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

Adipogenic differentiation was induced by culturing 3×105 cells/cm2 in adipogenic medium consisting of high glucose DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml strepto

Adipogenic differentiation was induced by culturing 3×105 cells/cm2 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 6×104 cells/cm2 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 2.5×105 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% 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

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

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 107 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 (107 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

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),

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

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)

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)

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

Cell morphology

Matrix-staining (metachromasia)

Normal (compared with host adjacent cartilage) 0

Surface regularity a

Thickness of cartilage b

Integration of donor with host adjacent cartilage

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)

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)

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

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)

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

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

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