosteochondral defects: At the implanted site, the restored subchondral bone
was covered by mixture of hyaline/fibrocartilage, in which the clusters(arrowhead) and columnar clusters (arrow) of the cells were seen (A, B, C, D).
In the control site, the surface was irregular, and the large fibrous tissue waspresented in the subchondral (area with no bone at the bottom half of thedefect (E, F, G, H)). Black dotted lines indicate the areas of osteochondral
defects immediately after the surgery. Images B and F are high-power fieldsof the red dotted square in images A and E, respectively. The small images
in sections B and F are high-power fields of white dotted squares in therespective images. The bars in the small images indicate 50 μm.
Additional file 2: ICRS gross grading scale and histological grading
scale in animal no. 3. Table S1. ICRS gross grading scale. Table S2.
ICRS histological grading scale.
Abbreviations
AGG: Aggrecan; ALP: Alkaline phosphatase; AP2: Adipocyte fatty acid-binding
protein 2; AT: Adipose tissue; AT-MSCs: Adipose tissue-derived mesenchymal stem
cells; BM: Bone marrow; BM-MSCs: Bone marrow-derived mesenchymal stem cells;
CCM: Complete culture medium; C-MYC: Cellular myelocytomatosis oncogene;
Col-II: Collagen type II; CT: Computed tomography; DMEM: Dulbecco’s modified
Eagle’s medium; EDTA: Ethylenediaminetetraacetate; FACS: Fluorescence-activatedcell sorting; FBS: Fetal bovine serum; KLF-4: Krüppel-like factor 4; MFI: Mean
fluorescence intensity; MMPigs: Microminipigs; MSCs: Mesenchymal stem
cells; NANOG: Homeobox protein NANOG; NBF: Neutral buffered formalin;
TGF-β3: Transforming growth factor beta 3; OA: Osteoarthritis;
OC: Osteocalcin; OCT-4: Octamer-binding transcription factor 4;
ON: Osteonectin; PBS: Phosphate-buffered saline; PPAR-γ2: Peroxisome
proliferator-activated receptor γ2; SB: Staining buffer; SOX-2: Sex-determining
region Y box 2; SOX-9: Sex-determining region Y-box 9.
Competing interests
KN is a co-founder of Cyfuse Biomedical K.K. TT is a full-time employee of thesame company. The other authors have no commercial, proprietary, or financial
interest in the products or companies described in this article.
Authors’ contributions
DM isolated and expanded AT-MSCs and performed genetic and molecularanalysis. ST assessed the osteochondral defects by CT. TT prepared 3D constructs
of AT-MSCs. HK assessed histopathologically. NM supervised histopathologicalassessment. MF implanted 3D constructs of AT-MSCs. KN conceived this study
and interpreted the data. KM designed this experiment and interpreted thedata. All authors approved the final version of the manuscript.
Acknowledgements
This study was supported by Japan Society for the Promotion of Science(grant no. 23380185 to KM). The study sponsor had no involvement in thisstudy design, collection, analysis, or interpretation of data; in the writing of
the manuscript; or in the decision to submit the manuscript for publication.
Author details
1
Veterinary Surgery, Department of Veterinary Clinical Science, Joint Faculty of
Veterinary Medicine, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima

Page 11 of 12

890-0065, Japan. 2Veterinary Teaching Hospital, Joint Faculty of Veterinary
Medicine, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima 890-0065,
Japan. 3Cyfuse Biomedical K.K., 1-1 Maidashi 3-chome, Higashi-ku, Fukuoka
812-8582, Japan. 4Veterinary Pathology, Department of Pathological andPreventive Sciences, Joint Faculty of Veterinary Medicine, Kagoshima University,
21-24 Korimoto 1-chome, Kagoshima 890-0065, Japan. 5Department of Advanced
Technology Fusion, Graduate School of Science and Engineering, Saga University,
Honjyo 1-chome, Honjyo-cho, Saga 840-8502, Japan.
Received: 25 August 2014 Accepted: 25 February 2015

References
1. Muraki S, Oka H, Akune T, Mabuchi A, En-Yo Y, Yoshida M, et al. Prevalenceof radiographic knee osteoarthritis and its association with knee pain in theelderly of Japanese population-based cohorts: the ROAD study. Osteoarthr
Cartil. 2009;17:1137–43.
2. Gelber AC, Hochberg MC, Mead LA, Wang NY, Wigley FM, Klag MJ. Joint
injury in young adults and risk for subsequent knee and hip osteoarthritis.
Ann Intern Med. 2000;133:321–8.
3. Fernandes JC, Martel-Pelletier J, Pelletier JP. The role of cytokines in osteoarthritis
pathophysiology. Biorheology. 2002;39:237–46.
4. Ding C, Cicuttini F, Scott F, Cooley H, Boon C, Jones G. Natural history ofknee cartilage defects and factors affecting change. Arch Intern Med.
2006;166:651–8.
5. Muraki S, Akune T, Oka H, Mabuchi A, En-Yo Y, Yoshida M, et al. Associationof occupational activity with radiographic knee osteoarthritis and lumbar
spondylosis in elderly patients of population-based cohorts: a large-scale
population-based study. Arthritis Rheum. 2009;61:779–86.
6. Mankin HJ. The response of articular cartilage to mechanical injury. J Bone
Joint Surg Am. 1982;64:460–6.
7. Lane JG, Massie JB, Ball ST. Follow-up of osteochondral plug transfers in agoat model: a 6-month study. Am J Sports Med. 2004;32:1440–50.
8. Szerb I, Hangody L, Duska Z, Kaposi NP. Mosaicplasty: long-term follow-up.
Bull Hosp Jt Dis. 2005;63:54–62.
9. Bentley G, Biant LC, Carrington RW, Akmal M, Goldberg A, Williams AM,
et al. A prospective, randomised comparison of autologous chondrocyte
implantation versus mosaicplasty for osteochondral defects in the knee.
J Bone Joint Surg (Br). 2003;85:223–30.
10. Fujisato T, Sajiki T, Liu Q, Ikada Y. Effect of basic fibroblast growth factor on
cartilage regeneration in chondrocyte-seeded collagen sponge scaffold.
Biomaterials. 1996;17:155–62.
11. Funayama A, Niki Y, Matsumoto H, Maeno S, Yatabe T, Morioka H, et al.
Repair of full-thickness articular cartilage defects using injectable type II collagengel embedded with cultured chondrocytes in a rabbit model. J Orthop Sci.
2008;13:225–32.
12. Diaz-Romero J, Gaillard JP, Grogan SP, Nesic D, Trub T, Mainil-Varlet P.
Immunophenotypic analysis of human articular chondrocytes: changes insurface markers associated with cell expansion in monolayer culture. J Cell
Physiol. 2005;202:731–42.
13. Tatebe M, Nakamura R, Kagami H, Okada K, Ueda M. Differentiation oftransplanted mesenchymal stem cells in a large osteochondral defect inrabbit. Cytotherapy. 2005;7:520–30.
14. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al.
Multilineage potential of adult human mesenchymal stem cells. Science.
1999;284:143–7.
15. Zuk PA, Zhu M, Ashjian P, De-Ugarte DA, Huang JI, Mizuno H, et al. Human
adipose tissue is a source of multipotent stem cells. Mol Bio Cell.
2002;13:4279–95.
16. Gurevitch O, Slavin S, Resnick I, Khitrin S, Feldman A. Mesenchymal
progenitor cells in red and yellow bone marrow. Folia Biol. 2009;55:27–34.
17. Stolzing A, Jones E, McGonagle D, Scutt A. Age-related changes in humanbone marrow-derived mesenchymal stem cells: consequences for cell therapies.
Mech Ageing Dev. 2008;129:163–73.
18. Asumda FZ, Chase PB. Age-related changes in rat bone-marrow mesenchymal
stem cell plasticity. BMC Cell Biol. 2011;12:44.
19. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al. Multilineage
cells from human adipose tissue: implications for cell-based therapies. TissueEng. 2001;7:211–28.
20. Nakamura T, Sekiya I, Muneta T, Hatsushika D, Horie M, Tsuji K, et al.
Arthroscopic, histological and MRI analyses of cartilage repair after aMurata et al. Journal of Orthopaedic Surgery and Research (2015) 10:35

Page 12 of 12

minimally invasive method of transplantation of allogeneic synovial
mesenchymal stromal cells into cartilage defects in pigs. Cytotherapy.
2012;14:327–38.
Ishihara K, Nakayama K, Akieda S, Matsuda S, Iwamoto Y. Simultaneous
regeneration of full-thickness cartilage and subchondral bone defects
in vivo using a three-dimensional scaffold-free autologous construct derived
from high-density bone marrow-derived mesenchymal stem cells. J Orthop
Surg Res. 2014;9:98.
Lu Z, Doulabi BZ, Huang C, Bank RA, Helder MN. Collagen type II enhanceschondrogenesis in adipose tissue-derived stem cells by affecting cell shape.
Tissue Eng Part A. 2010;16:81–90.
Yoon IS, Chung CW, Sung JH, Cho HJ, Kim JS, Shim WS, et al. Proliferationand chondrogenic differentiation of human adipose-derived mesenchymal
stem cells in porous hyaluronic acid scaffold. J Biosci Bioeng.
2011;112:402–8.
Arrigoni E, De-Girolamo L, Di-Giancamillo A, Stanco D, Dellavia C, Carnelli D,
et al. Adipose-derived stem cells and rabbit bone regeneration: histomorphometric,
immunohistochemical and mechanical characterization. J Orthop Sci.
2013;18:331–9.
Park CW, Rhee YS, Park SH, Danh SD, Ahn SH, Chi SC, et al. In vitro/in vivo
evaluation of NCDS-micro-fabricated biodegradable implant. Arch Pharm
Res. 2010;33:427–32.
Koga H, Shimaya M, Muneta T, Nimura A, Morito T, Hayashi M, et al. Local
adherent technique for transplanting mesenchymal stem cells as a potential
treatment of cartilage defect. Arthritis Res Ther.
2008;10:R84.
Dashtdar H, Rothan HA, Tay T, Ahmad RE, Ali R, Tay LX, et al. A preliminary
study comparing the use of allogenic chondrogenic pre-differentiated andundifferentiated mesenchymal stem cells for the repair of full thickness articularcartilage defects in rabbits. J Orthop Res.
2011;29:1336–42.
Lee JI, Sato M, Kim HW, Mochida J. Transplantation of scaffold-free spheroids
composed of synovium-derived cells and chondrocytes for the treatment ofcartilage defects of the knee. Eur Cell Mater. 2011;22:275–90.
Yoshioka T, Mishima H, Kaul Z, Ohyabu Y, Sakai S, Ochiai N, et al. Fate ofbone marrow mesenchymal stem cells following the allogeneictransplantation of cartilaginous aggregates into osteochondral defects ofrabbits. J Tissue Eng Regen Med. 2011;5:437–43.
Suzuki S, Muneta T, Tsuji K, Ichinose S, Makino H, Umezawa A, et al.
Properties and usefulness of aggregates of synovial mesenchymal stem cells
as a source for cartilage regeneration. Arthritis Res Ther.
2012;14:R136.
Kawaguchi H, Miyoshi N, Miura N, Fujiki M, Horiuchi M, Izumi Y, et al.
Microminipig, a non-rodent experimental animal optimized for life science
research: novel atherosclerosis model induced by high fat and cholesterol
diet. J Pharmacol Sci. 2011;115:115–21.
Takeishi K, Horiuchi M, Kawaguchi H, Deguchi Y, Izumi H, Arimura E, et al.
Acupuncture improves sleep conditions of minipigs representing diurnal
animals through an anatomically similar point to the acupoint (GV20)
effective for humans. Evid Based Complement Alternat Med.
2012;2012:472982.
Nakayama K. In vitro biofabrication of tissues and organs. In: Forgacs G, Sun
W, editors. Biofabrication: micro- and nano-fabrication, printing, patterningand assemblies. Oxford: William Andrew; 2013. p. 1–21.
Zola H, Swart B, Nicholson I, Voss E. Leukocyte and stromal cell molecules:
the CD markers. Hoboken: John Wiley & Sons Inc; 2007.
Chen J, Lu Z, Cheng D, Peng S, Wang H. Isolation and characterization of
porcine amniotic fluid-derived multipotent stem cells. PLoS One.
2011;6:e19964.
Guo KT, SchAfer R, Paul A, Gerber A, Ziemer G, Wendel HP. A newtechnique for the isolation and surface immobilization of mesenchymal
stem cells from whole bone marrow using high-specific DNA aptamers.
Stem Cells. 2006;10:2220–31.
Casado JG, Gomez-Mauricio G, Alvarez V, Mijares J, Tarazona R, Bernad A,
et al. Comparative phenotypic and molecular characterization of porcine
mesenchymal stem cells from different sources for translational studies in alarge animal model. Vet Immunol Immunopathol. 2012;147:104–12.
Riekstina U, Cakstina I, Parfejevs V, Hoogduijn M, Jankovskis G, Muiznieks I,
et al. Embryonic stem cell marker expression pattern in humanmesenchymal stem cells derived from bone marrow, adipose tissue, heartand dermis. Stem Cell Rev. 2009;5:378–86.

39. Windmolders S, De-Boeck A, Koninckx R, Daniëls A, De-Wever O, Bracke M,
et al. Mesenchymal stem cell secreted platelet derived growth factor exerts
a pro-migratory effect on resident cardiac atrial appendage stem cells. J Mol
Cell Cardiol. 2014;66:177–88.
40. Tang L, Yin Y, Zhou H, Song G, Fan A, Tang B, et al. Proliferative capacityand pluripotent characteristics of porcine adult stem cells derived from
adipose tissue and bone marrow. Cell Reprogram. 2012;14:342–52.

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22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.
35.

36.

37.

38.

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