Bone marrow derived mesenchymal stem cell (BM MSC) application in articular cartilage repair 2

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Adipogenic differentiation was induced by culturing 3×10
5
cells/cm
2
in
adipogenic medium consisting of high glucose DMEM supplemented with
10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco), 10% glutamax
(Gibco), 100 µg/ml insulin (Sigma), 500 µM 3-isobuthy-l-methylxanthine
(IBMX), 100 µM indomethacin (MW= 357.8 g) and 1 µM dexamethasone
(Sigma). The medium was changed every 3-4 days for three weeks.
Osteogenic differentiation was induced by culturing 6×10
4
cells/cm
2
in
osteogenic differentiation medium consisting of low glucose DMEM
supplemented by 10% FBS (Gibco), 100 U/ml penicillin, 100 µg/ml
streptomycin (Gibco), 10% L-Glutamine (Gibco), 50 µg/ml L-ascorbic acid 2-
phosphate sequimagnesium (Sigma), 100 µg/ml MEM sodium pyruvate
(Gibco), 0.1 µM dexamethasone (Sigma), and 100mM b-glycerophosphate.
Chondrogenic differentiation was induced by high-density pellet cell culture
system. 2.5×10
5
cells were centrifuged at 1,100 rpm for 5 minutes. The
aggregated cells in pellet were incubated with serum-free chondrogenic
differentiation medium consisting high glucose DMEM, 100 U/ml penicillin,
100 µg/ml streptomycin (Gibco), 10% L-Glutamine (Gibco), 50 µg/ml L-
ascorbic acid 2-phosphate sequimagnesium (Sigma), 100 µg/ml MEM sodium
pyruvate (Gibco), 40 µg/ml Proline (Sigma), 100 nM dexamethasone (Sigma),

ITS+Premix final concentration: 5.5 µg/ml transferring, 10 µg/ml bovine
insulin, 5 µg/ml sodium selenite, 4.7 µg/ml linoleic acid, and 500 µg/ml bovine
serum albumin)(BD Bioscience, Franklin Lakes, NJ), and 10 ng/ml TGF-β1
(R&D Systems, Minneapolis, MN). The medium was changed two to three
times a week.

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3.3.9 Histological evaluation
Adipogenic differentiation was determined using 0.3% oil red O stain for 15
minutes at room temperature to stain intracellular lipids, and counterstained
with hematoxylin.
Osteogenic differentiation was evaluated using Alizarin Red. Cells were
stained with the 2% Alizarin Red S solution (pH 4.1~4.3) for 5 minutes at
room temperature, and the reaction were observed microscopically. Cells
were washed with distilled water to remove the excess stains. Calcium
deposits in differentiated cells would produce red-orange stains.
Chondrogenic differentiation of pellet cultures was evaluated using Alcian blue
staining for sulphated proteoglycans and immunohistochemical staining for
collagen type II. The pellets were fixed with 10% formaldehyde overnight in
cold room (4°C), dehydrated by a series of titrated ethanol and embedded in
paraffin blocks. Pellets were sectioned at a thickness of 5 µm and transferred
to slides and incubated in 60°C for 1 hour. For histological staining, the
sections were rehydrated by series of graded ethanol. Samples were
incubated in 0.5% Alcian blue for 30 minutes at pH 1.0 to stain
glycosaminoglycans. To visualize the cytoplasm and the nucleus, sections
were counterstained with nuclear fast red for 5 minutes. For
immunohistochemical staining, sections of labeled and unlabeled pellets were
fixed on slides, and were pre-digested with pepsin (1 mg/ml in Tris–HCl, pH
2.0) for 30 minutes at room temperature, and incubated with the primary
antibody for 60 minutes at room temperature. Anti-collagen II (Chemicon,

1/500) and anti-collagen X (Quartett, 1/25) antibodies were used as primary

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antibodies. After washing steps, sections were incubated with biotinylated
goat anti-mouse for 30 minutes and after washing samples were incubated
with streptavidin peroxidase for 45 minutes at room temperature. Sections
were visualized using DAB chromogen and substrate. Then, sections were
counterstained with hematoxylin for 5 minutes. Negative control slides were
incubated with mouse serum IgG as a substitute for the primary antibody.
3.3.10 Animal model
Eight female mini-pigs (6-months-old, 12-18 kg) were used for this study. All
animals’ right knees used as experimental and left knees as control groups.
The experimental knee intra-articularly injected with 10
7
labeled MSCs mixed
in 1 ml hyaluronan (SYNVISC®), while 1 ml hyaluronan alone used for control
group.
3.3.11 Surgical procedure
The knee of an animal was opened through standard medial para-patellar
incision under general anesthesia (figure 3.1). A chondral defect of 6mm in
diameter 1-2 mm in depth was created in weight-bearing medial femoral
condyle. After washing the defect site to ensure that all the cartilage was
resected, the joint was closed and rested for one week. MSCs (10
7
cells)
labeled with 50µg/ml ferucarbotran (10pg/cell) were seeded in 1 ml
hyaluronan and used for intra-articular injection of the experimental knee and
1 ml hyaluronan alone was used for intra-articular injection of the control
knee. The lateral mid-patellar approach was used for these intra-articular
injections and local pressure on the injection site was performed to ensure

that the leakage did not occur. Moreover, to help the distribution of the cells in

52!
the joint, passive flexion/extension of the joint were performed. MRI scanning
was performed on the joint at 2- weekly interval for up to 6 weeks post-
operation. The animals were sacrificed at 6 weeks and histological analysis of
cartilage was correlated with MRI findings.
!
Figure 3-1. Mini-pigs as an animal model.
Mini-pig’s knees were opened under general anesthesia through standard
medial para-patellar incision (A, B) and a chondral defect of 6 mm in diameter
and 1-2 mm in depth (C) was created in weight-bearing of each medial
femoral condyle.
3.3.12 Preliminary MR imaging experiments
To optimize the imaging of mini-pig knee preliminary experiments were
performed to determine the scanning conditions. We imaged a normal knee
joint to find the best MR sequences so that we could visualize the cartilage in
a 1.5 Tesla (1.5T) clinical MR scanner (Signa, General Electrics (GE)). Proton
Density Fast Spin Echo with/without Fat Saturation (FSE PD, FSE PD FS),
2D/3D Gradient Echo (GRE), 3D Steady State (spoiled: SPGR (SPoiled
Gradient Recalled) and coherent: FIESTA (Fast Imaging Employing STeady
STtate Acquisition)), 2D MERGE (Multiple Echo Recombined Gradient Echo),

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SE MT (spin echo with magnetization transfer), and SE No MT (spin echo with
no magnetization transfer) sequences have been examined.
Moreover, in order to have a better picture of the MR images of the labeled
cells in the knee and to choose the best optimized MR sequences, we
performed a preliminary MRI of knee explants with chondral defects without /
with different fillings using the same scanner. Conditions include: blank defect

(no filling), defect with scaffold only (1% agarose) filling, and defects filled with
the agarose mixed with different amount of ferucarbotran (used as a
representative of different concentrations of the labeled cells, assuming 3 pg
Fe per cell).
3.3.13 MR imaging of live animals
The pig was wrapped to ensure it is immobilized, warm and secure with only
the knees to be imaged protruding (rear legs). The animal was in prone
position on the imaging table. A dual-surface coil was placed around the knee,
and secured by surgical tape. The animal was placed in the scanner and
images were acquired. MRI scanning was performed before cell injection,
immediately after injection and at 2 and 6 weeks post-injection. The optimized
MRI sequences were determined to be: 3D-FSE sequence: repetition time
(TR) 2500; echo time (TE) 14; echo train length (ETL) 8; matrix 328x256; field
of view (FOV) 8x8 cm; slice thickness (ST) 1mm; 3D-SPGR sequence: flip
angle (FA) 30°; TR 18; TE 3.4; matrix 320x256; FOV 8x8 cm; slice thickness
1mm; and 3D-GRE sequence: flip angle (FA) 20°; TE in phase; matrix
320x256; FOV 8x8 cm; slice thickness 1mm. The animals were sacrificed at 6
weeks and histological analysis of the repaired cartilage was correlated with

54!
MRI findings.
!
Figure 3-2. MR imaging of the mini-pigs' knee joint.
A dual-surface coil was placed around the knee and secured by surgical tape
(A). The mini-pigs were wrapped to ensure it is immobilized, warm and secure
with only the knees to be imaged protruding (rear legs)(B and C). The animal
was in prone position on the imaging table and MRI was performed in a
General Electrics (GE) Signa 1.5 Tesla (1.5T) MR scanner (D).
!

55!
3.3.14 Postmortem analysis
At 6-week post injection, animals were euthanized and femoral condyles and
surrounding tissues like surgical scars, synovium membrane, para-patellar fat
were excised, fixed and processed for histological evaluations. H&E staining
were performed for morphological evaluation of the repaired cartilage.
Masson's trichrome was used to stain the collagen fibers, and
immunohistochemical staining was carried out to check for the presence of
collagen type I and II. Toluidine blue and Safranin-O were used to detect the
presence of proteoglycans. The Wakitani histological grading scale for
cartilage repair (51) was used to compare the quality of the neo-cartilage
tissue (Table 3.1). The iron-labeled cells within the repaired defect were
visualized by Prussian blue staining (197). Localization, fate and possible
interaction of the iron-labeled cells with the surrounding tissue were deduced
by serial section Prussian blue staining of the harvested tissues (197).


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Table 3-1. Histological grading scale for cartilage repair.
(Adapted from (51)).
a
Total smooth area of the reparative cartilage compared with the entire area of the cartilage defect.
b
Average thickness of the reparative cartilage compared with that of the surrounding cartilage.



!
Category
Points

Cell morphology

Hyaline cartilage
0
Mostly hyaline cartilage
1
Mostly fibrocartilage
2
Mostly non-cartilage
3
Non-cartilage only
4


Matrix-staining (metachromasia)

Normal (compared with host adjacent cartilage)
0
Slightly reduced
1
Markedly reduced
2
No metachromatic stain
3


Surface regularity
a

Smooth (>3/4)
0
Moderate (>1/2–3/4)
1
Irregular (1/4–1/2)
2
Severely irregular (<1/4)
3


Thickness of cartilage
b


>2/3
0
1/3–1/2
1
<1/3
2


Integration of donor with host adjacent cartilage

Both edges integrated
0
One edge integrated
1
Neither edge integrated
2

Total maximum
14

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3.3.15 Statistical analysis
ANOVA test was used to compare the multiple groups of the in vitro data, and
paired Student’s T-test was performed to evaluate differences of in vivo
histological data of both treatment groups (scaffold only and stem cell seeded
scaffold). A p value of less than 0.05 was considered as significant differences
between both groups of hyaluronan only (control group) and MSC seeded
hyaluronan (experimental group).

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3.4 Results
3.4.1 Characterization of MSCs
Cells cultured from aspirated BM had characteristic of fibroblastic spindle-
shape morphology and flow cytometry confirmed that more than 96% of the
cells were positive for MSC characteristics, such as adhesion molecules
(CD29, CD44, and CD90), more than 98% of cells were negative for
hematopoietic markers (CD14, CD31, CD34, and CD45), and endothelial
marker (CD31) (figure 3.3).

59!
!
Figure 3-3. Flow cytometry analysis of the stem cells surface markers.
Harvested cells were positive for CD29, CD44, and CD90, and negative for
CD14, CD31, CD45, and CD34.
!

60!
3.4.2 Prussian blue staining of SPIO-labeled MSCs
SPIO labeling success was evaluated by Prussian blue staining (figure 3.4).
The cells appeared to contain more particles with increasing SPIO
concentrations. In the highest concentrations (100 and 125 µg/ml), some iron
clusters were observed attaching to the surface of the cells. Labeling
efficiency (percentage of cells with SPIO) was dependent on the SPIO
concentration in the medium and was > 90% when 50 µg/ml or higher SPIO
was used.
!
Figure 3-4. Prussian blue staining of the unlabeled and labeled MSCs.
Blue dots in the cells demonstrate the presence of iron particles in the cells;
Unlabeled (A), labeled with 25 µg/ml ferucarbotran (B), labeled with 50 µg/ml
ferucarbotran (C), labeled with 75 µg/ml ferucarbotran (D), labeled with 100
µg/ml ferucarbotran (E), labeled with 125 µg/ml ferucarbotran (F).
3.4.3 Transmission Electron Microscopy (TEM)
Since light microscopy cannot discriminate the location of iron particles, TEM
was used to confirm that SPIO were indeed in the cytoplasm of the labeled
MSCs (figure 3.5).

61!
!
Figure 3-5. Transmission electron microscopy of the labeled MSCs.
TEM confirmed the presence of the ferucarbotran particles in the cytoplasm of
the labeled cells.
!
3.4.4 Iron content quantification in labeled-MSCs
As shown in figure 3.6, mean iron content in labeled cells ranged from 3 to
141 pg per cell. The results showed that increasing the ferucarbotran

concentration in the culture media would increase the intracellular iron content
of MSCs.

62!
!
Figure 3-6. Iron content quantification.
Iron content quantification of the MSCs measured by Atomic absorption
spectrometry, the iron uptake by cells is linear up to 50µg/ml but it does not
follow the linear increase in higher concentrations (more than 75µg/ml), which
can be due to extra cellular aggregated iron nanoparticles.
3.4.5 Viability and proliferation of labeled MSCs
There is no significant decrease in cell viability in labeling concentration below
75 µg/ml SPIO. Viability was lower in 100 and 125 µg/ml, 89% and 85%
respectively, (Figure 3.7). MTS showed that labeling with all concentrations of
SPIO did not decrease proliferation rate compared with control (Figure 3.8).
0µg/
ml
25µg/
ml
50µg/
ml
75µg/
ml
100µg
/ml
125µg
/ml
Average iron content per
cell (pg/cell)
0 2 9 26 89 139

0
50
100
150
200
Average iron content per cell
(pg/cell)