Experimental animals
Twenty four male New
Zealand white rabbits, aged 6 months and weighing between 2.0 and 2.5 kg, were
used in this study. All rabbits were certified clinically healthy following
physical and blood profile examinations. The rabbits were housed in individual
cages, fed on commercial diet (Cargill) and drinking water was provided
addlibitum. Physical examination was done weekly during the study period, which
included rectal temperature, pulse and respiratory rate to ensure that they
were in healthy state. Prior to induction of degenerative joint disease,
radiographs of both stifle joints were taken to rule out any possible joint
disease.
Isolation and
characterization of rabbit bone marrow-derived mesenchymal stem cells (BM-MSCs)
The isolation of MSCs
was performed on euthanized rabbits as illustrated by Braga-Silva et al. [18].
This included anesthetizing the rabbits with ketamine-xylazine and subsequently
euthanizing them with sodium pentobarbital (Dolethal). An incision was then
made through the skin on the cranial thigh region and all muscles attached to
the femur were removed to allow for a brief immersion of the femoral bone in
70% alcohol. The femoral bone was later placed in a 50 ml falcon tube
containing media and both ends of the epiphysis were cut using a bone cutter.
Finally, bone marrow was flushed out into a 15ml falcon tube with 5ml media.
The collected bone
marrow was immediately mixed with 5 ml of 83% Dulbecco’s Modified Eagle’s
Medium Ham’s F12 (DMEM F12) that contained high glucose supplemented with 15%
fetal bovine serum (FBS), 1% penicillin/streptomycin (antibiotic) and 1%
amphotericin B (fungi zone) (GIBCO®, USA) as previously described [19,20]. Ten
ml of previously prepared media was placed in a T75 tissue culture flask and
bone marrow suspension was added. The flask was incubated at 37?C in 5% CO2 for 3 days in a
CO2 incubator. Non-adherent cells were removed together with the old
medium and replaced with a fresh medium. After 12 days of incubation, the
culture reached the semi-confluent stage (P0) and the monolayer cells were
washed twice with 2 ml of phosphate buffer saline (PBS) (pH 7.2). Then, two ml
0.2% trypsin in ethylene di amine tetra-acetic acid (EDTA) (Sigma, USA) was
added to the flask and gently mixed for equal distribution in the tissue
culture flask for 2 minutes in order to separate adhered cells from the culture
flask. The cells were examined under an inverted microscope (Olympic, Japan)
until the cells appeared rounded and the trypsin solution was then discarded.
DMEM F12 medium containing 10% FBS was added and gently tapped to detach the
cells from the flask. The trypsination process was repeated for another three
consecutive sub-cultures. The cells were harvested by discarding the medium,
washing with PBS and addition of trypsin to the tissue culture flask in order
to detach the cells. The trypsin solution was then replaced with 10 ml of fresh
DMEM F12. The medium and cells were collected in a test tube, centrifuged
(Hettich, Germany) at 1800 revolutions per minute (rpm) for ten minutes and the
supernatant was decanted to allow for resuspension of the pellet in 2 ml DMEM
F12. The number of cells in each culture flask was quantified using a
haemocytometer (Neubaur, Haemocytometer, Hawksleyand son. Ltd, England). Cell
suspension (0.1 ml) was removed in a sterile manner and added to a dilution
tube containing 0.8 ml of DMEM F12 and 0.1 ml of 0.4% Trypan Blue stain. The
mixture was gently mixed at room temperature and a small drop of the stained
cell suspension was transferred onto the haemocytometer and cover slip placed
on top. A Small drop of the cell suspension was removed aseptically using a
Pasteur pipette and placed on one side of the haemocytometer and examined under
the inverted microscope (Leica, Auterian). The total number of viable cells in
each four corners of the haemocytometer was counted. The total number of cells
harvested from the tissue culture flasks was determined using the following
equation: NCxDx104/Q, where NC=number of count vital cells (non-vital cell is
stained blue), D=sample dilution (10) and Q=number of squares used in haemocytometer
[21]. At 1st passage the stem cells were preserved using liquid nitrogen N2.
Since freezing can be lethal to cells due to the effects of damage by ice
crystals, alteration in the concentrations of electrolytes, dehydration and
changes in PH, a typical freezing medium containing 90% serum and 10% Dimethyl
sulfoxide (DMSO) was used, as reported by Fleming and Hubel, [22] and Linch et
al., [23]. The isolated cells were pre-characterized by their morphology,
multipotency and immunophenotyping characters of stem cells to ensure the
isolated cells were mesenchymal stem cell (MSCs) in nature.
Induction of
osteoarthritis
Rabbits were
anaesthetized using intra-muscular injection of Ketamine hydrochloride -
xylazine hydrochloride - acepromazine at the dose rate of 40 mg / kg, 5 mg / kg
and 1 mg / kg respectively. Adequate anesthesia depth was monitored based on
eyes and intra-digital reflexes, heart rate, respiratory rate and response to
stimuli. The hair over the left stifle joint was clipped and the skin was aseptically
prepared as is routine using chlorhexidine scrub, 70% alcohol and tincture
iodine. A 26–gauge 1 ½ inches hypodermic needle was used to inject 2.5 mg MIA /
0.3 ml NS intra-articularly. The needle was inserted into the mid-line and
advanced between the femoral epicondyles and menisci. Any resistance to
injection was taken as evidence that the needle was not being introduced into
the joint cavity, and in such cases the needle was re-positioned before
attempting to administer the MIA. The needle was withdrawn when the injection
was complete. Care was taken not to have any evidence of leakage through the
needle tract [24,25].
Protocol of treatment
The current methods of
treatments using both allogeneic and xenogeneic MSCs were explored for their
potential to regenerate damaged tissues by OA in MIA-induced model of OA in
rabbit’s stifle joint. Four treatment groups were used in this study (Six
rabbits were used in each group). The first group, the rabbit stem cell-treated
group (RSTG) was given a single intra articular injection of rabbit bone
marrow-derived MSCs at a density of 1.5X106 cells / 0.3 ml media (the cells
were in the second passage, and were derived from an anesthetized rabbit and
cryopreserved at -20 ?C).
The second group, the media stem cell-treated group (MSTG) received an
injection of the same volume of medium without cells into the osteoarthritic
stifle joints. The third group, the sodium hyaluronate-treated group (SHTG) was
treated with four injections of 0.3 ml 0.1% sodium hyaluronate at weekly
intervals starting 4 weeks post OA induction. Lastly, The fourth group (the
control group), the NS-treated group (NSTG) was given a single intra-articular
injection of 0.3 ml NS in the affected stifle joints.
Note: All rabbits were
exercised for 10 mins daily during the whole study period, except for the first
month after initiation of treatments, in which case the rabbits were rested.
Histopathology
evaluation
The stifle joints of
both legs were fixed in 10 % formalin for about two months, followed by
decalcification with EDTA + 12% hydrochloric acid for about one month. The
decalcification solution was changed twice per week. Samples of both tibia and
femur were firstly separated into medial and lateral parts and further
subdivided into two parts. The samples were dehydrated through an ascending
series of ethanol, followed by clearing with xylene and finally, impregnation
in paraffin using an automated tissue processing apparatus. After embedding in
paraffin blocking, sectioning was achieved using microtome, and the slides were
stained with Hematoxylin-Eosin (H and E) and Safranin O stains. Then,
histological images were captured using a microscope image analyzer (OLYMPUS)
According to Bancroft, [26]. Histopathological changes of the articular cartilage
and subchondral bone were evaluated after staining with H and E stain using the
scoring system described by Kobayashi et al. [27]. Briefly, the degree of
changes were graded as no changes (normal): 0, slight changes (mild): 1,
moderate changes (moderate): 2 and severe changes (severe or very severe): 3.
The grading items included chondrocyte cells loss, chondrocyte cloning and
hypertrophy, chondrocyte disorganization, surface irregularity of articular
cartilage, fibrillation of articular cartilage surface, Safranin O stain
reduction, degeneration/necrosis, marginal osteophyte formation and subchondral
changes (Table 1).
Normal (no changes)
indicates absence of OA lesion in articular cartilage and subchondral bone.
Mild changes denote only small or focal area (less than 50%) of the articular
cartilage or subchondral bone showing changes. Moderate changes showed that
about 50% of articular cartilage or subchondral region was affected. Severe
changes indicated histopathological changes of large area (more than 50%) of
articular cartilage or subchondral region.